Structural analyses of (1->3),(1->4)-beta-D-glucan of oats...

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Structural analyses of (13),(14)-β- D-glucan of oats and barley Liisa Johansson ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Auditorium 1041 in Viikki Biocenter, on April 7 th 2006, at 12 o’clock noon. University of Helsinki Department of Applied Chemistry and Microbiology General Chemistry Division Helsinki 2006

Transcript of Structural analyses of (1->3),(1->4)-beta-D-glucan of oats...

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Structural analyses of (1→3),(1→4)-β-D-glucan of oats and barley

Liisa Johansson

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in

Auditorium 1041 in Viikki Biocenter, on April 7th 2006, at 12 o’clock noon.

University of Helsinki Department of Applied Chemistry and Microbiology

General Chemistry Division

Helsinki 2006

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Custos: Professor Vieno Piironen Department of Applied Chemistry and Microbiology University of Helsinki Helsinki, Finland Supervisors: Professor Pertti Varo Department of Applied Chemistry and Microbiology University of Helsinki Helsinki, Finland

Ph.D., dos. Liisa Virkki Department of Applied Chemistry and Microbiology University of Helsinki Helsinki, Finland Reviewers: Ph.D. Peter J. Wood

Agriculture and Agri-Food Canada Guelph, Ontario, Canada Ph.D., dos. Bo Hortling KCL Espoo, Finland

Opponent: Dr. Henk Schols

Department of Agrotechnology and Food Sciences Wageningen University Wageningen, The Netherlands

ISBN 952-10-3000-3 (paperback) ISBN 952-10-3001-1 (PDF) ISSN 0355-1180 Yliopistopaino Helsinki 2006 Photo of front cover: Mikko Heikkinen

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Johansson, L. 2006. Structural analyses of (1→3),(1→4)-β-D-glucan of oats and barley (dissertation). EKT series 1354. University of Helsinki, Department of Applied Chemistry and Microbiology. 85 pp. ABSTRACT The structures of (1→3),(1→4)-β-D-glucans of oat bran, whole-grain oats and barley and processed foods were analysed. Various methods of hydrolysis of β-glucan, the content of insoluble fibre of whole grains of oats and barley and the solution behaviour of oat and barley β-glucans were studied. The isolated soluble β-glucans of oat bran and whole-grain oats and barley were hydrolysed with lichenase, an enzyme specific for (1→3),(1→4)-β-D-β-glucans. The amounts of oligosaccharides produced from bran were analysed with capillary electrophoresis and those from whole-grains with high-performance anion-exchange chromatography with pulse-amperometric detection. The main products were 3-O-β-cellobiosyl-D-glucose and 3-O-β-cellotriosyl-D-glucose, the oligosaccharides which have a degree of polymerisation denoted by DP3 and DP4. Small differences were detected between soluble and insoluble β-glucans and also between β-glucans of oats and barley. These differences can only be seen in the DP3:DP4 ratio which was higher for barley than for oat and also higher for insoluble than for soluble β-glucan. A greater proportion of barley β-glucan remained insoluble than of oat β-glucan. The molar masses of soluble β-glucans of oats and barley were the same as were those of insoluble β-glucans of oats and barley. To analyse the effects of cooking, baking, fermentation and drying, β-glucan was isolated from porridge, bread and fermentate and also from their starting materials. More β-glucan was released after cooking and less after baking. Drying decreased the extractability for bread and fermentate but increased it for porridge. Different hydrolysis methods of β-glucan were compared. Acid hydrolysis and the modified AOAC method gave similar results. The results of hydrolysis with lichenase gave higher recoveries than the other two. The combination of lichenase hydrolysis and high-performance anion-exchange chromatography with pulse-amperometric detection was found best for the analysis of β-glucan content. The content of insoluble fibre was higher for barley than for oats and the amount of β-glucan in the insoluble fibre fraction was higher for oats than for barley. The flow properties of both water and aqueous cuoxam solutions of oat and barley β-glucans were studied. Shear thinning was stronger for the water solutions of oat β-glucan than for barley β-glucan. In aqueous cuoxam shear thinning was not observed at the same concentration as in water but only with high concentration solutions. Then the viscosity of barley β-glucan was slightly higher than that of oat β-glucan. The oscillatory measurements showed that the crossover point of the G´ and G´´ curves was much lower for barley β-glucan than for oat β-glucan indicating a higher tendency towards solid-like behaviour for barley β-glucan than for oat β-glucan.

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PREFACE

This work was carried out at the Department of Applied Chemistry and Microbiology,

University of Helsinki. The work was financially supported by the University of

Helsinki, the National Technology Agency of Finland and the Jenny and Antti Wihuri

Foundation.

I want to express my gratitude, although too late, to our late professor Pertti Varo for

taking me into the staff, encouraging me to start this work and supporting me in my

efforts while doing it. I also thank prof. Vieno Piironen for her support and guidance

which she gladly offered whenever requested. Her critical comments greatly helped in

finishing of the thesis. Prof. Maija Tenkanen is warmly thanked for her support and

encouragement during the work.

I am deeply grateful for the pre-examiners, Ph.D. Peter Wood and dos. Bo Hortling

for their devoted work on the pre-examination of my work and for the constructive

comments they gave.

My sincerest and warmest thanks go to my supervisor, my closest colleague and my

dear friend dos. Liisa Virkki. No words can express the amount of help and support

and friendship she has given me throughout these years. Our co-operation started

when she said: We could do something together. That something turned out to be this

thesis.

I owe all those who have been members of the group during these years for the

wonderful co-operation, enthusiasm and great times together. Dos. Päivi Ekholm was

the first to join the group and started the other line of research concerning minerals

and dietary fibre. Dos. Päivi Tuomainen brought along her outstanding knowledge on

chromatography which was crucial for the introducing of the HPAEC-PAD-system.

M.Sc. Marja Lehto is greatly acknowledged for her skilful and enthusiastic work in

the laboratory. Special thanks go to M.Sc. Maija Ylinen for her diverse know-how,

responsible work and good spirits always. Our pro-gradu-student Heli Esselström I

want to thank for her outstanding work on the hydrolysis of β-glucan. Her enthusiasm

and positive attitude made it joyful to work with her.

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The co-operation with the Division of Cereal Technology was not only crucially

important but also greatly widened my knowledge of cereal chemistry. I thank them

all, prof. Hannu Salovaara, D.Sc. Tuula Sontag-Strohm and M.Sc. Heli Anttila for the

inspiring conversations and co-operation during the many phases of this work.

Prof. Sirkka Maunu and prof. Heikki Tenhu at the Laboratory of Polymer Chemistry

are not only co-authors but also my good, old friends. Our history goes back a great

number of years. I thank them warmly for their friendship and help and support

through the years. Special thanks go to Mikko Karesoja for his careful and

enthusiastic work on the rheology of β-glucans.

I cordially thank Ph.D. Hannu Rita for the statistical analyses and the many wonderful

conversations we had. They opened quite new perspectives for me into the world of

statistics.

I sincerely thank the personnel of the Division of General Chemistry for the warm and

friendly atmosphere that I have enjoyed ever since I joined the staff. Help has always

been available when I needed it.

Finally, I want to thank my mother Aili, my late father Veikko and my dear sisters,

Leena and Katri, for their love and support on all occasions of my life.

Last but not least I thank my wonderful daughters, Inka and Milla, for bringing joy to

my life.

Helsinki, February 2006

Liisa Johansson

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LIST OF ORIGINAL PUBLICATIONS The thesis is based on the following original publications which are referred to in the text by their Roman numerals. I Johansson L, Virkki L, Maunu S, Lehto M, Ekholm P, Varo P. 2000.

Structural characterization of water-soluble β-glucan of oat bran. Carbohydrate Polymers 42: 143-148.

II Johansson L, Tuomainen P, Ylinen M, Ekholm P, Virkki L. 2004. Structural analysis of water-soluble and -insoluble β-glucans of whole-grains of oats and barley. Carbohydrate Polymers 58: 267-274.

III Johansson L, Tuomainen P, Anttila H, Rita H, Virkki L. The effect of processing on the structure of β-glucan of oats. Cereal Chemistry, submitted. IV Johansson L, Virkki L, Anttila H, Esselström H, Tuomainen P, Sontag-

V Virkki L, Johansson L, Ylinen M, Maunu S, Ekholm P. 2005. Structural characterization of water-insoluble nonstarchy polysaccharides of oats and

barley. Carbohydrate Polymers 59: 357-366. VI Johansson L, Karesoja M, Ekholm P, Virkki L, Tenhu H. Comparison of the

solution properties of (1→3),(1→4)-β-D-glucans extracted from oats and barley. Food Science and Technology, submitted.

The papers are reproduced with kind permission from the publishers. Contribution of the author to papers I to VI I The author planned the study together with the other authors and was

responsible for the experimental work except for the NMR analyses. The author wrote the manuscript.

II The author planned the study together with the other authors and was

responsible for the experimental work except for the NMR analyses. The author wrote the manuscript.

III The author planned the study with the other authors. She performed the

experimental work and wrote the manuscript. PhD Rita carried out the statistical analyses and wrote the part of the manuscript concerning statistics together with the author.

Strohm T. Hydrolysis of β-glucan, Food Chemistry 97: 71-79.

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IV The author planned the study together with the other authors and was responsible for the experimental work together with the other authors. She wrote the manuscript in cooperation with the other authors.

V The author planned the study together with the other authors. She took part to

the finishing of the manuscript. VI The author planned the study together with the other authors. She wrote the

manuscript.

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CONTENTS ABSTRACT PREFACE LIST OF ORIGINAL PUBLICATIONS LIST OF ABBREVIATIONS 1 INTRODUCTION 11 2 REVIEW OF LITERATURE 13 2.1 Dietary fibre 13 2.2 Health effects of cereal β-glucan 14 2.3 Determination of the amount of β-glucan 17 2.4 Extraction of β-glucan 18 2.4.1 Extraction of soluble β-glucan 18 2.4.2 Extraction of insoluble β-glucan 21 2.5 Structure of β-glucan 21 2.6 Solution properties of β-glucan 25 2.7 Structural analysis methods 26 2.7.1 Hydrolysis 26 2.7.1.1 Acid hydrolysis 26 2.7.1.2 Methylation analysis 28 2.7.1.3 Enzymatic hydrolysis 28 2.7.2 Chromatographic and capillary electrophoresis methods 30 2.7.2.1 Liquid chromatography 30 2.7.2.2 Gas chromatography 31 2.7.2.3 Capillary electrophoresis 31 2.7.3 Spectroscopic methods 32 2.7.3.1 Nuclear magnetic resonance 32 2.7.3.2 Infrared spectroscopy 33 2.8 Molar mass 33 2.9 Effects of processing on β-glucan 35 3 AIMS OF THE STUDY 37 4 MATERIALS AND METHODS 38 4.1 Materials 38 4.2 Isolation of β-glucan and fractionation of water insoluble fibre 39 4.2.1 Isolation of soluble β-glucan 39 4.2.2 Fractionation of water insoluble dietary fibre and isolation

of insoluble β-glucan 41 4.2.3 Isolation of soluble and insoluble fibre and β-glucans from processed foods 41

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4.3 Hydrolysis of β-glucan 42 4.3.1 Acid hydrolysis 42 4.3.2 Lichenase hydrolysis 43 4.3.3 Determination of β-glucan content by the AOAC method 43 4.4 Chromatographic and capillary electrophoresis methods 44 4.4.1 Gas chromatography 44 4.4.2 Capillary electrophoresis 45 4.4.3 High performance anion exchange chromatography 46 4.4.4 Molar mass determination 47 4.5 Spectroscopic methods 47 4.5.1 Nuclear magnetic resonance spectroscopy 47 4.5.2 Infrared spectroscopy 48 4.6 Rheology 48 4.7 Statistical analysis 49 4.8 Summary of analytical methods and samples 50 5 RESULTS 50 5.1 Comparison of the soluble β-glucans of oat bran and whole-grain

oats 50 5.2 Comparison of the β-glucans of whole-grain oats and barley 52

5.3 The effects of processing 54 5.4 Comparison of the insoluble dietary fibre of whole-grain oats

and barley 55 5.5 Hydrolysis of β-glucan 57 5.6 Rheology of β-glucans of whole-grain oats and barley 59 6 DISCUSSION 60 6.1 Comparison of the soluble β-glucans of oat bran and whole-grain

oats 60 6.2 Comparison of the β-glucans of whole-grain oats and barley 61

6.3 The effects of processing 64 6.4 Comparison of the insoluble dietary fibre of whole-grain oats and barley 66 6.5 Hydrolysis of β-glucan 67 6.6 Rheology of β-glucans of whole-grain oats and barley 69 7 CONCLUSIONS 70 8 REFERENCES 72 ORIGINAL PUBLICATIONS I-VI

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LIST OF ABBREVIATIONS AACC The American Association of Cereal Chemists ANOVA analysis of variance AOAC Association of Official Analytical Chemists ATR attenuated total reflection CE capillary electrophoresis CP-MAS NMR cross polarization-magic angle spinning nuclear

magnetic resonance db dry matter basis DF dietary fibre DP degree of polymerisation FDA The Food and Drug Administration FT-IR Fourier-transform infrared spectroscopy G´ storage modulus G´´ loss modulus GC gas chromatography GC-MS gas chromatography with mass spectrometric detection HDL cholesterol high density lipoprotein cholesterol HPLC high-performance liquid chromatography HPSEC high-performance size exclusion chromatography HPAEC-PAD high-performance anion-exchange chromatography pulse amperometric detection HSQC heteronuclear single quantum coherence IR infrared LDL cholesterol low density lipoprotein cholesterol Mn number average molar mass Mw weight average molar mass Mp peak molar massMr relative molar mass NIDDM non-insulin dependent diabetes mellitus NMR nuclear magnetic resonance 2D-NMR two-dimensional NMR RI refractive index UV ultraviolet WIS water insoluble fibre

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1 INTRODUCTION

Many studies have shown the beneficial health effects of oats. Consuming oats lowers

the levels of blood cholesterol and attenuates postprandial glucose response (Braaten

et al., 1994; Wood et al., 1994a, b; Wood et al., 2000; Mälkki and Virtanen, 2001;

Cavallero et al., 2002). This is believed to be caused by the main component of

soluble dietary fibre of oats called (1→3),(1→4)-β-D-glucan also referred to as β-

glucan (Ripsin et al., 1992; Tappy et al., 1996; Drzikova, 2005). The positive health

effects are believed to be caused by the viscosity of β-glucan (Guillon and Champ,

2000; Morgan, 2000; Wood, 2004). Viscosity in turn is affected by concentration and

molar mass. Consequently, these factors must be taken into account when health

effects are to be concidered. Therefore extractability and the effects that processing

may have on it are important. An increase in faecal butyric acid and an improvement

of symptoms in patients with ulcerative colitis has been reported by Nyman (2003). β-

glucans from fungi and cereals have been shown to be immunostimulators. They bind

to receptors on macrophages and other white blood cells and activate them, thus they

enhance the resistance to infections (Brown and Gordon, 2001; Yun et al., 2003).

(1→3),(1→4)-β-D-glucan is found in the endosperm cell walls and in the subaleurone

layer of oats and barley (Miller and Fulcher, 1994; 1995). Oats contain 1.8-7.9% β-

glucan (Bhatty, 1992; Welch, 1995; Oscarsson, 1996; Saastamoinen et al., 2004). The

percentage varies with cultivar and environmental effects (Saastamoinen et al., 1992;

Miller et al., 1993). Barley contains 2.8-11% of β-glucan whereas in wheat and rye

the main component of soluble dietary fibre is arabinoxylan and the amounts of β-

glucan are small (Fincher and Stone, 1986) although Henry (1987) reports 2.5% of β-

D-glucan in rye.

Cereal mixed-linkage (1→3),(1→4)-β-D-glucan is a linear polysaccharide made up

entirely from glucose. Sequences of (1→4)-linked D-glucopyranosyl units are

separated by single (1→3)-β-linked units. (1→4)-linked sequences are mainly 3 or 4

glucose units long but sequences up to 13 in water soluble and up to 20 in water

insoluble β-glucans have been found (Izydorczyk, 1998a, b). The solubility of β-

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glucan is enhanced by the (1→3)-linkages compared to cellulose which only has

(1→4)-linkages. These linkages make the molecule less regular in shape than

cellulose and thus prevent it from packing into highly ordered structures (Fincher and

Stone, 1986). Some β-glucan is unextractable by water but the reason for this is not

known.

The molar mass of β-glucan has been widely studied, but the results vary greatly.

Autio (1996), in a review, reports values between 49 000 and 3x106 g/mol. The value

strongly depends both on the conditions of isolation and the method used for analysis

(Forrest and Wainwright, 1977; Woodward et al., 1983; Autio, 1996; Beer et al.,

1997a; Goméz et al., 1997a). The molar mass of extracted β-glucan may also vary

between cultivars (Jaskari et al., 1995; Beer et al., 1997a) and is effected by heat

treatments of the starting material (Beer et al., 1997b; Zhang et al., 1998; Nyman,

2003).

Soluble β-glucan can be isolated by extraction with water or a buffer at various

temperatures. Enzymatic hydrolyses of starch and protein and extraction of fat with

solvents can be included in the method. The method of Westerlund (1993) involves

defatting, extraction by water, enzyme hydrolyses of starch and proteins, precipitation

of polysaccharides by ethanol, solubilisation and precipitation of β-glucan by 30%

(NH4)2SO4 solution, dialysis and drying. Insoluble β-glucan can be extracted from the

water-insoluble fibre by water after arabinoxylans have been removed with Ba(OH)2

(Gruppen et al., 1992).

For analyses β-glucan can be hydrolysed to glucose by acids, the first step in

establishing structure. The released glucose is most commonly analysed by gas or

liquid chromatography or by oxidase or hexokinase. Enzymatic hydrolysis may yield

oligosaccharides which can be analysed by liquid chromatography and which can

provide structural information on sequence units, or which can be further hydrolysed

by enzyme to glucose for quantitation. NMR spectroscopy can be used for non-

destructive structural analysis.

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There are only a few studies on the composition of water-insoluble fibre of oats and

barley. Claye et al. (1996) studied the soluble and insoluble fibre content and

composition of five fibre sources, including oats. They found cellulose, lignin,

insoluble pectin and two fractions of hemicelluloses. Manthey et al. (1999) studied the

soluble and insoluble dietary fibre content and chemical composition of oats without

fractionating the fibre. Aalto and Varo (1988) reported the content of soluble and

insoluble fibre of Finnish barley varieties but the compositions were not studied.

2 REVIEW OF LITERATURE

2.1 Dietary fibre

Dietary fibre (DF), an essential part of the human diet consisting of many substances

of plant origin that are not digested in the human upper gastrointestinal tract includes

polysaccharides such as cereal β-glucans, arabinoxylans and cellulose. Starch is not a

part of DF because it is hydrolysed by enzymes and absorbed in the small intestine.

These components are located in the cell walls of the grain. The starchy endosperm is

the storage tissue of the grain. Outside of the endosperm are the aleurone and

subaleurone layers which protect the grain and provide the enzymes needed in the

germination. The outer layers, the seed coat and the pericarp contribute significantly

to the insoluble dietary fibre content of the grain (Fincher and Stone, 1986; Fulcher

and Miller, 1993).

According to Schneeman (2001) DF regulates the rate of nutrient digestion and

absorption, serves as a substrate for the microflora of the gut and promotes laxation.

The fibre affects gastrointestinal function through properties including viscosity,

water holding capacity, bulk, fermentability and binding of bile acids (Schneeman,

2001). Jenkins et al. (2001) state that an important feature of fibre is to reduce the rate

of absorption of nutrients. According to these authors, one effect of fibre is to convert

the carbohydrate components into a slow release form that requires less insulin and

also increases the elimination of bile acids. Fibre also alters the colonic short-chain

fatty acid profiles.

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What is included in dietary fibre has been under debate for a long time. The American

Association of Cereal Chemists has adopted a definition for dietary fibre (AACC,

2001). The definition is as follows:

“Dietary fiber is the edible parts of plants or analogous carbohydrates that are

resistant to digestion and absorption in the human small intestine with complete or

partial fermentation in the large intestine. Dietary fibre includes polysaccharides,

oligo-saccharides, lignin, and associated plant substances. Dietary fibres promote

beneficial physiological effects including laxation, and/or blood cholesterol

attenuation, and/or blood glucose attenuation.”

According to this definition, oat and barley β-glucans are components of dietary fibre

since they are plant polysaccharides resistant to digestion and absorption in the small

intestine, and they attenuate both blood cholesterol and glucose.

2.2 Health effects of cereal β-glucan

Oat β-glucan is known to reduce blood cholesterol levels. The Food and Drug

Administration of the USA has accepted a health claim in which it is stated that a

daily intake of 3 g of soluble oat β-glucan can lower the risk of coronary heart disease

(FDA, 1997). Oats also reduce the glucose and insuline responses.

Ripsin et al. (1992) showed in a meta-analysis that oat products in a diet cause a

modest reduction in blood cholesterol level. They concluded that a daily intake of 3 g

of oat soluble fiber was needed to reduce cholesterol 0.13-0.16 mmol/L. The

reduction was greater for those having a high initial cholesterol level. Lia et al. (1995)

studied the effect of β-glucan on the excretion of bile acids using breads baked with

oat bran, oat bran with β-glucanase, barley or wheat in the diet of ileostomy subjects.

They showed that the excretion of bile acids was 53% higher with the oat bran bread

than with the bread containing oat bran and β-glucanase, and also significantly higher

than with barley and wheat bread. The excretion of cholesterol was higher for barley

bread than for wheat or oat bran-β-glucanase bread. Braaten et al. (1994) showed that

isolated oat β-glucan mixed in a drink significantly reduced the total and LDL

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cholesterol levels of hypocholesterolemic adults but did not change the level of HDL

cholesterol. In a meta-analysis of studies on pectin, oat bran, guar gum and psyllium

Brown et al. (1999) showed that soluble fibre lowered cholesterol but the effect was

small. Sayar et al. (2005) studied the effect of oat flour on the binding of bile acids in

an in vitro digestion system. They showed that oat flour bound 7.5-14.8% of bile

acids which is higher than for flour from other grains. No significant correlation was

found between β-glucan content of the flour or molar mass of β-glucan and bile acid

binding. However, a significant correlation between bile acid binding and insoluble

fibre content was found. The authors conclude that the binding of bile acids is a

multicomponent-dependent process and also that physical conditions, such as pH,

viscosity and temperature, and physical properties of the dietary fibre preparation

influence the interaction. The in vitro studies of Drzikova et al. (2005) showed that

the binding of bile acids increased with increasing proportion of oat bran, total dietary

fibre, insoluble dietary fibre and β-glucan.

Wood et al. (1990) showed that both oat gum and guar gum significantly decreased

the postprandial glucose rise. Tappy et al. (1996) reported that approximately 5 g of

oat β-glucan present in extruded breakfast cereals caused a 50% decrease in

glycaemic response. Moreover, Tappy et al. (1996) found that when subjects with

NIDDM (non-insulin dependent diabetes mellitus) were fed oat β-glucan-enriched

breakfast cereals a 50% decrease in glycaemic response and a 35% decrease in insulin

response were observed.

Studies on barley β-glucan show similar health effects as with oat β-glucan. Kahlon et

al. (1993) showed that barley as well as oat β-glucan significantly lowered the

cholesterol level of hamsters. The work of Bourdon et al. (1999) showed that barley

β-glucan lowered the cholesterol concentration and attenuated the insulin response in

humans. Kalra and Jood (2000) showed that barley β-glucan lowered the levels of

total cholesterol, LDL-cholesterol and triglycerides in rats. Cavallero et al. (2002)

studied the effect of barley β-glucan on human glycaemic response. A linear decrease

in glycaemic index for increasing β-glucan content was found.

The mechanisms by which these effects are mediated are not well understood.

Edwards and Parrett (1996) suggested several possible mechanisms for lowering

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cholesterol levels. These include inhibition of fat digestion and absorption, increased

loss of bile acids and cholesterol, inhibition of cholesterol synthesis in the liver by

propionate or other bacterial products and the action of viscous non-starch

polysaccharides (NSP) on insulin and other hormone secretions. Kahlon (2001)

concluded that possible mechanisms of cholesterol reduction of oat bran include

increased faecal cholesterol and bile acid excretion, increased gastrointestinal tract

length and increased production of short-chain fatty acids. For barley Kahlon (2001)

suggested an additional mechanism, i.e. the inhibition of the rate-limiting enzymes for

cholesterol and bile acid synthesis. According to Mälkki and Virtanen (2001), high

viscosity retards the mixing of luminal contents which may retard the transport of

digestive enzymes to their substrates, impair the emulsification of lipids and retard the

transportation of nutrients to the absorbing surface and increase the thickness of the

unstirred layer on the absorbing surface. Morgan (2000) stated that the health effects

are believed to be caused by water soluble mixed-linkage β-glucan and its viscosity.

Lia et al. (1995) concluded that the increase in bile acid excretion probably explains

the effect of oat fiber in lowering of serum lipids. The work of Wood (1994) clearly

showed that there is a highly significant relationship between viscosity and the

glucose and insulin responses. According to Wood (2001; 2004) the properties

responsible for the health effects are flow viscosity and viscous gelling. These factors

are controlled by structure, molar mass and concentration, therefore the need for

characterizing of the β-glucan used was emphasized.

Health effects of oat fibre other than lowering of glucose and cholesterol have been

reviewed by Mälkki and Virtanen (2001). Oat fibre prolongs satiety after meals and

alleviates constipation. In the stomach and small intestine, it is mediated by a

viscosity effect that retards the transport of enzymes to their substrates. Increased

viscosity also retards the transport of nutrients to the absorbing sites and to the

unstirred layer on the absorbing surface. In the large bowel, oat fibre acts as a

substrate for fermentation enhancing the formation of butyric acid. Butyric acid is

believed to enhance the growth of normal colonic cells but decrease the growth of

carcinogenic cells. Karppinen et al. (2000) reported that the amounts of short-chain

fatty acids produced in fermentation were higher with oat bran than with rye and

wheat brans. This may assist in maintaining a healthy mucosa. According to Nyman

(2003) a diet rich in oats decreased the symptoms of ulcerative colitis by faecal

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butyric acid formation. However, Sundberg et al. (1996) showed that β-glucan

undergoes depolymerisation during transit through the upper gastrointestinal tract in

humans. They reported that 13-64% of β-glucan was lost when intake was compared

to excretion. Further, Johansen et al. (1993; 1997) reported a decrease of molar mass

of β-glucan in the upper gastrointestinal tract of pigs fed oat based diets.

The solution properties of molecules are affected by their structure, concentration and

molecular size. The amount of β-glucan is insufficient to determine the health effects

of β-glucan but extractability and molar mass must also be considered. These data are

not taken into account in many of the studies and so the results are often controversial.

In addition, there are few data about the effects of processing on the properties of β-

glucan.

2.3 Determination of the amount β-glucan

The amount of β-glucan is an important factor in considering health effects. In the

isolation processes some β-glucan may be lost. Thus, the total β-glucan content can

not be determined from the isolated β-glucan but instead must be done on the original

sample.

The most frequently used method for β-glucan determination is that of McCleary and

Codd (1991) which is also the Association of Official Analytical Chemists (AOAC)

995.16 method. This method involves the dissolution of β-glucan in a buffer,

hydrolysis with the lichenase enzyme to oligosaccharides and with β-glucanase to

glucose. Glucose is then analysed spectrophotometrically as a coloured substance

obtained with an oxidase/peroxidase reagent (Saastamoinen et al., 1992; Zygmunt and

Paisley, 1993; Wood, 1994; Åman, 1995; Lambo et al., 2005). The official method is

designed for samples within a certain range of β-glucan concentrations and needs

adjustment when the concentration is high. Another way is the use of dyes. Wood et

al. (1983) reported that Calcofluor and Congo Red are specific dyes for β-glucans and

can be used for locating β-glucan in cell walls. Wood and Weisz (1984) precipitated

oat β-glucan with Calcofluor and hydrolysed the β-glucan-Calcofluor-complex by

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acid to glucose which was then analysed by HPLC. Suortti (1993) has used post-

column dying with Calcofluor and fluorescence detection in HPSEC-analyses of β-

glucans in order to measure the molar mass and the amount of β-glucan with minimal

sample preparation. Åman and Graham (1987) found close correlations between an

enzymatic method and a fluorometric method with Calcofluor binding and flow

injection analysis for determination of oat and barley β-glucans.

2.4 Extraction of β-glucan

2.4.1 Extraction of soluble β-glucan

β-glucan can be extracted in many ways. The yield of extraction depends mainly on

the method used due to variables such as solvent and temperature of extraction.

Moreover, genotype and growing-year of the crop affect the β-glucan content

(Manthey et al., 1999). Water at different temperatures dissolves molecules of

different sizes and gives different yields. Izydorczyk et al. (1998a) extracted barley β-

glucan at 40 °C and 60 °C. The yields were 1.4 and 1.3% of the total barley grist,

respectively. They report small structural differences, higher molar mass and limiting

viscosity number for the 60 °C extract than for the 40 °C extract. Robertson et al.

(1997) extracted barley β-glucan using 0.15 M NaCl at 38, 65 and 100 °C and

obtained yields of 28%, 49% and 55%, respectively. Wood et al. (1991b) used water

and sodium carbonate at pH 10 at temperatures of 44, 60 and 80 °C. At 80 °C the

yield of β-glucan in carbonate buffer was 88% of the total amount, at 60 °C the yield

was 73% and at 45 °C it was 44%. In water extracts the yields were lower, i.e. 46, 37

and 33% respectively. Bhatty (1995) extracted β-glucan with 0.25 M, 0.5 M, 0.75 M

and 1 M NaOH at room temperature and obtained yields 77%, 88%, 98% and 97%,

respectively. Carr et al. (1990) extracted β-glucan from food products and cereal

grains with water at 100 °C for 1 h to obtain the water soluble β-glucan and with 1M

NaOH at 20 °C for 16 h for total extraction. Cui et al. (2000) extracted β-glucan from

wheat bran with 1 M NaOH at 25 °C. They report that a decrease of pH from 7 to 4.5

significantly increased the solubility of wheat β-glucan. Bhatty (1992) extracted β-

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glucan from oats with an acidic buffer (pH 1.5) at room temperature. The yield was

23% of the total, with water the yield was 39% and at pH 10.0 it was 26%. Åman and

Graham (1987) report that considerably more β-glucan from oats and barley was

solubilised with water at 38 °C than with acidic buffer. Rimsten et al. (2003)

compared four extraction methods for oats, barley, rye and wheat. The alkaline

extractions were those of Wood et al. (1991a) using Na2CO3 and of Suortti (1993)

using NaOH. The third method involved hot water and heat-stable α-amylase. The

fourth method used water and different xylanases. The highest yields were obtained

by the NaOH extraction for most cereals. However, the Na2CO3 method gave the

highest yields for oat bran. Beer et al. (1997b) used an in vitro system to extract β-

glucan from oat bran and rolled oats. The procedure involves the use of human

salivary α-amylase, porcine pepsin and pancreatin. The temperature was kept at 37 °C

throughout the process. The yield was 12-33%. The same samples were also extracted

with hot water and NaOH also. With the hot water extraction methods, the yield was

50-65% for bran and 70% for rolled oats. With the NaOH method at 22°C the yield

was 98%. Sayar et al. (2005) used the same procedure as Beer et al. (1997b) with

minor changes. The overall digestibility was 76.9-81.4%.

Very pure β-glucan can be produced using the method of Westerlund et al. (1993). It

involves defatting with propan-2-ol (isopropanol, IPA) and petroleum ether,

dissolution in water at 96 °C and hydrolysis of starch with heat-resistant α-amylase.

Proteins are hydrolysed with pancreatin. The polysaccharides are precipitated with

60% ethanol at 4 °C and the precipitate is dissolved in water. The solution is treated

with 30% (NH4)2SO4, which specifically precipitates β-glucan but leaves

arabinoxylans in solution. The precipitate is dissolved in water and dialyzed against

water at room temperature. The resulting β-glucan solutions are freeze dried. The

different extraction methods are shown in Table 1.

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Table 1. Methods for extraction of soluble β-glucan.

Material Extraction solvent Temperature °C Yield Reference

Barley water 40, 60 n.r. Izydorczyk et al., 1998a

Oats, barley wheat, malt, breakfast cereals

water Na2CO3 pH 10 44, 60, 80 33%, 37%, 46%

44%, 73%, 88% Wood et al., 1991b

Food products, cereal grains

water 1 M NaOH

100 20

n.r. total Carr et al., 1990

Barley and oat brans NaOH 0.25 M, 0.5 M, 0.75 M, 1 M room temp. 77%, 88%, 98%, 97% Bhatty et al., 1995

Oat meal acidic buffer, pH 1.5 water pH 10

room temp. 23% 39% 26%

Bhatty, 1992

Oats, barley water acidic buffer, pH 1.5 38 considerably more with

water Åman and Graham, 1987

Oats, barley, rye, wheat

20 mM Na2CO3 50 mM NaOH water + α-amylase water + xylanases

60 room temp, hot 40

14-98% 19-96% 7-52% n.r.

Rimsten et al., 2003

Wheat bran 1 M NaOH 25 n.r. Cui et al., 2000 Oat milling fractions water 96 n.r. Westerlund et al.,

1993 90 50-70%water

5% NaOH (1.25 M) 22

98%Oat bran, rolled oats 20 mM sodium phosphate buffer, pH

6.9 (in vitro) 37 12-33%Beer et al., 1997b

n.r. = not reported

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2.4.2 Extraction of insoluble β-glucan

There are studies on insoluble β-glucan of oats and barley (MacGregor and Fincher,

1993; Vasanthan et al., 2002) but the concept of insoluble β-glucan varies. Heims and

Steinhart (1991) treated oat grain and bran with enzymes after defatting to remove

starch and proteins. Insoluble fibre was then obtained by centrifuging. Claye et al.

(1996) used cold and hot water extraction to remove soluble substances and

enzymatic treatment to remove starch and protein. The method of Gruppen et al.

(1992) was developed for fractionating wheat insoluble fibre. This method involves

isolation of water soluble and insoluble fibre by incubation with water at 63 °C

(Gruppen et al., 1990). The precipitate, i.e. the insoluble fibre fraction, is treated with

Ba(OH)2 to extract arabinoxylans. Ba2+-ions keep β-glucan insoluble but some β-

glucan is still coextracted. The main part of β-glucan can then be extracted from the

precipitate with water. Moreover, Izydorczyk et al. (1998b) applied the method of

Gruppen (1992) after extraction of water soluble components from barley. After

Ba(OH)2 – extraction, water and 1 M NaOH were used to fractionate the insoluble

fibre. The Ba(OH)2 extract contained arabinoxylans. The water extract contained β-

glucan and some arabinoxylan. The monosaccharide analysis of the NaOH extract

showed arabinose, xylose and glucose. Wood et al. (1994a) used the precipitate left

after treatment of oat gum with lichenase as the insoluble β-glucan. Manthey et al.

(1999) analysed the content and composition of both soluble and insoluble dietary

fibre from six oat genotypes harvested over four growing-seasons. The samples were

incubated in sodium acetate at 100 °C with α-amylase. After treatment with

amyloglucosidase the samples were centrifuged. The precipitate thus obtained was

used as insoluble fibre.

2.5 Structure of β-glucan

Oat β-glucan is a linear polysaccharide that consists only of β-D-glucopyranosyl

units. These units are joined by either (1→3)- or (1→4)-β-D-linkages, hence the name

mixed-linkage (1→3),(1→4)-β-D-β-glucan. The distribution of the individual (1→3)-

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and (1→4)-linkages is not random, nor is it regular. The (1→4)-β-links occur mostly

in groups of two or three and are separated by a single (1→3)-link as first reported by

Parrish et al. (1960) and later on by others (Staudte et al., 1983; Buliga and Brant,

1986; Wood, 1993; Wood et al., 1994a). Woodward et al. (1983) first showed the

presence of cellulose like sequences and single (1→3)-linkages as also reported by

Vårum et al. (1988). The building blocks, 1,3-linked cellotriosyl and –cellotetraosyl

units constitute over 90% of the molecule. The (1→3)-link prevents close packing of

the molecule and makes the molecule partly soluble in water, unlike cellulose which

is built entirely of β-(1→4)-linked D-glucanosyl units and is capable of close packing

to crystalline structures (Woodward et al., 1983; Böhm and Kulicke 1999a; Morgan,

2000). According to Åman and Graham (1987) 20% of oat and 46% of barley β-

glucan is insoluble. Miller and Fulcher (1995) found 20-25% of β-glucan insoluble in

water. Fig. 1 shows the 3-O-β-cellobiosyl-D-glucose section of the molecule.

O

O

OOO O

OOO

OOn

CH2OH

CH2OH

CH2OHCH2OH

CH2OH

Figure 1. Structure of β-glucan.

Barley is also rich in (1→3),(1→4)-β-D-glucan. Barley β-glucan has been widely

studied because of the problems it causes in beer making (Bamforth, 1982; Grimm et

al., 1995; Wang et al., 2004). The structure of barley β-glucan is believed to be

similar to that of oat β-glucan (Wood et al., 1994a; Beer et al., 1997a; Cui et al., 2000;

Tosh et al., 2004a; Papageorgiu et al., 2005). However, comparisons between the

results of different studies of the two β-glucans are difficult because the methods of

isolation and analyses differ from experiment to experiment. Extraction conditions

strongly affect the type of molecules that will be obtained and the results of analysis

depend on the methods used.

Structural sequence analysis is often carried out by breaking the β-glucan to

oligosaccharides. This is best achieved with the enzyme (1→3),(1→4)-β-D 4-

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glucanohydrolase, i.e. lichenase (EC 3.2.1.73), that specifically cleaves the (1→4)-

linkage next to a (1→3)-linkage at the reducing end as shown in Fig. 2. The products

are (1→4)-linked oligosaccharides with one (1→3)-linked glucose unit as an end

group at the reducing end. The oligosaccharide with DP3, i.e. 3-O-β-cellobiosyl-D-

glucose, is the main product and DP4, i.e. 3-O-β-cellotriosyl-D-glucose, comes

second. These two constitute over 90% of the total β-glucan content (Wood et al.,

1994a). The remaining oligosaccharides contain longer sequences of (1→4)-linkages,

again terminated by a (1→3)-linked glucose at the reducing end. In soluble β-glucans

oligosaccharides of up to DP13 and in insoluble β-glucan up to DP20 have been

identified in the hydrolysis products (Izydorczyk et al., 1998a; b). DP9 is the most

abundant of sequences with DP > 5 (Wood et al., 1994a).

Figure 2. The mode of action of lichenase on β-glucan, showing linkages hydrolysed. Horizontally arranged open circles represent 1,4-β-linked blocks of D-glucopyranosyl units and oblique lines represent 1,3-β-linkages.

An often used indicator of structural differences of β-glucans is the ratio DP3:DP4

(Wood et al., 1991a; Izydorczyk et al., 1998a; b). The higher the ratio found in the

hydrolysis reaction products, the more there are cellotriosyl sequences. According to

many authors this ratio is lower for oat than for barley β-glucans. Wood et al. (1994a)

reported values 2.1 - 2.4 (calculated as mol-%) for soluble oat β-glucans and 2.8-3.3

for barley β-glucans. Izydorczyk et al. (1998a, b) obtained values 1.76 and 2.13 for

soluble barley β-glucan extracted at 40 °C and 65 °C, respectively and for insoluble β-

glucan they reported values of 2.07-2.43. For wheat β-glucan the ratio is 3.1 - 4.5

(Wood, 1994b; Cui et al., 2000; Lazaridou et al., 2004). Wood et al. (1994a) reported

a value of 3.0-3.2 and Roubroeks et al. (2000) values of 2.31 and 1.94 for rye.

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Structural differences have also been reported to exist between soluble and insoluble

β-glucans the ratio DP3:DP4 being higher for insoluble than for soluble β-glucans

(Izydorczyk et al. 1998a, b). Again comparison of results is difficult because the

concept of insoluble β-glucan differs from study to study. Tvaroska et al. (1983)

showed in their work on lichenan and barley β-glucan that the more there are (1→3)-

linkages in a molecule, the greater is the possibility of consecutive cellotriosyl units.

These units can form helices and cause insolubilisation through aggregation. There

are works to support this theory. Böhm and Kulicke (1999a) studied the rheology of

barley β-glucan in concentrated solutions. They found a decreasing tendency for

gelation as the number of cellotriose units decreases in the order of lichenan > barley

> oat. Cui et al. (2000) investigated the structures of wheat, barley and oat β-glucans

and found DP3:DP4 ratios of 4, 3 and 2, respectively. Wheat having more

trisaccharides than the other cereals has a more regular structure, is less soluble and

has more gelling ability than the others. Tosh et al. (2004a) measured the storage

modulus G´ for β-glucans isolated from lichenan, oat, barley, rye and wheat. They

found a strong positive correlation between elasticity and the amount of cellotriosyl

units. This strongly supports the theory in which consecutive cellotriose units form

the junction zones in the gel network. Ajithkumar et al. (2006) hydrolysed barley β-

glucan with different enzymes one of which selectively degraded cellotetraosyl units

leaving the cellotriosyl units unhydrolysed. They showed that small but significant

amounts of cellotriosyl blocks were present in the β-glucan in which the number of

cellotriosyl units could reach 40 units. These blocks can be involved in the formation

of junction zones.

Another possible explanation for insolubility and gelation is the number of cellulose-

like sequences with high DP. These may act as junction zones to build a physical

network which causes aggregation and gel formation (Woodward et al., 1983; Fincher

and Stone, 1986; MacGregor and Fincher, 1993). Izydorczyk et al. (1998a, b) found

that the alkali-extractable β-glucans of barley had higher amounts of long β-(1→4)-

segments, higher ratios of β-(1→4)/(1→3)-linkages and higher ratios of

cellotriosyl/cellotetraosyl units than the water-extractable β-glucans. Doublier and

Wood (1995) studied the rheology of partially hydrolysed oat β-glucans. They found

that the hydrolysed β-glucans showed a more pronounced gel-like behavior than the

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unhydrolysed β-glucan. They concluded that this is not likely to be caused by the

small number of cellulose-like sequences but rather the lower molar mass which gives

the molecules a higher mobility. Moreover, Tosh et al. (2004a) found no correlation

between elasticity and the content of DP6-9 which suggests that this theory does is not

valid. To conclude, two possible explanations for insolubility have been proposed but

it still remains uncertain which is the prevailing one.

2.6 Solution properties of β-glucan

Oat β-glucan has a capacity to form highly viscous solutions. This is believed to be

the key to the health promoting effects of oat β-glucan (Morgan, 2000; Wood, 2004).

At concentrations below ~ 0.3% β-glucan solutions behave like Newtonian liquids.

This is the critical concentration where overlapping or entanglement of chains begins

(Wood, 2004). Above this concentration the solutions are non-Newtonian and show

shear thinning behaviour (Doublier and Wood, 1995). In addition to entanglements,

high viscosity is caused by strong association of β-glucan in water solutions (Grimm

et al., 1995; Wood, 2001) but the mechanism of association is under debate.

The rheological properties of both oat and barley β-glucans have been studied.

However, no reports concerning the rheology of oat and barley β-glucan were found

where the β-glucans were isolated using the same method. With different methods of

extraction isolates with different characteristics are produced and thus their

rheological properties are also different. Lazaridou et al. (2004) suggested that the

viscosity and viscoelastic properties of barley β-glucan were mostly influenced by the

size of the molecule and less so by the fine structure. Vaikousi et al. (1994) stated that

molecular size is the major factor affecting viscoelastic properties of barley β-glucan.

Böhm and Kulicke (1999a, b) studied the flow behaviour and gelation of barley β-

glucan. They found a transition from viscoelastic behaviour to gel-like properties in

6% solutions over several hours. Goméz et al. (1997c) analyzed the viscoelastic and

flow behaviour and the formation of aggregates of barley β-glucan. They found that

β-glucan forms structured solutions by aggregation through temporary links between

short chain segments. Lazaridou et al. (2003) studied the effect of molar mass on the

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rheological properties and gelation of oat β-glucan. They found that the gelation rate

decreased and the gelation time increased with increasing molar mass. The work of

Doublier and Wood (1995) dealt with the flow behaviour of unhydrolysed and

partially acid hydrolyzed oat β-glucans at different concentrations. Their results

showed that unhydrolysed β-glucan behaves like a noninteracting polysaccharide

whereas the hydrolyzed β-glucans show gel-like behaviour. Tosh et al. (2004b)

studied the gelation of β-glucans partially hydrolyzed by acid, lichenase and cellulase.

They showed that the method of hydrolysis had an effect on gel formation and

concluded that it is the (1→3)-linked cellotriose segments that form the junction

zones needed for gelation rather than the cellulose-like sequences. The mechanism of

aggregation is still under debate.

2.7 Structural analysis methods

2.7.1 Hydrolysis

2.7.1.1 Acid hydrolysis

Polysaccharides are susceptible to acid hydrolysis which is used for determining the

monosaccharide content of polysaccharides in various matrices. The most commonly

used acids are trifluoroacetic acid (TFA), sulfuric acid (H2SO4) and hydrochloric acid

(HCl). Olson et al. (1988) analysed the monosaccharides of foods, dietary fibres and

fecal residues by hydrolysis with 2.0 N TFA at 121 °C for one hour. The same

method was used by Manthey et al. (1999) to analyse the monosaccharide content of

dietary fibre of oats. Salvador et al. (2000) used both TFA and H2SO4 for

monosaccharide analyses of potato, sweet potato and cassava. TFA is not able to

significantly hydrolyse the (1→4)-glycosidic bonds of cellulose but has the advantage

of simple removal by evaporation unlike sulphate-ions which usually require removal

prior to analysis or chromatography (Lebet et al., 1997). For cellulose a

presolubilisation using strong H2SO4 is needed. The procedure, known as the Saeman

hydrolysis (Saeman et al., 1954), starts with a presolubilisation step at room

temperature and is followed by a secondary step with the acid diluted. This method is

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commonly used for total hydrolysis of polysaccharide samples containing cellulose

and insoluble hemicelluloses. The decomposition is primarily due to the secondary

hydrolysis (Saeman et al., 1954). Typically (e.g. Lebet et al., 1997) samples are

dissolved in 12 M H2SO4 at room temperature after which the acid solutions are

diluted to 1 M and incubated at 100 °C for 3 h. Theander and Åman (1979) and also

Miron et al. (2001) similarly used a two step hydrolysis with H2SO4 to hydrolyse

foodstuffs.

Houben at al. (1997) used 4 M HCl at 100 °C to determine the pentosan content of

wheat flour, commercial starch and gluten. HCl was chosen because H2SO4 is difficult

to remove and evaporation of TFA is time-consuming. In this method the effect of

chloride-ions on chromatographic retention times after neutralisation and dilution was

found to be negligible so there was no need to remove the acid. Partial hydrolysis of

β-glucan to reduce the molar mass of the molecules can be accomplished by HCl as

described by Doublier and Wood (1995) and Tosh et al. (2004b) who studied the

rheological behaviour of partially hydrolysed β-glucans of oat.

Ip et al. (1992) used hydrofluoric acid (HF) in addition to TFA to analyze capsular

polysaccharides. These polysaccharides contain phosphodiester groups which are not

hydrolyzed with TFA alone.

Under conditions of hot strong acid, glucose can undergo structural changes to

products such as 5-hydroxymethylfurfural (Johnson et al, 1969; Kaar et al., 1991;

Puls, 1993; Sjöström, 1993). Monosaccharides can also undergo decomposition and

reversion reactions under the conditions of acid hydrolysis (Biermann, 1988).

However, Selvendran et al. (1979) showed that neutral sugars are stable up to two

hours and that five hours of heating caused only minor degradation for arabinose,

xylose and galactose. Reversion or formation of new glycosidic linkages can be

avoided by using low monosaccharide concentrations (Biermann, 1988). According to

Puls (1993) acid hydrolysis is often a compromise between possible sugar destruction

and incomplete hydrolysis. After TFA and especially after HCl hydrolysis some

amount of sample may be unhydrolysed or structurally changed while with H2SO4 the

hydrolysis to neutral sugars is complete but part of the monomer units may degrade.

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Puls (1993) suggests using as mild conditions as possible and to process the standards

through the hydrolysis process so that loss factors can be established.

2.7.1.2. Methylation analysis

Methylation has been used for linkage analysis in polysaccharides for a long time

(Aspinall 1982). In this method the free hydroxyl groups of the polysaccharide are

completely methylated. Glycosidic linkages are not broken because they are mostly

stable in the basic conditions needed for alkylation. The polysaccharide is then

hydrolysed with acid. New hydroxyl groups will be formed during the hydrolysis in

place of the former glycosidic linkages. These hydroxyl groups identify the places of

glycosidic linkages in the original polysaccharide. The methylated sugars can be

analysed by GC after reduction and derivatisation. Aspinall and Carpenter (1984)

have used methylation to analyse the β-glucan extracted from oat bran. The acetylated

sugars produced were analysed by GC- mass spectrometry. The results showed that

oat β-glucan contains unbranched chains of 4-O- and 3-O-substituted β-D-

glucopyranose residues in the ratio 2.6:1. Wood et al (1991a) analysed the methylated

oligo-saccharides produced by lichenase hydrolysis of β-glucan from oat as alditole

acetates using GC with flame ionization or mass spectrometry detection. They found

that the main components of β-glucan were β-(1→3)-linked cellotriosyl and

cellotetraosyl units but small amounts of regions containing 4-8 consecutive (1→4)-

linked units. Laine et al. (2002) have used methylation to analyse the structures of

wood polysaccharides in a somewhat different manner than above. After methylation

the samples were subjected to acid methanolysis, derivatized by silylation and

analysed by GC equipped with mass spectrometric detection (GC-MS).

2.7.1.3 Enzymatic hydrolysis

Enzymatic hydrolysis is a specific and gentle method for fragmentation of many

polysaccharides. For the mixed-linkage (1→3),(1→4)-β-D-glucan, lichenase (EC

3.2.1.73), is widely used for sequence analyses (Wood et al., 1991a; Wood, 1994a;

Miller and Fulcher, 1995; Izydorczyk 1998a, b; Roubroeks et al., 2000; Colleoni-

Sirghie et al., 2003a). It is an (1→3),(1→4)-β-D-glucan 4-glucanohydrolase that

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specifically cleaves the (1→4)-bond adjacent to a (1→3)-bond at the reducing end.

The products are oligosaccharides consisting of β-(1→4)-linked glucose units with

one (1→3)-linked glucose at the reducing end as described in section 2.5.

Roebroeks et al. (2001) used also an (1→4)-β-D-glucan 4-glucanohydrolase (EC

3.2.1.4) to hydrolyse β-glucan in addition to lichenase. This enzyme breaks the

internal (1→4)-linkages next to a 4-substituted residue in a random fashion. The main

products thus produced were glucose, cellobiose, laminaribiose, 4-O-β-laminaribiosyl

D-glucose and 4-O-β-laminaribiosyl D-cellobiose. The authors stated that the primary

targets of this enzyme are the longer sequences of consecutive (1→4)-linkages which

indicates that the enzyme is site specific. These sequences are located within the β-

glucan since viscosity dropped during the treatment. Moreover, Tosh et al. (2004b)

used an endo-1,4-β-glucanase (cellulase) in addition to lichenase and acid in order to

study the gelation mechanism of oat β-glucan. Isolated β-glucan was partially

hydrolyzed with cellulase or HCl. The oligo-saccharide fingerprint after partial

lichenase treatment (DP3-DP9) was, not unexpectedly, the same as for the

unhydrolysed β-glucan. Both acid and cellulase treated samples produced more

cellobiose and laminaribiose when digested with lichenase than the unhydrolysed β-

glucan. Further, cellotriose was obtained which was not produced from the

unhydrolysed β-glucan.

The AOAC 995.15 method (McCleary and Codd, 1991) for determination of β-glucan

involves two enzymes. Lichenase hydrolyses the molecule to oligosaccharides and β-

glucosidase hydrolyses the oligosaccharides to glucose. Glucose is then analysed

spectrophotometrically as a coloured substance which is produced by treatment with a

glucose oxidase/peroxidase reagent (McCleary and Codd, 1991).

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2.7.2 Chromatographic and capillary electrophoresis methods

2.7.2.1 Liquid chromatography

Carbohydrate analyses can also be performed using liquid chromatography. Partition

chromatography on cation-exchange resins uses water as the eluent and refractive

index detection. The disadvantage is that acid hydrolysates must be deionized. Borate-

complex anion-exchange uses borate solutions as the mobile phase. In this method the

separation is based on the different strenghts of borate-carbohydrate complexes.

Deionization is not necessary but a special after-column reagent is needed for

detection (Puls, 1993). Liquid chromatography with reversed-phase columns has been

used for analysis of carbohydrates. Underivatized saccharides are usually eluted with

water. Monosaccharides are eluted first but are not resolved. Double peaks

corresponding to α- and β-anomers are produced. Derivatisation overcomes the

problems associated with anomarisation. Derivatized carbohydrates can be separated

using gradient elution with increasing concentration of acetonitrile (El Rassi, 1995).

Jiang and Vasanthan (2000) used an evaporative light scattering detector combined

with a HPLC system and equipped with a polyamine column to analyse the

oligosaccharides produced by lichenase treatment of barley β-glucan. According to

Lebet et al. (1997) the drawbacks of HPLC are poorer resolution as compared to GC

and non-specific detection for neutral sugars. The drawback with refractive index

detection is that gradient elution cannot be used.

High-performance anion-exchange chromatography with pulse-amperometric

detection (HPAEC-PAD) is presently a widely used technique for both oligo- and

monosaccharides (Izydorczyk et al., 1998; Cheng and Kaplan, 2001; Panagiotopoulos

et al., 2001; Talaga et al., 2002). PAD is suitable for carbohydrates due to its

sensitivity and specificity for compounds with hydroxyl groups. Elution is done at

high pH where carbohydrates are partially ionized and are separated as their

oxyanions (Huber and Bonn, 1995). The drawbacks are lower resolution and

reproducibility of retention times as compared to GC (Lebet et al., 1997). The benefit

of HPAEC-PAD over GC in monosaccharide analysis is that derivatisation is not

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needed. Jiang and Vasanthan (2000) state that HPAEC-PAD has a higher sensitivity

than HPLC equipped with evaporative light scattering detector. Salvador et al. (2000)

achieved complete separation for monosaccharides with HPAEC-PAD. However,

when using a HPLC system with derivatisation the resolution was not satisfactory.

Wood et al. (1994a) studied the structural differences between β-glucans of oats,

barley and rye through oligosaccharides produced by treatment with lichenase

analysed using HPAEC-PAD. Likewise, Izydorczyk et al. (1998a, b) examined water-

soluble and alkali-soluble barley β-glucans and Roubroeks et al. (2000) studied the

structure of rye β-glucan with HPAEC-PAD after lichenase hydrolysis.

2.7.2.2 Gas chromatography

Gas chromatography (GC) is a sensitive and reproducible method and it has long been

used to analyse the monosaccharide content of polysaccharides after hydrolysis with

strong acids such as trifluoroacetic acid and sulphuric acid (Blakeney et al., 1983;

Olson et al., 1988; Pettersen and Schwandt, 1991). Monosaccharides are not volatile

and must therefore be derivatized for GC analysis. A common method is to first

reduce them to alditols with NaBH4 and then acetylate them with 1-methylimidazole

and acetic anhydride (Blakeney et al., 1983; Olson et al., 1988). Reducing the

aldehyde group to an alcoholic hydroxyl simplifies the chromatogram since the α- and

β-forms of monosaccharides no longer exist.

2.7.2.3 Capillary electrophoresis

In capillary electrophoresis (CE) separation is based on differences in solute velocity

in an electric field. The separation is usually performed in a capillary filled only with

buffer. CE provides a sensitive and selective analysis method for the structural

components of β-glucan received through enzymatic hydrolysis. Carbohydrates do not

absorb UV-radiation therefore derivatisation is needed for analyses with UV-

detection. Rydlund (1995) has used capillary electrophoresis (CE) for analysing

mono- and oligosaccharides from wood samples. The derivatisation was through

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reductive amination with 6-aminoquinoline. Soga and Serwe (2000) determined

underivatized mono- and disaccharides in foods by CE with indirect UV-detection.

Larsson et al. (2001) used CE with mass spectrometric detection to determine

carbohydrates from maize starch hydrolysate after reductive amination. Moreover,

Chiesa and Horvath (1993) analysed malto-oligosaccharides after reductive amination

using UV-detection. Arentoft et al. (1993) and Hoffstetter-Kuhn et al. (1991) analysed

carbohydrates as borate complexes using UV-detection.

2.7.3 Spectroscopic methods

2.7.3.1 Nuclear magnetic resonance

Nuclear magnetic resonance (NMR) spectroscopy is a nondestructive method that

provides direct information on the chemical structure of polysaccharides. It has been

used to obtain structural information on high-molar mass molecules and their building

blocks (Bock et al., 1991).

One-dimensional liquid state 1H- and 13C-NMR spectra have been used to study

anomeric protons and carbons of β-glucan and arabinoxylans and for comparison of

β-glucans of different origins (Dais and Perlin, 1982; Westerlund et al., 1993; Wood

et al., 1994a). Two-dimensional NMR (2D-NMR) provides detailed information of

the structural features of β-glucan (Dawkins, 1994; Ensley et al., 1994). Cui et al.

(2000) used two-dimensional NMR spectroscopy to study the structure of wheat β-

glucan. They used direct and long-range homo (1H/1H) and hetero (13C /1H) nuclear

shift correlations to make complete assignments of both the 13C and 1H spectra and to

confirm sequences and linkage sites. They confirmed the assignments of Dais and

Perlin (1982) and showed that the (1→3)-linkages are not consecutive. Roebroeks et

al. (2000) used two-dimensional NMR to elucidate the structural features of rye β-

glucan.

The problem of low solubility of polysaccharides can be avoided using solid-state

NMR analysis but resolution is poorer than with liquid state NMR. Cross polarization

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–magic angle spinning (CP-MAS) 13C-NMR enables the investigation of

conformations and static interactions. It was used by Bowles et al. (1996) to examine

barley β-glucan and its interaction with bile acids. Dudley et al. (1983) used CP-MAS

NMR for analyses of cellulose oligomers and the structure of cellulose II. Morgan et

al. (1999) analysed the structure of β-glucan extract from barley with CP-MAS NMR.

2.7.3.2 Infrared spectroscopy

Fourier transform-infrared (FT-IR) spectroscopy has also been successfully used for

the structural analysis of polysaccharides. It is rapid and sensitive and enables

conformational and configurational analyses and fingerprinting of carbohydrates in

various physical states (Kačuráková and Wilson, 2001). It can be used to monitor

developmental and compositional changes in cell walls and is able to distinguish the

α- and β-conformations of saccharides (Kačuráková et al., 2000). Sekkal et al. (1995)

used FT-IR to investigate glycosidic linkages of oligosaccharides. Kačuráková et al.

(2000) studied model compounds with FT-IR to identify plant cell wall

polysaccharides such as pectin and hemicelluloses. Barbosa et al. (2003) studied the

(1→3,1→6)-β-D-glucan produced by the ascomyceteous fungus, Botryosphaeria sp.

Hromádková et al. (2003) studied the influence of the drying method on the physical

properties and immunomodulatory activity of (1→3)-β-D-glucan from

Saccharomyces cervisiae.

2.8 Molar mass

The molar mass of a polysaccharide is an important characteristics of the molecule

that determines its physicochemical properties such as viscosity. However,

polysaccharides are polydisperse molecules. Therefore the molar masses of

polysaccharides are averages and the distribution of molar mass plays an equally

important role. The most frequently used molar mass averages are the number-

average molar mass Mn and the weight-average molar mass Mw. The definitions are as

follows (van Krevelen, 1990):

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∑∑=

i

iin n

MnM =

∑∑

ii

i

Mww/

∑∑=

ii

iiw Mn

MnM

2

= ∑∑

i

ii

wMw

where Mi = molar mass of the component molecules of kind i

ni = number-fraction of the component molecules i

wi = weight-fraction of the component molecules i

Methods of analysis include various chromatographic techniques which require

calibration with standards and absolute techniques like light scattering where

calibration is not required and no assumptions are made concerning the conformation

of the molecule (Harding et al., 1991). This is a benefit in analysis of cereal β-glucans

because β-glucan standards are not commercially available. High performance size

exclusion chromatography (HPSEC) equipped with a light scattering detector (LS) or

post-column dying with Calcofluor and fluorescent detection are often used

techniques to determine the molar masses of cereal β-glucans.

The reported values of the molar mass of cereal β-glucans vary between 49 000 and

3x106 g/mol (Autio, 1996) and even as high as 4x107 daltons has been reported

(Forrest and Wainwright, 1977). The value strongly depends on the methods of

extraction and analysis, which makes comparisons difficult. Beer et al (1997a)

extracted β-glucan from a series of cultivars of oat and barley in water at 90 °C. They

report peak molar masses (Mp) obtained using HPSEC with both refractive index (RI),

viscosity and LS detection and showed that the Mp was higher for oats than for barley

and that the Mp depended on both cultivar and extraction method. Zhang et al. (1998)

extracted β-glucan at 40, 65 and 100 °C from oat grains. The analysis of relative

molar mass (Mr) was done using HPSEC with RI detection. The Mr were 118-1024

kDa for 40 °C, 985-1919 kDa for 65 °C and c. 2300 kDa for 100 °C. Small variations

of molar masses between cultivars have been reported by Beer et al. (1997a) and

Colleoni-Sirghie et al. (2003a, b).

Rimsten et al. (2003) studied the molar mass of β-glucans extracted from cereals.

They used HPSEC with post-column Calcofluor delivery and fluorescent detection.

The system was calibrated with β-glucan standards made by fractionating a purified

β-glucan sample with a HPSEC-system equipped with multi-angle laser light

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scattering and refractive index (RI) detectors. Also Suortti (1993) has applied post-

column dying with Calcofluor and fluorescence detection in HPSEC-analyses of β-

glucans in order to measure the molar mass of β-glucans of barley, malt and oat.

Wood et al. (1991b) used Calcofluor detection after HPSEC for determining the molar

mass of β-glucans of oats, barley, wheat, rye and malt samples. The molar mass of oat

β-glucan was the highest (3 x 106 g/mol) and that of rye the lowest (1.13 x 106 g/mol).

For barley and malt the values were 2.14 x 106 and 1.11 x 106 g/mol, respectively.

Wheat β-glucan was not sufficiently solubilised for detection. Jaskari et al (1995)

extracted β-glucan from oat bran at 95 °C and analysed it by gel permeation

chromatography and Calcofluor detection. The value obtained was 8.4x105 g/mol.

Åman et al. (2004) studied the effect of processing on the molar mass of β-glucan of

oat-based foods using HPSEC and Calcofluor detection. The molar masses of β-

glucans of oats, rolled oats, oat bran and oat bran concentrate were in the range 2.06-

2.3 x 106 g/mol. The molar masses after baking, fermentation and pasta preparation

were 240 000-1.67 x 106 g/mol, respectively, showing extensive degradation of β-

glucan during these processes.

The molar mass of linear polymers can also be determined using the Mark-Houwink-

Sakurada equation if the necessary parameters in a given solvent at a given

temperature are known. This method, unlike chromatographic methods, is based on

viscosity measurements. Vårum et al. (1988) determined the parameters in aqueous

NaCl and LiI at 20 °C for oat β-glucan whose molar masses were determined by

osmotic pressure measurements. Gómez et al. (1997b) determined the parameters in

water at 25 °C for barley β-glucans using β-glucans characterized for their molar

masses by light scattering.

2.9 Effects of processing on β-glucan

From the perspective of health effects, both the quantity of dietary fibre or of β-glucan

and physicochemical characteristics, or quality, must be controlled. The quality can be

affected by various processes when preparing foods. Changes can be caused by

thermal, mechanical or enzymatic mechanisms (Poutanen, 2001). Viscosity is

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believed to be the key factor in the lowering of cholesterol and glycaemic index.

Therefore it is important to know how solubility and extractability of β-glucan are

influenced by processing (Wood, 2001).

The effects that processing may have on the extractability and Mw have been studied,

but the effects on structure have not received much attention. The only work so far is

that of Andersson et al. (2004) who showed that neither dough making nor baking

affected the cellotriosyl/cellotetraosyl ratio of barley β-glucan. They also concluded

that β-glucan may be degraded by endogenous enzymes in the barley and/or wheat

flour used in baking. Beer et al. (1997b) showed that baking of muffins increased the

extractability but decreased the Mw of β-glucan. In their study, frozen storage

decreased the extractability but did not change the Mw of β-glucan. They also found

that cooking of porridge did not affect the extractability or the Mw of β-glucan.

Vaikousi and Biliaderis (2004) studied the effect of freeze-thaw cycling on the

rheology of barley β-glucan and found that β-glucan underwent cryogelation during

repeated cycling processes. Kerkhoffs et al. (2003) found that the baking of bread and

cookies decreased the effectiveness of β-glucan in the lowering of cholesterol. They

also found that the Mw of β-glucan was decreased by baking but not by freezing.

Sundberg et al. (1996) found that the Mw of β-glucan decreased during bread making

and also in the upper gastrointestinal tract of humans in an ileostomic model. Rimsten

et al. (2003) studied barley β-glucan in an in vitro model. In this study the molar mass

of β-glucan was decreased during digestion. Johansen et al. (1997) reported a decrease

of molar mass in the gastrointestinal tract of pigs that were fed diets based on oat

bran. Robertson et al. (1997) reported that cooking and digestion in the upper gut

increased the extractablity of barley β-glucan. Lambo et al. (2005) reported a decrease

in the amount of β-glucan and loss of soluble and insoluble fibre in oats caused by

fermentation. In the study of Degutyte-Fomins et al. (2002) solubility of β-glucan was

increased by fermentation of oat bran by a rye sourdough starter. Karppinen et al.

(2000) reported that the quantity of short-chain fatty acids produced in fermentation

was higher with oat bran than with the other brans. Åman et al. (2004) studied the

effect of processing on the molar mass of oat-based foods. Baking, fermentation and

pasta preparation caused extensive degradation of β-glucan during these processes.

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3 AIMS OF THE STUDY

The general aim of the thesis was to study the structure of oat β-glucan. Therefore β-

glucan was isolated from different samples in a pure form that enables structural

analysis using chromatographic and spectrometric methods. Moreover, β-glucan was

isolated from barley by the same methods as those for oats to allow comparison of

structures, solution properties and molar masses between oat and barley β-glucans.

The compositions of water insoluble fibre fractions of oats and barley were analysed.

The effect of processing on the structure and extractability of oat β-glucan was

studied. This is important because extractability affects viscosity and viscosity seems

to be connected to the health effects of β-glucan.

The main objectives were:

• To analyse the structure of purified (1→3),(1→4)-β-D-glucan of oat

bran and whole-grain oats and barley (I, II).

• To study the effects of processing on oat β-glucan (III).

• To test different hydrolysis methods and methods for structural

analysis (IV).

• To characterize the water insoluble dietary fibre of oats and barley (V).

• To study the solution properties of β-glucans of oats and barley (VI).

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4 MATERIALS AND METHODS

The details of materials and methods can be found in papers I-VI.

4.1 Materials

The samples used were a commercial oat β-glucan, oat bran and whole-grain oats and

barley. In addition, processed foods and their starting materials were studied.

Oat bran was obtained as a commercial product of Melia, Finland (9.5% β-glucan) (I).

Whole-grain oats (Yty) and barley (Saana) grown and harvested in 1997 were

obtained from Boreal Plant Breeding Ltd (Jokioinen, Finland) (II, V, VI). The grains

were manually dehulled and milled (Cyclotec 1093, Tecator AB, Höganäs, Sweden)

to a particle size of < 0.5 mm. The commercial β-glucan of oats was of high-viscosity

grade (> 97%) and obtained from Megazyme International (Wicklow, Ireland) (IV).

The starting materials for the processed samples were commercial oat bran (8.0% β-

glucan), flakes (5.7% β-glucan) and oat bran high in β-glucan (12.3% β-glucan)

obtained from Finn Cereal Ltd (Finland). Fermented oat bran was taken from

commercial processing before flavouring (Bioferme Oy, Finland, Avenly Oy,

Finland) (III).

The processed samples were porridge, bread, fermentate both fresh and dried. Oat

porridge was prepared by cooking the flakes (11% by total volume) in water for 10

min. Oat bread (β-glucan content 1.7% db) was baked using commercial wheat flour,

oat bran, salt, yeast and water. The soluble and insoluble fibre fractions were isolated

from fermented oats, porridge and bread, all as fresh and after drying overnight at 60

°C as described in section 4.2.3. Dried sample materials were ground (Tecator

Cyclotec® mill) to pass a 0.5 mm screen before extraction of fibre fractions (III).

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4.2 Isolation of β-glucan and fractionation of water insoluble fibre

4.2.1 Isolation of soluble β-glucan

The method of Westerlund et al. (1993) with minor changes was used for isolating

water-soluble β-glucan from oat bran and whole-grain oats and barley. A schematic

representation of the process is shown in Figure 3. The ground grains were defatted,

using Soxhlet extraction with hot isopropanol and petroleum ether (40-60 °C). The

polysaccharides were solubilised in water at 96 °C for two hours and starch was

hydrolysed using Termamyl 300 L DX, a heat-stable α-amylase from Novo Nordisk

(Bagsvaers, Denmark). The insoluble fraction was separated using centrifugation.

Proteins in the supernatant were hydrolysed using pancreatin (8 x U.S.P. Sigma

Chemical Co., St. Louis, MO). The polysaccharides were precipitated using 60%

ethanol at 4 °C. The precipitate was dissolved in water at 70-80 °C. β-Glucan was

precipitated using 30% (NH4)2SO4 and separated using centrifugation. The β-glucan

precipitate was dissolved in Milli-Q water (Millipore Corporation, Bedford, MA,

USA) at 80 °C and dialysed (CelluSep T1, MW cutoff 3500, Membrane Filtration

Products, Inc., San Antonio, Texas, USA) against purified water at room temperature

for 3 x 24 h. The resulting β-glucan solution was freeze-dried (I, II, VI).

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flour

defatting centrifugation

water + Termamyl 96 °C, 2 h

centrifugation precipitate

pancreatine 40 °C, 3 h

60% ethanol, 4 °C, over night

centrifugation supernatant

dissolution in water 30% (NH4)2SO4, 4 °C, 72 h

centrifugation supernatant

dissolution at 80 °C dialysis 3 x 24 h, room temp.

centrifugationfreeze drying

Figure 3. The isolation of soluble β-glucan

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4.2.2 Fractionation of insoluble dietary fibre and isolation of insoluble β-glucan

The isolation of insoluble fibre (WIS) was carried out using the method described by

Asp et al. (1983). The method of Gruppen et al. (1992) was used to fractionate the

WIS (Fig. 4). The WIS was first extracted with saturated Ba(OH)2 to remove

arabinoxylans (Fraction 1). The precipitate was then extracted with water (Fraction 2).

This fraction contained most of the β-glucan and is referred to as the insoluble β-

glucan (OIS for oats and BIS for barley) (II). Fraction 3 was obtained by extraction

with 1 M KOH and fraction 4 with 4 M NaOH. Fraction 5 was the residue left after all

extractions (V).

WISExt. with sat. Ba(OH)2

Fr.1

Ext. with waterFr. 2

Ext. with 1 M KOH

Fr. 3 Ext. with 4 M NaOH

Fr. 4

Fr.5

Figure 4. The fractionation of the water-insoluble dietary fibre of whole grain oats and barley.

4.2.3 Isolation of soluble and insoluble fibre and β-glucans from processed foods

Soluble fibre was extracted from the processed sample materials with the modified

method of Asp et al. (1983). Starch was hydrolysed using heat-stable amylase

Termamyl 300 L (Novo Nordisk) in a boiling-water bath and protein with pepsin (EC

3.4.23.1, Sigma, St. Louis, MO) and pancreatin (EEC 232-468-9, Sigma). The soluble

fibre extract was separated by centrifugation and the insoluble pellet washed four

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times with 100 mL of MilliQ-water and centrifuged to remove all the remaining

soluble materials. The soluble β-glucan was isolated from this fraction. The insoluble

pellet was dried over night at 70 °C. The insoluble β-glucan was isolated from the

pellet (III).

The soluble β-glucans of the processed foods were precipitated from the soluble fibre

fractions with aqueous ethanol. Ethanol was slowly added with stirring to give a final

concentration of 60% and the solution was kept overnight at 4 °C and centrifuged.

The precipitate was washed twice with water, centrifuged and freeze-dried (III).

The insoluble β-glucans of processed foods were isolated from the insoluble fibre

fractions as described in section 4.2.2. The fractions were first extracted with

Ba(OH)2 to remove the arabinoxylans. The precipitates were further extracted by

water to dissolve the β-glucans. The solutions were dialysed extensively against water

and freeze-dried (III).

4.3 Hydrolysis of β-glucan

4.3.1 Acid hydrolyses

The different β-glucans were acid hydrolysed with trifluoro acetic acid (TFA),

hydrochloric acid (HCl) and sulfuric acid (H2SO4) to determine their monosaccharide

content.

The acid hydrolysis of β-glucan from oat bran and whole-grain oats and barley was

done by heating in 3 M TFA at 120 °C in an oven for one hour. The solutions were

neutralised by concentrated ammonia and analysed by GC (I, II). The non-cellulosic

polysaccharides of the insoluble fibre fractions of whole-grains of oats and barley

were determined by hydrolysis using 2 M TFA at 120 °C for one hour. The two-step

Saeman hydrolysis was used for the monosaccharide analysis of all polysaccharides,

in which samples were treated first with 72% H2SO4 at room temperature for one

hour, followed by dilution to 1 M H2SO4 and heating at 120 °C for three hours (V).

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The acid hydrolyses of whole-grain oats and barley and processed foods were carried

out using 1.5 M H2SO4 at 120 °C in an autoclave (Steris Finn-Aqua 46-E,

Wilmington, DW) for one hour. The glucose obtained was analysed by HPAEC-PAD

using hydrolysed glucose as standard. Free sugar residues were corrected for by the

factor of 0.9 to anhydro sugars, as present in polysaccharides (McCleary and Codd,

1991) (II, III).

The hydrolysis experiments on commercial β-glucan were carried out at two

concentrations: 3 M and 0.1 M for HCl and TFA and 1.5 and 0.05 M for H2SO4.

Three temperatures were used: 120 °C in an autoclave, 70 °C and 37 °C in a water

bath. Three durations of hydrolysis, i.e. 1 h, 5 h, 12 h, were used (IV).

4.3.2 Lichenase hydrolyses

The β-glucans isolated from bran, whole-grains oats and barley and processed foods

and the commercial β-glucan were degraded with lichenase enzyme (EC 3.2.1.73,

Megazyme, Wicklow, Ireland) to study their structural building blocks. The method

was modified from the method of McCleary and Codd (1991) (AOAC method

995.16). Samples of β-glucan were dried at 70 °C for three hours. The samples (60-

110 mg) were weighed accurately and pretreated with aqueous ethanol (50%),

dissolved in NaH2PO4 buffer (4.0 ml, 20 mM, pH 6.5) in a boiling water bath and

stirred using a vortex mixer. The samples were then incubated with lichenase (100 U)

at 60 °C for 2 h. The resulting solution was incubated in a boiling water bath for 10

min and then diluted with water. The hydrolysis products (oligosaccharides) were

analysed either with CE or HPAEC-PAD (I-IV).

4.3.3 Determination of β-glucan content by the AOAC method

The β-glucan contents of the samples were determined by the AOAC 995.15 method

(McCleary and Codd, 1991) using an assay kit (Megazyme). The method allows

samples with low β-glucan content to be analysed reliably. The method involves

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dispersing the samples in a phosphate buffer (4.0 ml, 20 mM, pH 6.5) and incubating

with lichenase (EC 3.2.1.73), at 50 °C for 60 min. A second step with β-glucosidase

(EC 3.2.1.21) at 50 °C for 20 min hydrolyses the oligosaccharides to glucose. Glucose

is then derivatized to a coloured substance and analysed spectrophotometrically at 510

nm with a Perkin Elmer Lambda 25 Spectrometer (Perkin Elmer, Shelton, CT) .

For samples with a high β-glucan content a modified AOAC 995.15 method was used.

The samples were treated with isopropanol prior to analyses at room temperature over

night, then dried, first in an oven at 70 °C and then in a vacuum oven at 80 °C for at

least 4 h. 30 mg of sample was accurately weighed into test tubes. The samples were

wetted with 50% ethanol, 8 ml of buffer (pH 6.5) was added and the samples were

incubated in a boiling water bath for 3 min after which they were placed in a water

bath at 50 °C. 0.2 ml (10 U) of lichenase was added and the samples were incubated

for 3 hours with magnetic stirring. Lichenase was inactivated by addition of 5 ml of

buffer (pH 4.0). The tubes were centrifugated for 10 min at 1000 G. The supernatant

was diluted four fold and an aliquot of 0.1 ml was taken into another tube. 0.1 ml of

β-glucosidase was added and the samples were incubated for 10 min at 50°C. 3 ml of

the derivatisation reagent was added and the samples incubated for an additional 20

min. The glucose produced was determined spectrophotometrically at 510 nm (I-V).

4.4 Chromatographic and capillary electrophoresis methods

4.4.1 Gas chromatography

GC was used to identify monosaccharides obtained from the acid hydrolyses of β-

glucans. GC analyses were carried out using a Micromat HRGC 412 chromatograph

(Orion Analytica, Finland) equipped with a flame ionization detector. The column

was a NB-17 fused silica capillary column (25m × 0.32 mm i.d., film thickness 0.25

µm, HNU Nordion Ltd Oy, Helsinki, Finland). Helium was used as carrier gas

(pressure 0.7 bar). The injector temperature was 225 °C and the detector temperature

280 °C. The column oven was programmed from 190 °C (4 min hold) to 230 °C (6

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min hold) at a rate of 4 °C/min. Total separation of monosaccharides was achieved

under these conditions.

The monosaccharides obtained from the hydrolysis step of β-glucan and the standards

(glucose, arabinose, xylose) (Fluka, St. Gallen, Switzerland) were reduced with

NaBH4 (Fluka) at 40 °C for 90 min and acetylated with 1-methyl-imidazole (Fluka)

and acetic anhydride (Merck, Darmstadt, Germany). Dichloromethane (Fluka) was

added to dissolve the alditole acetate produced. The monosaccharides were identified

according to their retention times and quantified using an internal standard method of

myo-inositol (Sigma). Standard curves were produced for glucose, arabinose and

xylose at three concentrations between 2.5 and 10 mg/mL. The correlation

coefficients were at least 0.998 in all cases. The sample concentrations were in the

linear region of the standard curves. The detection limit was not analysed. All

analyses were made in duplicate (I, II, V).

4.4.2 Capillary electrophoresis

Capillary electrophoresis (CE) was used to analyse the products obtained by lichenase

hydrolysis of β-glucans of oat bran and whole-grain oats and barley. The analyses

were performed with a Hewlett Packard3D (Waldbronn, Germany) capillary

electrophoresis system with UV-detection at 245 nm. An uncoated fused silica

capillary column was used (total length 60 cm, effective length 51 cm, 50 µm i.d.) at

25 °C. Injections were performed hydrodynamically (50 mbar, 2s). The applied

voltage was 21 kV. The running electrolyte was an alkaline borate buffer (pH 9.2, 420

mM H3BO3 / 220 mM NaOH). Samples and buffer were filtrated through a 0.2 µm

filter. The standards were aqueous solutions of glucose and cello-oligosaccharides

with DP2-DP5. The oligosaccharides were derivatized through reductive amination

using sodium cyanoborohydride and 6-aminoquinoline. The reagents were added and

the mixtures were heated at 40 °C for two hours (I, II). The oligosaccharides were

identified according to their migration times. Quantification was carried out for

glucose and oligosaccharides with DP3-5 with the internal standard method involving

arabinose. Standard curves were produced for all standards at three concentration

levels. The correlation coefficients were at least 0.97 in all cases. The concentrations

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of the samples were in the linear region. All analyses were made in triplicate. The

detection limit was 7.5 µg/ml (I, II).

4.4.3 High performance anion exchange chromatography

HPAEC-PAD was used to analyse the products of both acid and lichenase hydrolyses

of β-glucans. Monosaccharide analyses were performed isocratically in NaOH. With

oligosaccharides, gradient elution with NaOH and NaOH/sodium acetate was needed

to shorten the retention times (Johnson and Lacourse, 1995) (II-IV).

The analyses of glucose from acid hydrolyses were performed isocratically with 8

mM NaOH. The system was equipped with Waters 515 HPLC pumps and Waters

Automated Gradient Controller (Waters Corporation, Milford, MA, USA). The

analytical column used was a Dionex (Sunnyvale, CA, USA) CarboPac PA1 (4 x 250

mm) and the guard column was a PA1 (3 x 25 mm), maintained at 30 °C. A Decade

detector with gold electrode was used (Antec Layden, The Netherlands). The pulse

potentials and durations were: E1 = 0.15 V, t1 = 400 ms, E2 = 0.75 V, t2 = 120 ms, E3

= -0.8 V, t3 = 300 ms, ts = 20 ms. The flow rate was 1 ml/min. All samples were

filtered before analysis (0.2 µm, Acrodisc 13 GHP filter; Pall, Corporation, Ann

Arbor, MI, USA). A standard curve for glucose (J.T. Baker, Devender, Holland)

hydrolysed in the same way as the samples, was produced for quantification. The

correlation coefficient was better than 0.99 in all cases. The sample concentrations

were in the linear region of the standard curve. All analyses were performed in

triplicate. The detection limit was 2 µM. Reagent blanks were analysed and showed

no peaks. The repeatability of the column was followed by frequently running a

solution of standards (IV).

The oligosaccharides produced using lichenase treatment were also analysed with

HPAEC-PAD. The system was the same as for monosaccharide analyses but with

gradient elution. The eluents were A: 150 mM NaOH and B: 500 mM sodium acetate

in 150 mM NaOH. The gradient was from 90% A - 10% B to 100% B in 15 min. The

pulse potentials and durations were the same as for the monosaccharides. All samples

were filtered through a 0.2 µm filter before analysis. Quantification was performed

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with malto-oligosaccharides with a degree of polymerisation (DP) 3-6 (Sigma). A

standard curve was determined for each oligosaccharide at a concentration range

covering the range of the samples. The masses of the oligosaccharides released from

β-glucan samples were calculated using the Millenium32 software. The sample

concentrations were in the linear region of the standard curve. The correlation

coefficient was at least 0.99 in all cases. All analyses were performed in triplicate.

The detection limit was 2 µM for all standards. Reagent blanks were analysed and

showed no peaks. The repeatability of the gradient was followed by frequently

running a solution of standards (II-IV, VI).

4.4.4 Molar mass determination

The molar masses were measured for the oat bran β-glucans and the whole-grain β-

glucans of oats and barley using HPSEC with NaOH elution (Suortti, 1993;

Wilhelmson et al., 2001). Light-scattering detection was used for soluble β-glucans.

Calcofluor postcolumn dye-binding with fluorescence detection was used for

insoluble β-glucans because the arabinoxylans present did not allow light-scattering

detection to be used (I, II). The analyses were performed by Suortti at VTT

Biotechnology, Espoo, Finland.

4.5 Spectroscopic methods

4.5.1 Nuclear magnetic resonance spectroscopy

Liquid state nuclear magnetic resonance (NMR) spectra of purified β-glucan of oat

bran were obtained using a VarianUNITYINOVA 300 spectrometer (Varian NMR

Systems, Palo Alto, CA, USA) operating at 300 MHz for 1H and 75 MHz for 13C. β-

glucan samples were dissolved in DMSO-d6 by heating and stirring at 80 °C for

several hours. The samples totally dissolved and there was no need of filtering.

Sample concentrations were 0.8-1.0% (w/w). All measurements were carried out at 80

°C. Chemical shifts were referenced to solvent signal 2.5 ppm for 1H and 39.9 ppm

for 13C relative to TMS. One-dimensional 13C NMR measurements were done using

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pulse length of 6.5 µs (60°) and with 2 second delays time. The inverse detected 1H-13C heteronuclear correlation NMR spectrum (HSQC) was measured using 256

increments of 80 scans (I).

The 1H NMR spectra of soluble (BS) and insoluble β-glucans of barley (BIS) were

obtained using a VarianUNITY500 NMR spectrometer operating at 500 MHz for

protons. The measurements were performed at 80 °C. The samples were dissolved in

D2O and acetone-d6 (δH = 2.225 ppm) was used as the internal reference (II).

The solid state 13C CP-MAS spectra of β-glucans isolated from whole-grain oats and

barley were obtained by a VarianUNITYINOVA 300 NMR spectrometer operating at 75

MHz for carbons. A 5-kHz spinning speed and 7-mm rotor were used. The contact

time was 0.5 ms, acquisition time 20 ms and delay between pulses 3 s. The chemical

shifts were adjusted using external secondary referencing (hexamethylbenzene,

methyl line set to 17.3 ppm). The same spectral window was used during the sample

measurements (II, V).

4.5.2 Infrared spectroscopy

Fourier-transform infrared (FT-IR) spectroscopy was used for purified β-glucans from

whole grains of oats and barley. The spectra were recorded with a Perkin Elmer

Spectrum One FT-IR instrument (Perkin Elmer Ltd, Bucos, England) equipped with a

Universal ATR (attenuated total reflection) sampling accessory and a MIR TGS

detector. A total of 200 scans were run for each spectrum from 4000 to 650 cm-1 at a

resolution of 8 cm-1 (II, V).

4.6 Rheology

The viscosities of commercial oat β-glucan solutions were measured for water

solutions, for acidified aqueous solutions and for acidic solutions prepared at 70 °C.

For viscosity measurements of water solutions and acidified β-glucan samples, a 2.5%

aqueous stock solution of commercial oat β-glucan (CB) was prepared by incubating

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at 70 °C. A dilution series (series A) was made thereof with water to give

concentrations between 0.3 and 1.8%. Another series of solutions (series B) was also

prepared from the stock solution. These solutions were diluted with water to give the

same concentrations as for pure water solutions and acidified to pH 1 with 0.1 M HCl.

Both the water solutions and the acidified solutions were then incubated at 37 °C for 5

hours. A third set of samples (series C) with the same concentrations as the two other

sets was prepared by incubating β-glucan in 0.1 M HCl at 70 °C. The viscosities of all

solutions were measured with a ThermoHaake RheoStress 600 (Thermo Electron

GmbH, Dreieich, Germany) using a cone and plate geometry with 35 mm diameter

and an angle of 2°. Three parallel samples were each analysed in triplicate at 37 °C

(IV).

Rheological measurements β-glucans isolated from whole-grain oats and barley were

performed using a Bohlin VOR Rheometer (Malvern Instruments Ltd.,

Worcestershire, UK) at 20 °C both in water and in aqueous cuoxam. Viscosity was

measured at shear rates of 0 – 400 1/s using a cone-and-plate geometry with a cone

angle of 2.5°. The moduli G´ and G´´ of water and aqueous cuoxam solutions were

determined by oscillation measurements performed over the range of 0.01 – 100 Hz.

All the measurements were conducted in the linear viscoelastic range, which was

confirmed by the measured strain sweep. Amplitude in the oscillation measurements

was 30% (VI).

4.7 Statistical analysis

In all analyses at least three parallel samples were analysed to obtain increased

precision of results (I-V). Statistical significance tests were performed using students

t-test and one-way Analysis of Variance (ANOVA) with fixed 5% risk level;

correspondingly, 95% confidence intervals were used (II).

In paper III statistical analyses were tailored to evaluate the similarity of three

hydrolysis methods were used. Three methods for the determination of the quantity of

β-glucan (acid hydrolysis = AH, a modification of the AOAC method 995.15 = MH,

lichenase hydrolysis = LH) were compared for samples of soluble β-glucans isolated

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from fresh and dry porridge, bread and fermentate as well as their starting materials,

i.e. flakes, bran concentrate and bran. Moreover, the effects of processing (i.e.

cooking, baking, fermentation and drying) on the amount of extractable β-glucan were

studied for the raw materials vs. products as well as for fresh vs. dried products. All

analyses were carried out using Statistix 8.0 software. (III).

4.8. Summary of analytical methods and samples. Table 2. A summary of the materials and analytical methods used in the studies.

Work Sample Hydrolysis method Analysis method Acid hydrolysis GC Lichenase hydrolysis CE I Oat bran β-glucan Not hydrolysed NMR

Not hydrolysed HPSEC Acid hydrolysis GC, HPAEC-PAD Lichenase hydrolysis CE, HPAEC-PAD II Whole grain β-glucan of oats

and barley Not hydrolysed NMR, FT-IR Not hydrolysed HPSEC

Acid hydrolysis HPAEC-PAD Lichenase hydrolysis HPAEC-PAD III β-glucan of processed foods Lichenase + β-glucosidase SpectrophotometryAcid hydrolysis HPAEC-PAD Lichenase hydrolysis HPAEC-PAD IV Commercial oat β-glucan Lichenase + β-glucosidase SpectrophotometryAcid hydrolysis GC V Whole grain β-glucan of oats

and barley Not hydrolysed FT-IR, NMR

VI Whole grain β-glucan of oats and barley Not hydrolysed Rheology

5 RESULTS

5.1 Comparison of the soluble β-glucans of oat bran and whole-grain oats

Two kinds of β-glucans were obtained from oat bran: the S-type that dissolved readily

in water and the G-type that remained as gel lumps in water even at temperatures up

to 80 °C. No separation into two types occurred during isolation from whole-grains of

oats. In acid hydrolysis only glucose was detected. The isolated β-glucans were

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treated with lichenase and the oligosaccharides obtained were analysed by CE for

bran β-glucan and with both CE and HPAEC-PAD for whole-grain oat β-glucan. The

molar masses (Mw) were determined using HPSEC with NaOH elution and light-

scattering detection. Table 3 shows the DP3:DP4 ratios based on w-% calculated from

the masses (db) of oligosaccharides released by lichenase hydrolysis, the Mw and the

Mw /Mn ratio. The S- and G-types of bran did not differ from whole-grain β-glucan in

structure as shown by the DP3:DP4 ratio. The Mw of the G-type was higher than that

of the S-type. The Mw of whole-grain β-glucan was much lower than that of bran β-

glucans. The Mw /Mn ratio, a measure of the polydispersity and the molar mass

distribution is much broader for bran than for whole-grain β-glucans (I, II).

Table 3. The DP3:DP4 ratios based on the w-% analysed by HPAEC-PAD after lichenase hydrolysis, the Mw and the Mw /Mn of oat bran and whole-grain soluble β-glucans. β-glucan sample DP3:DP4 Mw (g/mol) Mw /Mn

Bran, S-type 1.2 1100 x 103 3.6

Bran, G-type 1.3 1600 x 103 2.5

Soluble whole-grain oats 1.4 510 x 103 1.3

NMR spectroscopy was used to evaluate the structural features of the β-glucans of

oats and barley. Liquid state 1H and 13C NMR, two-dimensional 1H-13C correlation

spectrum (HSQC) and 13C CP-MAS spectra were recorded. For 13C spectroscopy the

sensitivity is low. However, with the HSQC measurements spectra can be obtained in

a fairly short time even for low concentration solutions. The 13C spectrum obtained

from oat bran β-glucan contained 15 resonances. The spectrum shows the high purity

of the β-glucan and suggests a structure of mixed linked β-glucan with 3-O- and 4-O-

substituted residues. The 1H-spectra showed the typical proton signals of glucose

polysaccharides and the signals of the anomeric protons in the region 4.5-4.8 ppm (II,

Fig. 1). The two-dimensional inverse detected 1H-13C heteronuclear NMR correlation

spectrum (HSQC) of oat bran β-glucan (I, Fig. 3) showed the signals of C-3 (87.3

ppm), C-5 (76.5 ppm), C-2 (72.5 ppm) and C-4 (68.6 ppm) in 3-O-linked glucose

residues. Other signals originated from 4-O-linked residues. The 13C CP-MAS spectra

of the β-glucans showed broad resonances in the region 50-110 ppm which are

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partially overlapping signals of the carbons of β-glucan and arabinoxylan. The signals

in the region 10-40 only seen in the spectrum of the insoluble oat β-glucan, are

probably from fat residues (Fig. 5).

5.2 Comparison of the β-glucans of whole-grain oats and barley

The soluble and insoluble β-glucans of oats and barley isolated from whole-grains

were hydrolysed to oligosaccharides with lichenase and the oligosaccharides were

analysed using CE and HPAEC-PAD. HPAEC-PAD proved to be more reproducible

than CE and only the HPAEC-PAD results are discussed (II). Fig. 5 shows a typical

HPAEC-PAD chromatogram of the oligosaccharides obtained from β-glucan. In acid

hydrolysis only glucose was detected from soluble glucans. From insoluble glucans

small quantities of arabinose and xylose were also detected.

mV

0

200

400

600

800

min2 4 6 8 10 12 14

3

4

5 6

Figure 5. A HPAEC-PAD chromatogram of oligosaccharides obtained from β-glucan after lichenase hydrolysis. Numbers refer to DP of oligosaccharides.

Table 4 shows the DP3:DP4 ratios, the Mw and the Mw /Mn ratios of the soluble and

insoluble β-glucans of oats and barley. The DP3:DP4 ratio was significantly (P<0.05)

higher for barley than for oat for both soluble and insoluble β-glucans. The ratio was

also higher for insoluble than for soluble β-glucans (P<0.05). The Mw of the β-

glucans of oats and barley were similar for both soluble β-glucans and again for

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insoluble β-glucans. The Mw of the soluble β-glucans were higher than the Mw of

insoluble β-glucans. The Mw /Mn ratio was the same for soluble β-glucans of oats and

barley (II).

Table 4. The DP3:DP4 ratios based on w-% analysed by HPAEC-PAD after lichenase hydrolysis , the Mw and the Mw /Mn of whole-grain β-glucans.

β-glucan sample DP3:DP4 Mw (g/mol) Mw /Mn

Soluble oat 1.4 (1.9)a 510 x 103 1.3

Soluble barley 1.6 (2.2) 470 x 103 1.3

Insoluble oat 1.7 (2.3) < 200 x 103 n.d.

Insoluble barley 1.8 (2.4) < 200 x 103 n.d.

n.d. = not determined a values in parentheses are based on mol-% The total contents of β-glucan in the flours and the WIS were analysed by the

Association of Analytical Chemists (AOAC) 995.15 method. Oat flour contained

4.0% (db) and barley flour 3.7% (db). The content of β-glucan in the WIS of oats was

11.5% and barley 6.7%. Based on these figures and yields of WIS 18% of the total β-

glucan was insoluble in oats and 25% in barley.

The FT-IR analyses of the β-glucans showed the signals typical of β-glucans. The

spectra of the β-glucans of oats and barley were similar and the spectra of the soluble

β-glucans were similar to those of the insoluble ones.

Liquid-state 1H NMR and solid-state 13C CP-MAS were used to analyse the structures

of β-glucans of whole-grain oats and barley. The 1H spectra show the typical

anomeric signals of β-glucans (II, Fig. 1). The spectrum of barley β-glucan was

similar to that of the oat β-glucan. The 13C CP-MAS spectra of oats and barley were

also similar (II, Fig. 2). Figure 6 shows the spectra of the soluble β-glucans of oats

and barley. Partially overlapping broad signals were seen in the region of 50-110

ppm. These signals originate from β-glucan and arabinoxylan. Additional signals

were seen in the spectrum of insoluble oat β-glucan in the region 10-40 ppm

originating from fat residues (II).

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barley

oat

Figure 6. The 13C CP-MAS NMR spectra of soluble β-glucans from oats and barley.

5.3 The effects of processing

Soluble and insoluble β-glucans extracted from processed foods and their starting

materials were hydrolysed using three different methods: acid hydrolysis, lichenase

hydrolysis and hydrolysis with both lichenase and β-glucosidase. Table 5 shows the

sums of the amounts of oligosaccharides DP3-6 and the DP3:DP4 ratios obtained after

lichenase hydrolysis for both soluble and insoluble β-glucans. The quantity of

oligosaccharides released from β-glucan from insoluble dry porridge was

considerably lower than the other samples and clearly erroneous.

The results of the three hydrolysis methods were compared statistically to analyse the

similarity between the methods and to reveal the effects of processing on the

extractability of β-glucans (III). These will be discussed in section 5.5.

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Table 5. The yields and DP3:DP4 ratios of β-glucans from processed and unprocessed foods and their starting materials based on w-% as analysed by HPAEC-PAD after lichenase hydrolyses.

Soluble Insoluble total (w-%) DP3:DP4 total (w-%) DP3:DP4 Flake 43.6 ± 5.3 1.6 39.2 ± 9.8 1.8 Porridge 66.4 ± 2.5 1.5 41.3 ± 10.3 1.5 Porridge dry 77.7 ± 0.8 1.5 4.9 ± 3.3 2.0 Bran concentrate 62.6± 9.1 1.5 48.5 ± 7.9 1.6 Bread 46.6 ± 4.1 1.6 24.7 ± 7.3 2.0 Bread dry 37.6 ± 0.6 1.5 20.0 ± 3.7 2.2 Bran 46.6 ± 6.6 1.5 39.9 ± 9.2 2.1 Fermentate 71.3 ± 7.4 1.5 22.4 ± 9.5 2.2 Fermentate dry 56.0 ± 6.6 1.8 39.3 ± 7.9 1.5

All processes affected the extractability of β-glucans. Cooking increased the amount

of extractable β-glucan whereas baking decreased it. The results for fermentation are

uncertain due to large standard deviation (III, Table V) but the effect appeared to be

increasing. Drying decreased the amount of extractable β-glucan for bread and

fermentate but increased it for porridge. Processing did not cause differences in the

distribution of DP3-rich β-glucans as shown by the DP3:DP4 ratio except for dried

fermentate where the ratio was higher (1.8) than for other samples (1.5-1.6).

The DP3:DP4 ratio was lower in insoluble β-glucan from porridge than from flakes or

dried porridge. This ratio for insoluble β-glucan from oat bran concentrate was lower

than for bread or dried bread. For the fermented sample, the ratio was lower for the

dried sample than for the starting material or the fresh sample. The ratio was again

higher for insoluble than for soluble samples as it was for whole-grains. The only

exception was dry fermentate where the ratio was higher for soluble β-glucan than for

insoluble β-glucan and higher than for any other sample (III).

5.4 Comparison of the insoluble dietary fibre of whole-grain oats and barley

The proportion of WIS was higher in barley (13.7%) than in oats (6.1%) (V, Table 1)

but the contents in the five fractions obtained by sequential extractions did not differ

greatly between oats and barley. The amount of fat in oats (8.5%) was twice the

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amount in barley (4.4%) (V, Table 1). Some protein and minerals were also found in

the fractions. The monosaccharide contents of the fractions were analysed with acid

hydrolysis. The cellulosic and noncellulosic glucose could be distinguished by using

both TFA and H2SO4. The monosaccharide residues identified in each of the five

fractions after acid hydrolyses were glucose, arabinose and xylose. The proportions of

total sugars were similar, 57.9% in oats and 60.1% in barley and the proportions of

noncellulosic sugars were 48.0% and 55.1%, respectively. The β-glucan contents of

the grains were also similar, 4.0% in oats and 3.7% in barley. However, in the

insoluble fibre the contents of β-glucan differed being 11.5% for oats and 6.7% for

barley. The arabinose:xylose ratio was calculated for all fractions. The ratios were

similar for oats and barley but differences between the fractions were found. The

arabinose:xylose ratio decreased from 0.59 for fractions 1 and 2 to 0.50 for fractions 3

and 4. This indicates a more branched structure for fractions 1 and 2 than for fractions

3 and 4. In fraction 5 the ratio was close to 1 indicating a highly branched

arabinoxylan. (V).

The FT-IR spectra of oat and barley β-glucans show characteristic broad absorption

bands typical to polysaccharide structures. The spectra were similar for oats and

barley (V).

The solid-state 13C CP-MAS spectra were recorded for water insoluble fibre (WIS)

and derived fractions designated 1, 2, 3 and 5. The amount of fraction 4 was too small

for spectral analysis. The sample weights were low as was their solubility. With solid

state NMR the problems of measuring solutions with low concentration was

overcome. The solid state 13C CP-MAS NMR showed the characteristic signals of

polysaccharides. All the spectra of WIS and its fractions of both oats and barley

showed broad resonances in the region 50-110 ppm which are partly overlapping

signals of polysaccharide carbons (V, Fig. 3). The fractions of WIS (Fr 1-Fr 5) are

mixtures of arabinoxylans, β-glucan and cellulose. Fraction 5 (the residue) contains

mostly cellulose. Additional resonances in the region 10-40 ppm originate from fat

and protein residues (Pizzoferrato et al., 2000; Davies et al., 2002). The resonance at

173 ppm originates from the carbonyl carbon of proteins (Davies et al., 2002). It was

absent from the spectrum of fraction 2 which was the β-glucan-rich fraction (V).

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5.5 Hydrolysis of β-glucan

The monosaccharide content of β-glucan isolated from bran was analysed after acid

hydrolysis with TFA at 120 °C using GC and that of β-glucan isolated from whole

grain oats and barley after H2SO4 hydrolysis at 120 °C. Only glucose was detected.

The yield of glucose with GC was 86% (I, II). In subsequent analyses of HPAEC-

PAD was the chromatographic method of choice since it proved to be reproducible

and there is no need for derivatisation.

For the hydrolysis of commercial β-glucan, three acids were tested, i.e. HCl, TFA and

H2SO4 under several different conditions described in section 4.3. The products were

analysed with HPAEC-PAD. All acids hydrolysed commercial β-glucan with

recoveries of 88-89% of anhydroglucose when the acid concentrations were high and

the temperature was 120 °C. With β-glucan isolated from whole grain the recovery

was also 89%. These recovery data were obtained when the standard glucose was run

through the same hydrolysis process as the samples. Use of unhydrolysed glucose as

standard indicated that some of the glucose was lost. The loss was greatest with HCl,

about 73%. With TFA it was 37% and with H2SO4 25% (IV, Fig.2.).

Hydrolysis of commercial β-glucan with low concentrations of HCl, TFA and H2SO4

at 120 °C in an autoclave for one hour produced a series of products. Glucose was the

main product but cellobiose was also found in all samples. The amount of glucose and

also the total amount of both glucose and cellobiose decreased but the amount of

cellobiose increased in TFA when compared to HCl and further increased when

hydrolysed by H2SO4. The hydrolysis with TFA and H2SO4 produced a series of

oligosaccharides with DP3 or higher but with HCl no oligosaccharides were detected

(IV).

Hydrolysis with low concentration acids at 70 °C for 5 h and 12 h yielded a complex

series of products the main component being glucose. With a hydrolysis time of five

hours the percentages of glucose obtained for HCl, TFA and H2SO4 hydrolysis were

0.13%, 0.12% and 0.09%, respectively. The oligosaccharide region of the

chromatograms showed partially overlapped peaks and was too complicated to be

analysed. When the hydrolysis time was increased to 12 h, the percentages of glucose

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obtained increased to 0.27%, 0.20% and 0.15% for HCl, TFA and H2SO4, respectively

(IV).

When hydrolysis was carried out under stomach-mimicing conditions (pH 1, 37 °C),

as expected, no hydrolysis products were detected for any of the acids during the 5 h

or 12 h incubation times. The solutions which were acidified and then incubated at

37 °C retained their viscosity as compared to water solutions. In contrast, the viscosity

of the solution incubated with HCl at 70 °C declined considerably (IV).

When hydrolysis was performed using lichenase, and products analysed by HPAEC-

PAD, summation of masses indicated 100% purity for β-glucans isolated from bran,

whole-grain oats and barley and commercial β-glucan (I-II, IV). For processed foods

lichenase gave the highest recoveries of the three hydrolysis methods used (III).

The enzymatic AOAC 995.15 method for the determination of the β-glucan content

could not be applied as described for samples with very high β-glucan content and

poor solubility. The recoveries were about 30% (IV, Table 3.). The optimisation of

the method requires dilution of the sample solution for both the lichenase and the β-

glucanase hydrolysis steps. Moreover, a longer incubation time of three hours was

essential for lichenase hydrolysis. With the modified method the recoveries were 91%

for commercial β-glucan and 75-86% for isolated β-glucans. The isolated β-glucans

were treated with isopropanol and dried in a vacuum oven to improve their solubility

(IV).

Statistical analyses were used to compare the hydrolysis methods and the effects of

processing (III). In comparing the methods two hypotheses were made: 1) the results

of the acid hydrolysis (AH) and the modified AOAC method 995.15 (MH) would be

similar, because they are both based on total hydrolysis to glucose 2) that the

similarity between the AH and the lichenase hydrolysis (LH) methods would not be as

close as between AH and MH because in LH the hydrolysis products are

oligosaccharides instead of glucose.

The AH and MH methods had the closest similarity for the starting materials of

processed foods and fresh products, whereas for dried products the methods showed

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greatest variation. Comparison of AH and LH showed somewhat differing results,

especially for dried products. Methods AH and MH thus show closer similarity than

AH and LH, except for dried products. However, LH gave the highest recoveries

(III).

5.6 Rheology of β-glucans of whole-grain oats and barley

The viscosities of the samples of commercial oat β-glucan in water and in water

solutions subsequently acidified by HCl were measured after incubation at 37 °C. The

solutions showed the shear-thinning behaviour typical of polysaccharides (IV, Fig. 5).

The viscosity dropped rapidly at low shear rates and levelled off to a plateau, the

value of which differed with the concentration. Small differences were seen between

water solutions and acidified solutions at low shear rates, especially at a concentration

of 1.8% where the shapes of the viscosity curves differed. For samples incubated at 70

°C with HCl, the viscosity dropped to the level of the lowest concentration (IV).

The viscosities of the β-glucans of whole-grain oats and barley were measured in

water and in aqueous cuoxam (VI, Fig. 1. and 3.). Aqueous cuoxam is a solvent

traditionally used in cellulose chemistry, and has been shown to dissolve various

polysaccharides without the formation of molecular aggregates (Seger and Burchard,

1994; Grimm et al., 1995). Copper complexates with the hydroxyls of sugar residues.

The forming of complexes prevents hydrogen bond formation between the

macromolecular chains (VI). Shear thinning was stronger in aqueous oat β-glucan

solutions than in barley β-glucan solutions. In cuoxam solutions the viscosities were

much lower than in water.

The oscillatory measurements of the water solutions were not reproducible, regardless

of polymer concentration. However, with samples dissolved in aqueous cuoxam, the

oscillation measurements were reproducible. The difference of the solution behaviour

is clearly observed in the viscoelastic properties of the solutions. The cross-over point

of the G´ and G´´ curves was about 10 Hz for oat β-glucan but only 1.5 Hz for barley

β-glucan (VI, Fig. 4. and 5.).

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6 DISCUSSION

6.1 Comparison of the soluble β-glucans of oat bran and whole-grain oats

The structural differences between soluble β-glucans of oat bran and whole-grain oats

were studied comparing their DP3:DP4 ratios. A higher ratio indicates a higher

number of cellotriosyl blocks in the molecule. The molar ratios found in our work

were similar, i.e. 1.5 and 1.7 for the two types of β-glucans from bran compared with

1.8 for the β-glucan from whole-grain (I, Table 3, II, Table 4). The results were lower

than those reported by Wood et al. (1994a) and Cui et al. (2000), 2.1 and 2.2,

respectively. However, our ratios were in agreement with those (1.8 to 2.1) of

Izydorczyk et al. (1998a) and with those (1.45 to 1.86) of Miller and Fulcher (1995)

for different oat cultivars.

The liquid state 13C NMR and two-dimensional 1H-13C correlation spectrum of oat

bran β-glucan showed the typical signals for polysaccharides and the analyses

confirmed the structural features previously reported for β-glucans (Dais and Perlin,

1982; Dawkins, 1994; Ensley et al., 1994). The glucose units are joined by either 1,4-

or 1,3-linkages. No evidence of consecutive (1→3)-linkages were found in the

spectrum (I). The solid-state 13C CP-MAS NMR and the liquid-state 1H NMR spectra

of soluble β-glucan from whole-grain oats also showed the typical signals of

carbohydrates (II).

The lower Mw of the S-type bran β-glucan than that of the G-type is reflected in the

solubilities, the S-type being more readily soluble than the G-type. The Mw of whole-

grain β-glucan was much lower than that of the bran. The separation of bran from

grains includes a hydrothermal treatment called kilning which inactivates enzymes.

The whole-grains had not received heat treatments and so any endogeneous β-

glucanase enzymes of the grains would remain active and would be able to hydrolyse

the β-glucans as soon as water was added to the samples, thus causing the low Mw.

The Mw/Mn ratio was much higher for bran β-glucans than for whole-grain β-glucan

indicating a broader molar mass distribution, (I, II).

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In conclusion, the structures of bran and whole-grain oat β-glucans are similar.

However, the molar masses of β-glucans of bran could not be compared with those of

whole-grain due to the different thermal history of the bran and the whole-grains. The

structure with 1,4-linked blocks of glucose units joined with single 1,3-linkages was

confirmed.

6.2 Comparison of the β-glucans of whole-grain oats and barley

The DP3:DP4 ratios found in this work for soluble β-glucans were lower than those

reported by Wood (1994), Böhm and Kulicke (1999a) and Cui et al. (2000) but in

accordance with those of Izydorczyk et al. (1998a, b) and Colleoni-Sirghie et al.

(2003a).

The comparison between oat and barley β-glucans and between soluble and insoluble

β-glucans revealed differences. A higher DP3:DP4 ratio was found for barley β-

glucan than for oat β-glucan (II, Table 4). This has also been reported by Böhm and

Kulicke (1999b), Cui et al. (2000) and Tosh et al. (2004a). A higher DP3:DP4 ratio

was also found for insoluble than for soluble β-glucans (II, Table 4). This is in

agreement with the results of Izydorczyk et al (1998a, b) for barley β-glucan even

though their isolation process was different from the one used in this work.

Mixed-linkage β-glucan is only partly soluble in water. It was shown in this work that

18% of oat β-glucan and 25% of barley β-glucan remained insoluble of the total β-

glucan contents (II). This is in agreement with the 20-25% reported by Miller and

Fulcher (1995) for whole-grain oat β-glucan. Åman and Graham (1987) reported that

54% of barley β-glucan and 80% of oat β-glucan was soluble. Subtracting these

figures from 100% gives the percentages of insoluble β-glucan of barley and oats, i.e.

46 % and 20%, respectively. For oats the amount is well in accordance with the

results of our work but not for barley.

The significance of the structural differences of β-glucans to their solubility and

solution behaviour have been discussed in the literature. It has been generally

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believed that the more β-glucans contain long sequences of (1→4)-bonds, the higher

is their tendency to associate, owing to the rigidity of these cellulose-like segments

(Woodward et al., 1983; Fincher and Stone, 1986; MacGregor and Fincher, 1993;

Izydorczyk et al., 1998a; Doublier and Wood, 1995). Another explanation has been

presented by Tvaroska et al. (1983) who studied lichenan and barley β-glucan.

Lichenan is a (1→3), (1→4)-β-D-glucan isolated from the lichen, Iceland Moss, that

consists almost entirely of β-(1→3)-linked cellotriose-units. Tvaroska et al. (1983)

showed that a high value for the DP3:DP4 ratio increases the probability that

cellotriosyl units would appear regularly, which causes aggregation through the

formation of helices. Böhm and Kulicke (1999b), in their work on lichenan and β-

glucans of oats and barley, reported an increased tendency to gel formation with

increased amounts of consecutive cellotriose units. In their work the DP3:DP4 ratio

was 23 for lichenan, 2.5 for barley and 1.9 for oat β-glucan. The rate of gel formation

was extremely high for lichenan and orders of magnitude lower for barley β-glucan.

The rate of gel formation for oat β-glucan was lower than for barley β-glucan. They

concluded that consecutive cellotriose units rather than cellulose like sequences are

responsible for the association. Oat β-glucan has the least regular structure of the

three glucans studied and is therefore less able to gelatinize than barley β-glucan and

much less so than lichenan. Goméz et al. (1997b) studied the flow and viscoelasticity

of barley β-glucan to elucidate the aggregation of this molecule. They concluded that

barley β-glucan forms structured solutions through aggregation involving links

between short chain segments. Storsley et al. (2003) studied β-glucans of barley. They

suggest that an increase in the DP3:DP4 ratio contribute to a more extended

conformation of the molecules. Moreover, this might predispose the β-glucans of

barley to intermolecular association and decreased solubility. The results of Tosh et

al. (2004b) on the gelation of β-glucans that were partially hydrolyzed by acid,

lichenase and cellulase support the conclusion that it is the (1→3)-linked cellotriose

sections that form the junction zones needed for gelation and not the cellulose-like

sequences. The results of our work support the hypothesis of cellotriosyl junction

zones imparting gel forming properties to β-glucan assuming that the basis of

insolubility is the same as for gelation; insoluble β-glucans had higher DP3:DP4

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ratios than the soluble β-glucans. In addition, in barley the DP3:DP4 ratio was higher

than in oats and also more β-glucan remained insoluble in barley than in oats.

The IR spectra of oat and barley β-glucans showed the typical signals of

polysaccharides as described by Sekkal et al. (1995), Kačuráková et al. (2000) and

Proniewicz et al. (2001). The signal at about 895 cm-1shows the β-configuration of the

glycosidic bonds (Gutierrez et al., 1996) (II).

Liquid-state 1H NMR spectra were measured to study the anomeric structures of β-

glucans. Solid-state 13C CP-MAS NMR shows the purity of the samples. Again the

spectra were similar for the β-glucans and no differences could be detected between

those of oat and barley nor between soluble and insoluble β-glucans. A more detailed

discussion of CP-MAS spectra is given in section 6.4.

The molar mass analyses showed that the Mw and the Mw /Mn ratio of the β-glucans of

oats and barley were similar. However, differences between soluble and insoluble β-

glucans were clear. The Mw of the soluble β-glucans were about 5 x 105 g/mol but for

the insoluble β-glucans the Mw were much lower, less than 2 x 105 g/mol (II). The

reason for such a surprising difference is not clear. Forrest and Wainwright (1977)

showed that β-glucan is not covalently bonded to pentosans in barley cell walls.

Woodward et al. (1983) suggest non-covalent interactions between β-glucan and other

components of the cell wall. Miller and Fulcher (1995) suggested that β-glucan is

entangled and hydrogen-bonded with other components of the cell wall. Roubroeks et

al. (2000), in their work on rye β-glucan, suggested that intermolecular associations

present in water are avoided in alkali, making β-glucan more soluble. Buckeridge et

al. (2004) suggest that β-glucan is highly associated with the cellulose microfibrils of

the cell wall and interlaced with glucuronoarabinoxylans. Thus it appears that β-

glucan in the insoluble fibre fraction is non-covalently bound to arabinoxylan and

therefore remains insoluble despite of its low molar mass. When arabinoxylan is

removed using Ba(OH)2 extraction, β-glucan is released and becomes water-

extractable (II).

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It can be concluded that the differences in the structures of oat and barley β-glucans

can only be ascertained by the DP3:DP4 ratio and that their molar masses are the

same. More β-glucan remains insoluble in barley than in oats. These results support

the suggestion of a higher frequency of cellotriose junction zones causing the

aggregation of β-glucan.

6.3 The effects of processing

Comparing the proportions of soluble and insoluble β-glucans for the processed and

unprocessed samples shows that cooking increased the amount of soluble β-glucan

but baking decreased it. The increase in extractable β-glucan was not significant for

fermented samples.

β-glucan is integral with cellulose and other noncellulosic polysaccharides in the cell

wall (Buckeridge et al., 2004) and cooking releases it from the matrix (Fincher and

Stone, 1986; Beer et al., 1997b; Robertson et al., 1997). Further, the results of our

work showed increased extractability after cooking. For baking our results showed

decreased extractability. This is in agreement with Andersson et al. (2004) who

showed that the enzymes of wheat flour used in baking of bread can cause the loss of

β-glucan. However, Beer et al. (1997b) showed that baking of bread without wheat

flour decreased the Mw of β-glucan and increased its extractability by in vitro

digestion. When wheat flour was added, the amount of soluble β-glucan did not

change. For fermentation our results showed increased extractability. The results

agree with those of Degutyte-Fomins et al. (2002) who showed that fermentation of

oat bran by rye sourdough starter increased the solubility. These findings are,

however, not in agreement with those of Lambo et al. (2005), who showed that

fermentation decreased the amount of β-glucan for both oats and barley. Mårtensson

et al. (2002) studied the fermentation of oat products with three probiotic strains. Only

one of them, the Lactobacillus bifidum DSM 20456 strain, decreased the β-glucan

content, the others had no effect.

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Drying decreased the amount of extractable β-glucan in bread and fermentate. The

observation is in agreement with Beer et al. (1997b), who showed that freezing, by

which water is repelled from the polysaccharide structures, decreases the amount of

soluble β-glucan in muffins. However, for porridge the amount of soluble β-glucan

increased after drying (III).

The DP3:DP4 ratio was 1.5-1.6 for all soluble β-glucans except for the dried

fermentated sample for which it was 1.8 (III, Table 3). A higher DP3:DP4 ratio was

found for insoluble than for soluble β-glucans of processed foods. This was also the

case for whole-grain oats and barley. These findings are in agreement with Andersson

et al. (2004) who found no change in the DP3:DP4 ratio of β-glucan of dough made of

barley and wheat flour after fermentation as compared to β-glucan of the flour blends.

The only exception was the dried fermented sample in which the ratio was higher for

the soluble than for the insoluble sample. This indicates that drying of fermentate

caused more DP3-rich β-glucans to be concentrated in the soluble fraction than in the

insoluble one (VI).

For insoluble β-glucans the values of the DP3:DP4 ratio vary. These findings indicate

changes in the distribution of DP3-rich insoluble β-glucans depending on the process

used. Cooking decreased the content of DP3-rich insoluble β-glucans but baking and

fermentation increased it. Drying increased the contents of DP3-rich insoluble β-

glucans in porridge and bread but decreased them in fermentate. The DP3:DP6 ratio

ranged between 20-40 for all samples except for the dry fermented sample where the

value was only 6.7 (III).

The lowering of cholesterol in the blood and glycaemic response is related to the

viscosity of soluble β-glucan. Thus the solubility and extractability are important

factors when considering the health effects of processed foods (Wood, 1994; Wood,

2001). In our work it was shown that the extractability of β-glucan was increased by

cooking and decreased by baking (III). A higher extractability increases the

concentration of β-glucan which further increases the viscosity of the solution. The

conclusion therefore is that if the health effects of processed foods are to be retained,

cooking is the most and baking the least favourable process.

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6.4 Comparison of the insoluble dietary fibre of whole-grain oats and barley

The chemical compositions of the oat and barley grains showed that the amounts of

total sugars and noncellulosic sugars were similar for the whole-grains of oats and

barley. The greatest differences were in the contents of water insoluble fibre and fat of

the grains whose amounts were well in agreement with values reported in the

literature (Åman and Newman, 1986; Aalto et al., 1988; Wilhelmson et al, 2001). The

contents of β-glucan were about the same in the whole-grains, a finding which was

similarly reported by Henry (1987). However, in the WIS the content of β-glucan was

considerably higher in oats than in barley (V, Table 1).

The water insoluble fibre (WIS) was divided into five fractions using sequential

extractions with different bases. The monosaccharide contents of the fractions were

analysed by GC (V, Table 4). The glucose found in the water insoluble dietary fibre

originates from β-glucan and cellulose; arabinose and xylose are from arabinoxylan.

The highest quantities of arabinose and xylose were found in fraction 1 which was

extracted with saturated Ba(OH)2. This fraction contained most of the arabinoxylans

but also a small proportion of the β-glucans. The main quantity of β-glucan was found

in fraction 2 which was extracted by water. This fraction also contained small

amounts of arabinoxylane. The release of β-glucan after Ba(OH)2 extraction was

discussed in section 6.2. Fraction 3 contained somewhat lower amounts of

polysaccharides than fraction 2 but the quantities of protein were the highest of all

fractions. This was also seen in the 13C CP-MAS spectra of the fractions where

resonances typical of proteins where found. Glucose was the only monosaccharide

obtained in fraction 5 and the quantity increased considerably with H2SO4-hydrolysis

compared to that of TFA indicating the presence of cellulose (V).

The CP-MAS spectra of polysaccharides differed only slightly from each other (V,

Fig. 2 and 3). The assignment of signals was based on the data from the literature for

the solid and solution state spectra of arabinoxylans, β-glucan and cellulose (Dais and

Perlin, 1982; Dudley et al., 1983; Bengtson and Åman, 1990; Bock et al., 1991;

Davies et al., 2002) and reference spectra run from model compounds of β-glucan and

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arabinoxylan. No structural differences between the β-glucans of oats and barley

could be distinguished. Signals from the residual fat and protein can be seen in the

spectra in regions 10-40 ppm and 173 ppm, except for fractions 2 and 5 (Pizzoferrato

et al., 2000; Davies et al., 2002) (V).

The IR spectra were assigned according to data of Kačuráková et al. (1994), Sekkal et

al. (1995), Kačuráková et al. (2000) and Proniewicz et al. (2001). The spectra of oats

and barley showed similar broad absorption bands typical to polysaccharide

structures. The β-configuration of the glucan linkages was shown as a sharp

absorption at about 895 cm-1 (Gutiérrez et al.,1996) (V).

6.5 Hydrolysis of β-glucan

Enzymatic hydrolysis of β-glucans to oligosaccharides can be achieved using

lichenase. Total recovery for isolated β-glucan samples was obtained for oat bran and

for whole-grain oats and barley (I-II). Together with monosaccharide analysis of acid

hydrolysates showing only glucose, this reflects the purity of the isolated β-glucan.

Total quantities of cereal β-glucan can be determined enzymatically, without losses

caused by isolation, using two enzymes, i.e. lichenase to obtain oligosaccharides and

β-glucosidase to hydrolyse the oligosaccharides to glucose. The glucose can then be

analysed spectrophotometrically as a coloured substance (McCleary and Codd, 1991).

This is the 995.16 method of AOAC which has been used in many studies (Wood et

al., 1994a; Izydorczyk et al., 1998a, b; Cui et al., 2000; Skendi et al., 2003). The

method is suitable for samples with low β-glucan concentration, e.g. flour. In our

work, modification of the method was found necessary for samples with high β-

glucan concentration and poor solubility. A longer incubation time together with

double the quantities of buffer in the lichenase step and a 4-fold dilution of the sample

solutions in the β-glucosidase step were needed to obtain acceptable results (IV).

Cereal β-glucans were easily hydrolysed to glucose with mineral acids, contrary to

cellulose. High concentration and a temperature of 120 °C were needed for total

hydrolysis. However, under these conditions glucose can undergo structural changes

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to products such as 5-hydroxymethylfurfural (Johnson et al, 1969; Biermann, 1988;

Kaar et al., 1991; Puls, 1993; Sjöström, 1993). Thus the recoveries of glucose were

only about 75% for H2SO4, 63% for TFA and 27% for HCl indicating the amounts of

glucose lost were highest for HCl and lowest for H2SO4. With hydrolysed standards

remarkably higher results were obtained the recoveries being about 90% for the acids

used. Therefore the use of hydrolysed standards is needed for quantitative

determinations as also pointed out by Puls (1993). (IV).

Hydrolyses by low concentration acids produced small amounts of glucose and

cellobiose. The quantities of these differed with the acid used. Moreover,

oligosaccharides with DP ≥ 3 were detected for TFA and H2SO4. This procedure is

not suitable for structural analysis because the recovery is poor and the

oligosaccharides cannot be analysed.

Hydrolysis under stomach mimicing conditions was investigated to see if any

hydrolysis would occur. As expected under these conditions, no hydrolysis products

were seen. Further, the viscosity of the β-glucan solution did not change under these

conditions (IV). This is consistent with the results of Wood et al. (1990) for kinematic

viscosity of solutions of oat gum in water and in 0.1 M HCl. It was therefore shown

that no hydrolysis occurs in the conditions prevailing in the human stomach.

Comparing the hydrolysis methods statistically revealed that the glucose producing

methods, i.e. acid hydrolysis and the modified AOAC method, gave similar results

whereas the similarity between the oligosaccharide producing lichenase hydrolysis

and the other methods was worse. Nevertheless, the highest recoveries were obtained

by the lichenase hydrolysis from the processed food samples.

The HPAEC-PAD proved to be more reproducible and sensitive method than CE for

the analyses of both mono- and oligosaccharides. It has the advantage over GC and

CE that derivatisation is not needed. In this work, good reproducibility of retention

times was obtained through regeneration and stabilisation of the column after each

injection.

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To conclude, lichenase hydrolysis together with HPAEC-PAD is the method of choice

for analysis of the content of β-glucan in purified samples. Hydrolysis with mineral

acids at 120 °C is a good alternative for the AOAC 995.16 method for purified β-

glucan samples (III).

6.6 Rheology of β-glucans of whole-grain oats and barley

The solution properties of β-glucans of whole-grain oats and barley were studied both

in water and in aqueous cuoxam solution. Typical shear thinning behaviour of

polysaccharides was revealed in the water solutions. In water polysaccharides form

entanglements above a critical concentration. These structures break down when the

sample is subjected to shear and consequently the viscosity drops (Goméz et al.,

1997c; Burchard, 2001; Williams et al., 2002; Lefebre and Doublier, 2005). At a

concentration of 15 mg/ml oat β-glucan showed stronger shear thinning than barley β-

glucan solutions (VI).

In contrast, no shear thinning occurred in aqueous cuoxam solutions at a

concentration of 15 mg/ml. The viscosities for the β-glucans of oats and barley were

the same but degrees of magnitude lower than those in pure water at low shear rates

and approximately the same at high shear rates (VI). The low viscosity and lack of

shear thinning show that indeed no aggregation occurred in cuoxam solutions. When

the polymer concentration was increased to 50 mg/ml shear thinning was observed,

the viscosities increased, and the viscosity of barley β-glucan was slightly higher than

that of oat β-glucan. Thus, differences in the solution behaviour were revealed

between β-glucans of oats and barley. As the isolation procedure and the molar mass

were the same, this difference must originate from the structures of the molecules

(VI).

Solution-like behaviour was shown for both samples since the loss modulus (G´´) was

greater than the storage modulus (G´) at low frequencies. A crossover of the moduli

curves occurred after which the G´ was greater than G´´. The crossover point is

expected to decrease with increasing molar mass and with long chain branching

(Ferry, 1980). The lower cross-over point indicates a more solid-like behaviour for

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70

barley β-glucan (VI). The values are in agreement with those of Lazaridou et al.

(2003).

The β-glucans of oats and barley had the same molar masses as shown in section 6.2.

These findings and the fact that β-glucan is not branched but linear suggest that the

changes in viscoelastic properties are caused by the structural difference indicated by

the DP3:DP4 ratios.

7 CONCLUSIONS

The β-glucans of oat and barley were shown to consist mostly of cellotriosyl- and

cellotetraosyl-blocks separated by single 1,3-linkages as previously reported. The β-

glucans of oats and barley had similar molar masses but differed in the

trisaccharide:tetrasaccharide ratio. This ratio was higher for barley than for oats

indicating a higher number of the trisaccharide. The soluble β-glucan of oat bran was

similar to the soluble β-glucan of whole-grain oats. The insoluble β-glucans of the

whole-grains had a higher DP3:DP4 ratio than the soluble ones. It was also shown

that a higher proportion of barley β-glucan remained insoluble compared to oat β-

glucan.

Processing changed the extractability of β-glucans of processed foods. Cooking

seemed to be the most and baking the least favourable processing method if health

effects are to be realized. Drying decreased the amount of soluble β-glucan in bread

and fermentate. However, in porridge the amount of soluble β-glucan increased after

drying.

Total hydrolysis of β-glucan to glucose required strong acids and a high temperature.

Hydrolysis under mild conditions produced small quantities of oligosaccharides with

glucose as the main product. No hydrolysis occurred under human stomach mimicing

conditions. High-performance anion-exchange chromatography with pulse-

amperometric detection is a good tool for analysing the hydrolysis products. It is

sensitive and reproducible and derivatisation is not required. Together with lichenase

hydrolysis HPAEC-PAD appears to be the best choice for structural analysis of

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71

purified samples of mixed-linkage β-glucan. Acid hydrolysis is a good alternative for

quantitative analysis of β-glucan.

The whole-grains of oats and barley differed in the proportions of water insoluble

dietary fibre which was higher in barley than in oat grains. The amounts of total and

noncellulosic sugars were about the same. The amounts of β-glucan were similar in

the grains but in the insoluble fibre the amount was considerably higher in oats than in

barley.

At room temperature the rheological behaviour of the soluble β-glucans of oats and

barley were similar in pure water with a strong tendency to form unstable aggregates.

Oat β-glucan showed stronger shear thinning than barley β-glucan solutions. In

aqueous cuoxam differences in viscoelastic behaviour were observed. The crossover

point of the G´ and G´´ curves was much lower for barley β-glucan than for oat β-

glucan. This indicates a higher tendency towards solid-like behaviour for barley than

for oat β-glucan. The isolation method and the Mw were the same. Therefore this

difference must be caused by the small structural difference revealed by the DP3:DP4

ratio.

The findings of this work on the structure and properties of β-glucans of oats and

barley support the theory that a higher prevalence of the 3-O-β-cellobiosyl-D-glucose-

blocks increases association and insolubility.

The health benefits related to oat and barley consumption depend on the β-glucan and

its properties. Not the amount alone but also the molar mass and the viscosity must be

known and controlled. There is an understanding of the amount of β-glucan needed to

lower blood glucose and cholesterol levels. However, the relationship between molar

mass and viscosity of β-glucan with the health effects is not known. In the future, the

important areas of research are the relationship between the health effects and the

properties of β-glucan.

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