Arabinoxylan and arabinoxylan oligosaccharides in response to

Master’s Thesis
Arabinoxylan and arabinoxylan oligosaccharides
in response to treatment with cell wall degrading
enzymes in wheat grain fractions
Cecilie Toft Vangsøe
20117590
Aarhus University
Title: Arabinoxylan and arabinoxylan oligosaccharides in response to treatment
with cell wall degrading enzymes in wheat grain fractions
Cecilie Toft Vangsøe
20117590
Master’s thesis (60 ECTS)
Molecular Nutrition and Food Technology
May 2016
Supervisor
Knud Erik Bach Knudsen
Professor and head of research unit
Department of Animal Science
Aarhus University
Front page photo (Stockvault, 2016)
PREFACE
The present Master’s thesis “Arabinoxylan and arabinoxylan oligosaccharides in response to treatment with
cell wall degrading enzymes in wheat grain fractions” amounted to 60 ECTS and completed the Master’s
Programme “Molecular Nutrition and Food Technology”. The work was carried out at the laboratory of Molecular nutrition and reproduction, Department of Animal Science, Aarhus University, Foulum from September 2015 to May 2016 under the supervision of Professor and Head of research unit Knud Erik Bach
Knudsen.
The aim of this study was to investigate the response incurred by the treatment cell wall degrading enzymes
with emphasis on alterations in arabinoxylan and arabinoxylan oligosaccharides. Furthermore this study
aimed at identifying the wheat grain fractions with the most potential to produce an optimal yield of AX and
AXOS along with exploring additional alterations induced by the treatment
The thesis is divided into 7 chapters comprehending a background review, aim and experimental approach,
materials and methods, results, discussion, conclusion, and finally future perspectives. Ahead of this is an
introduction clarifying the motivation behind this study and the relevance of it.
Acknowledgements
I would like to thank my supervisor Knud Erik Bach Knudsen for great guidance, supervision, and support
throughout the process of conducting this thesis. Furthermore I wish to thank the laboratory technicians
Winnie Østergaard Thomsen for countless hours of guidance and help working with the HPAEC-PAD and
Stina Greis Handberg for valuable guidance in the laboratory. Also a special thanks to Mette Skou Hedemann, Senior scientist, for assistance and guidance with the LC-MS metabolomics.
At last, thanks to my colleagues at research unit Molecular nutrition and reproduction for a pleasant working
environment.
Cecilie Toft Vangsøe
________________________
1
ABSTRACT
Recently wheat bran derived arabinoxylan (AX) and arabinoxylan oligosaccharides (AXOS) have been
shown to exert health-promoting effects with the potential to work as prebiotics. This study comprised six
different wheat grain fractions, which were subjected to treatment with cell wall degrading enzymes. The
objective was to investigate the response incurred by the treatment with emphasis on alterations in AX and
AXOS. Furthermore this study aimed at identifying the wheat grain fractions with the most potential to produce an optimal yield of AX and AXOS, along with exploring additional alterations induced by the treatment. To address this, the carbohydrate portion of the fractions was analysed according to monosaccharide
composition using gas liquid chromatography after subjecting the fractions to acid hydrolysis. This resulted
in the identification of soluble and insoluble high molecular weight polysaccharides, as well as low molecular weight oligosaccharides. The oligosaccharides were further identified using high-performance anionexchange chromatography with pulsed amperometric detection. Finally non-targeted LC-MS metabolomics
were used to elucidate the liberation of non-carbohydrate metabolites induced by the treatment. The results
revealed that the response of treatment mainly depended on degree of arabinose substitution. This was reflected in the relative increase in AXOS being inversely associated with the arabinose/xylose ratio in the
fractions comprising bran tissues. An arabinose/xylose ratio of ~1 was found to set the limit for further AX
degradation. The total AX extraction yield was greatest in the aleurone-rich fractions, where the most pure
aleurone fraction yielded 16.4% AX of the dry matter. Conversely, the pericarp rich fraction was largely resistant to modifications with regard to AX. The identification of the AXOS produced in response to treatment revealed that some were degraded completely to monomers. This was ascribed to the action of endogenous enzymes. Further experiments should be conducted involving the inactivation of endogenous enzymes
prior to treatment in order to confirm this. Ferulic acid was liberated in response to enzyme treatment. The
liberation appeared to depend on the degradation of AX to AXOS. In conclusion this study showed that the
wheat grain fractions rich in aleurone tissue results in the highest AX yield upon enzyme treatment, while
causing a liberation of the potentially bioactive phenolic acid, ferulic acid.
2
LIST OF ABBREVIATIONS
A/X
AX
AXOS
CHO
COS
DAS
DF
DP
ESI
GH
GLC
GOS
HMW
HPAEC-PAD
LC-MS
LMW
mXyl2
mXyl3
NCP
NDC
NSP
PCA
SCFA
UA
uXyl
WE-AX
WU-AX
XOS
XOScorr
Arabinose/xylose
Arabioxylan
Arabinoxylan oligosaccharides
Carbohydrate
Cellooligosaccharides
Degree of arabinose substitution
Dietary fibre
Degree of polymerisation
Electrospray ionisation
Glycoside hydrolase
Gas liquid chromatography
Glucooligosaccharides
High molecular weight
High-performance anion-exchange chromatography with pulsed amperometric detection
Liquid chromatography mass spectrometry
Low molecular weight
O2 monosubstituted xylose
O3 monosubstituted xylose
Non-cellulosic polysaccharides
Non-digestible carbohydrates
Non-starch polysaccharides
Principal component analysis
Short chain fatty acids
Uronic acids
Unsubstituted xylose
Water-extractable arabinoxylan
Water-unextractable arabinoxylan
Xylooligosaccharides
Xylooligosaccharides corrected for the presence of monomeric xylose
3
TABLE OF CONTENTS
PREFACE ......................................................................................................................................................... 1
Acknowledgements ....................................................................................................................................... 1
ABSTRACT ...................................................................................................................................................... 2
LIST OF ABBREVIATIONS ............................................................................................................................. 3
TABLE OF CONTENTS ................................................................................................................................... 4
INTRODUCTION ............................................................................................................................................. 6
Chapter 1 ................................................................................................................................................................... 7
Background........................................................................................................................................................... 7
1.1 WHEAT GRAIN ANATOMY AND PROCESSING .................................................................................... 8
1.1.1 Grain anatomy ...................................................................................................................................... 8
1.1.1.1 Germ ............................................................................................................................................. 8
1.1.1.2 Endosperm .................................................................................................................................... 9
1.1.1.3 Seed coat ....................................................................................................................................... 9
1.1.1.4 Pericarp ......................................................................................................................................... 9
1.1.2 Germination ........................................................................................................................................ 10
1.1.3 Grain processing ................................................................................................................................. 10
1.1.3.1 Milling ........................................................................................................................................ 11
1.2 THE CELL WALL MATRIX ..................................................................................................................... 11
1.2.1 Chemical composition ......................................................................................................................... 11
1.2.2 The structural components................................................................................................................... 11
1.2.2.1 Carbohydrates ............................................................................................................................. 11
1.2.2.1.1 Arabinoxylan ....................................................................................................................... 12
1.2.2.1.2 Cellulose .............................................................................................................................. 13
1.2.2.1.3 β-glucan ............................................................................................................................... 13
1.2.2.1.5 Pectins ................................................................................................................................. 14
1.2.2.1.6 Xyloglucan .......................................................................................................................... 14
1.2.2.1.7 Mannans .............................................................................................................................. 14
1.2.2.2 Additional cell wall components .................................................................................................. 14
1.2.2.1.4 Lignin .................................................................................................................................. 14
1.2.2.2.1 Phenolic acids ...................................................................................................................... 15
1.2.2.2.2 Acetic acid ........................................................................................................................... 15
1.2.3 The cell wall ....................................................................................................................................... 16
1.2.3.1 The tissues................................................................................................................................... 16
1.2.3.1.1 The starchy endosperm ......................................................................................................... 16
1.2.3.1.2 The aleurone tissue ............................................................................................................... 17
1.2.3.1.3 The maternal tissues ............................................................................................................. 18
1.2.3.2 Architecture and polymer interaction ........................................................................................... 20
1.3 NUTRITIONAL PROPERTIES OF ARABINOXYLANS AND ARABINOXYLAN
OLIGOSACCHARIDES .................................................................................................................................. 22
1.3.1 Dietary fibre definition ........................................................................................................................ 22
1.3.2 Dietary fibre in the gastrointestinal tract .............................................................................................. 22
1.3.3 Health related effects of dietary fibres ................................................................................................. 23
1.3.3.1 Arabinoxylan as dietary fibre ....................................................................................................... 24
1.3.3.2 Arabinoxylan oligosaccharides as a prebiotic ............................................................................... 25
Chapter 2 ................................................................................................................................................................. 27
Aim and Experimental Approach ...................................................................................................................... 27
2.1 Aims .......................................................................................................................................................... 28
2.2 Experimental approach ............................................................................................................................... 28
Chapter 3 ................................................................................................................................................................. 30
Materials and Methods ....................................................................................................................................... 30
3.1 WHEAT FRACTIONS............................................................................................................................... 31
3.2 PRODUCTION OF WHEAT FRACTIONS................................................................................................ 31
3.3 PRETREATMENT OF ISOLATED WHEAT FRACTIONS ....................................................................... 31
3.3.1 Enzyme treatment ............................................................................................................................... 31
3.3.2 Sample preparation ............................................................................................................................. 32
3.4 STARCH ................................................................................................................................................... 32
3.5 POLYSACCHARIDES AND LIGNIN ....................................................................................................... 32
4
3.5.1 Enzymatic hydrolysis of starch ............................................................................................................ 34
3.5.2 Acid hydrolysis of starch free residues and direct samples ................................................................... 34
3.5.3 Production of alditol acetate derivates for gas liquid chromatography................................................... 34
3.5.3.1 Data processing ........................................................................................................................... 35
3.5.4 Colorimetric determination of uronic acids .......................................................................................... 35
3.5.5 Klason lignin ...................................................................................................................................... 35
3.5.6 β-glucan ............................................................................................................................................. 36
3.6 OLIGOSACCHARIDES ............................................................................................................................ 36
3.7 NON-TARGETED LC-MS METABOLOMICS ......................................................................................... 37
3.7.1 Ultra high performance liquid chromatography-mass spectrometry ...................................................... 37
3.7.2 Data pre-processing and multivariate data-analysis .............................................................................. 38
3.8 CALCULATIONS ..................................................................................................................................... 38
Chapter 4 ................................................................................................................................................................. 40
Results ................................................................................................................................................................. 40
4.1 POLYSACCHARIDES AND LIGNIN ....................................................................................................... 41
4.1.1 Whole fractions................................................................................................................................... 41
4.1.2 Separated fractions .............................................................................................................................. 45
4.1.2.1 Supernatants ................................................................................................................................ 46
4.1.2.2 Pellets ......................................................................................................................................... 48
4.2 OLIGOSACCHARIDES ............................................................................................................................ 51
5.2.1 Whole fractions................................................................................................................................... 51
5.2.2 Supernatants ....................................................................................................................................... 58
4.3 NON-TARGETED LC-MS METABOLOMICS ......................................................................................... 61
Chapter 5 ................................................................................................................................................................. 66
Discussion ........................................................................................................................................................... 66
5.1 RESPONSE TO ENZYME TREATMENT ................................................................................................. 67
5.2 PROPERTIES AND EXTRACTION YIELD OF ARABINOXYLAN AND ARABINOXYLAN
OLIGOSACCHARIDES .................................................................................................................................. 70
5.3 FURTHER ALTERATIONS ...................................................................................................................... 71
Chapter 6 ................................................................................................................................................................. 74
Conclusion .......................................................................................................................................................... 74
Chapter 7 ................................................................................................................................................................. 76
Future Perspectives ............................................................................................................................................ 76
REFERENCES........................................................................................................................................................ 79
5
INTRODUCTION
There has been an alarming rise in the frequency of metabolic syndrome in affluent societies, which is now
affecting 20-25% of the adult population in the western world (Cornier et al., 2008). Metabolic syndrome is a
clustering of the metabolic risk factors: abdominal obesity, insulin resistance, dyslipidaemia, and hypertension. Metabolic syndrome increases the risk of developing cardiovascular disease three-fold and type 2 diabetes five-fold (O'Neill and O'Driscoll, 2015). Metabolic syndrome constitutes thus a major threat to public
health and welfare, and creates a significant economical burden to society.
A high consumption of energy dense foods, with high fat and/or sugar and low dietary fibre (DF) contents is
considered a central environmental factor responsible for this development (Bray et al., 2004; Stanhope,
2012). Conversely, the consumption of foods rich in DF has proven beneficial to alter these conditions by
improving serum lipid concentrations (Brown et al., 1999), lowering blood pressure and glucose (Anderson
et al., 2004; Whelton et al., 2005), and reducing risk of diabetes (Montonen et al., 2003) amongst others
(Anderson et al., 2009).
Cereals are the common designation of the grains of cultivated grasses. These are a widespread crop
throughout the temperate and tropical regions of the world, where the species wheat, rice, and maize provide
over 50% of the world’s plant-derived food energy. Among cereals, wheat is the most widely grown, accounting for 17% of the total cultivated land worldwide (Awika, 2011).
A major by-product in the production of white wheat flour is bran, which is discarded during the milling
process. The main outlet of the bran fraction, however, remains as feeds for livestock’s (Rosenfelder et al.,
2013). There exists thus a great potential for the use of bran in human nutrition, as it is commercially available in large quantities (Amrein et al., 2003).
The bran fraction is rich in DF, in particular arabinoxylans (AX), and pose a potential use as ingredient in
health promoting foods. Recently, indications states that enzymatic modification of the DF-fraction renders
the DF sources more effective in modifying the metabolic health effects (Cloetens et al., 2010). The application of an enzyme modified bran fraction could consequently improve the suggested health effects, and thus
potentially more efficiently mitigate metabolic syndrome and risk of cardiovascular disease. Previous studies
have shown that AX and arabinoxylan oligosaccharides (AXOS) produced by enzyme modifications in particular exerted positive physiological effects when consumed (François et al., 2012; François et al., 2014;
Johansson Boll et al., 2015; Maki et al., 2012). It is therefore of great relevance to explore the susceptibility
of different wheat grain tissues to enzyme degradation and production of AXOS. Furthermore it is of interest
to explore the possibilities of different enzymes, exerting different specificities towards different AX structures. This knowledge could lead to the most efficient production of an ideal DF profile.
6
Chapter 1
Background
7
1.1 WHEAT GRAIN ANATOMY AND PROCESSING
1.1.1 Grain anatomy
The cereal grain is botanically described as a caryopsis – a single seeded fruit formed from a single carpel
with no particular method of opening to access the seed. Once pollinated and fertilised, the ovary develops
into a fruit enclosing the seed or fertilised ovule. The seed accounts for the greater part of the whole fruit
when mature and is constituted of the embryonic axis, scutellum, endosperm, nucellus, testa or seed coat,
and a surrounding pericarp (Fig. 1.1). Quantitatively, the starchy endosperm accounts for 80-85%, and thus
by far the largest part of the grain. The bran, production-wise, consisting of the pericarp, seed coat, and aleurone layer constitutes 12-18%. Lastly the embryo constitutes about 3%. The following review of these components describes the general composition of wheat grains.
Figure 1.1. Cross-section of a wheat grain showing the location of component tissues. Adapted from Surget
and Barron (2005)
1.1.1.1 Germ
The germ is the cereal embryo. It is comprised of the embryonic axis, constituting the plant of the next generation, and the scutellum, providing the requirements for the embryonic axis. Once germination occurs, the
scutellum exchanges water and solutes with the starchy endosperm; secreting hormones and enzymes and
absorbing solubilised nutrients, supporting the growth and development of the embryonic axis. An example
of such is the release of the hormone gibberellin from the embryonic axis through the scutellum, causing
production and release of hydrolytic enzymes from the aleurone cells (Evers et al., 1999). Although embedded in the starchy endosperm, the embryo is eliminated during conventional milling along with the bran. It
is, however, present in organic flour and whole grain flour.
8
1.1.1.2 Endosperm
The endosperm constitutes the majority of the cereal grain and is composed of two components, of which the
dominating one is the starchy endosperm, and the minor one is the aleurone layer. The starchy endosperm
comprises cells densely packed with starch granules embedded in a protein matrix. The organisation and
structure of both is largely dependent on the cereal species. Along with minor constituent nutrients, starch
and protein can be solubilised and mobilised to support the growth the embryo during germination through
the action of hydrolytic enzymes, released from either the scutellum or the aleurone cells. Although the cells
of the starchy endosperm show a high degree of similarity, a gradient of increasing protein towards the periphery of the starchy endosperm exists, resulting in a reverse gradient in starch concentration. Along with
increasing protein concentration, the cell size decreases, while the cell wall thickens. The cell wall composition varies with the cereal species, where wheat mainly is composed of AX, and barley and oat mainly
(1 3) and (1 4)-𝛽-D glucan. The distribution of cellulose is sparse in in the cereal cell wall of this tissue
(Evers et al., 1999).
The aleurone tissue is a continuous one cell thick layer, surrounding the starchy endosperm. The aleurone
cells display thick walls, dense contents, and prominent nuclei. This layer is rich in protein, lipids, vitamins,
and minerals, stored and ready for solubilisation and mobilisation to support the growth of the embryonic
axis. During grain development, aleurone cells divide and differentiate to starchy endosperm cells, while
they during germination release hydrolytic enzymes. At maturity the thick aleurone cell wall become distinctly two-layered, with occasional knobby, irregular thickenings associated with the transfer of metabolites
to the starchy endosperm (Rost and Lersten, 1970). In the crease region, adjacent to the major vascular bundle in the pericarp, aleurone cells exist in a modified form, termed transfer cells (Burton and Fincher, 2014).
These cells are thought to assist in solute uptake (Thompson et al., 2001; Wang et al., 1994).
1.1.1.3 Seed coat
Immediately surrounding the endosperm lies the seed coat, composed of two distinct tissues; the nucellar
epidermis (also termed hyaline) and the testa, developing from the integuments surrounding the nucellus.
Throughout the grain, the seed coat peripherally adheres to the underlying endosperm, except in the crease
region, where a void, termed the endosperm cavity, exists. The nucellar epidermis is a single layer of compressed, empty cells remaining from the nucellus after fertilisation, where the embryo and endosperm expand
at the expense of this tissue. A thin cuticle in most cereals surrounds this tissue. Externally to this layer, lies
the testa, consisting of two cell layers in wheat. Within this layer, accumulation of corky substances occur,
contributing to both colour and reduced permeability (Evers and Millar, 2002). A dense cuticle surrounds the
testa and contributes in the regulation of water and gaseous exchange.
1.1.1.4 Pericarp
During the growth and development of the grain, the pericarp acts to protect and support the endosperm and
embryo. It comprises a multi-layered structure with both complete and incomplete cell layers. Outgoing from
the endosperm, these layers are the inner epidermis or tube cells, cross cells, parenchyma or intermediate
cells, hypodermis, outer epidermis, and ultimately the cuticle of the outer epidermis. The tube cells constitute
9
a discontinuous layer, covered by a continuous layer of
cross cells, adhering to the underlying cuticle of the testa. Through grain maturation and expansion of the
starchy endosperm, the pericarp becomes progressively
more compressed with empty cells, while some looses
integrity, and become deformed or crushed until the
layer is finally dry (Fig. 1.2). Also during maturation
some loss of the parenchyma occurs, causing firm attachment of this layer only to remain in the crease region at the ventral side of the grain. Some patchy parenchyma cells remain, now regarded as intermediate
cells. Upon drying, the cuticle becomes leaky and less
able to control water movements. The absorption of
water confirms the loss of parenchyma tissue, as the
pericarp existing on each site of this layer is easily sep-
Figure 1.2. Cross section of the periphery of the
wheat grain at maturity; pericarp (p), cross cells
(crc), and aleurone (al). Fasga staining of the mature
grain. Safranin is used in combination with alcain
blue. Safranin is a red, basic, cationic dye and reacts
with lignin. Lignified tissues stain red, and non- or
poorly lignified tissues stain blue.
Adapted from Saulnier et al. (2012)
arated during gentle abrasion. This fraction of the outer
pericarp is termed beeswing (Evers and Millar, 2002).
At the distal end of the grain, opposite to the embryo, hairs radiate. These are collectively termed brush and
vary with the cereal species (Bennett and Parry, 1981).
1.1.2 Germination
The initiation of germination is triggered in a viable grain when it absorbs sufficient water. The embryo ultimately grows roots and shoots, supported by reserves of energy and metabolites liberated by activated, endogenous enzymes. Under wet conditions, germination can occur before harvest, where the grain is still attached to the parent plant. This is termed premature germination or sprouting. Hydrolytic enzymes, particularly 𝛼-amylase, activated during germination, are utilised in the process of malting, where starch is hydrolysed to large quantities of maltose. Also the aleurone layer is enzymatically degraded during seed germination, along with the starchy endosperm, to support the growth of the embryo. This is probably the reason of
the less cross-linked, cellulosic, and lignified cells walls of these tissues (Rhodes et al., 2002).
1.1.3 Grain processing
The majority of nutritional uses of wheat involve refinement of the grain, in the sense of concentrating the
starchy endosperm by removal of other grain components. The refinement is typically combined with milling, resulting in fine particle flour. The use of wheat bran as an isolated product or in whole grain flours is
limited within human nutrition, why it in many cases is considered a co-product with few economical prospects. Consequently, feed for livestock remains the main outlet of bran.
10
1.1.3.1 Milling
The objective of milling is to isolate the starchy endosperm, by separating it from the bran and germ, and
subsequently to reduce the particle size of the isolated starchy endosperm, to obtain fine particle flour. In
order to optimise the isolation of starchy endosperm, the wheat grain is tempered or conditioned prior to
milling. This process includes soaking in water until a beneficial moisture level is reached, allowing the most
efficient separation. The water slowly enters the grain through the micropyle, which is a pore admitting water to the embryo prior to germination. This alters the grain structure and promotes deformation. The bran
layers, containing multiple capillaries, toughen upon the addition of water, accelerating the deformation of
the grain layers, and thus the separation from the starchy endosperm. In addition, it assures the retention of
an intact bran fraction, and hence prevents shattering and contamination of the starchy endosperm fraction.
Conversely, the starchy endosperm softens upon water entry, improving the milling performance. The following milling process includes repeatedly grinding and sifting to form the final product (Bass, 1988).
1.2 THE CELL WALL MATRIX
1.2.1 Chemical composition
A common feature of cereal grains is their general high content of carbohydrates, and especially nondigestible carbohydrates (NDC) in the form of fibre and lignin. Carbohydrates are typically classified according to their degree of polymerisation (DP); starting at monosaccharides (1 unit), disaccharides (2 units),
oligosaccharides (2-10 units), and finally polysaccharides (>10 units). The monosaccharides are the simplest
form of carbohydrates, meaning that they cannot be further reduced. The pentoses and hexoses, containing 5
and 6 carbon atoms, are the most common. These can be present in a number of isomeric forms, as either
aldoses or ketoses in either L- or D-form according to their chirality. They occur naturally in a ring form of
either a five-membered ring with four carbons and one oxygen atom (furanose), or a six-membered ring with
five carbons and one oxygen atom (pyranose). Finally, they adapt an 𝛼- or 𝛽-configuration according to the
direction of the anomeric hydroxyl-group. The individual residues can be linked through glycosidic bonds at
any of their hydroxyl-groups in either 𝛼- or 𝛽-configuration according to the orientation. While the digestible carbohydrate starch is distributed in granules within the cytoplasm of starchy endosperm cells, the NDC
and lignin are primarily present in the primary and secondary plant cell walls (Bechtel et al., 1990; Carpita
and Gibeaut, 1993; McDougall et al., 1996; Selvendran, 1984; Vincken et al., 2003). The following will focus on cell wall components as the structure of starch is beyond the scope of this thesis.
1.2.2 The structural components
1.2.2.1 Carbohydrates
Cell wall carbohydrates are composed of the pentoses arabinose and xylose; the hexoses glucose, galactose,
and mannose; the 6-deoxyhexoses rhamnose and fucose; and the uronic acids glucuronic and galacturonic
acid or their methyl ethers. Due to the complexity and large number of permutations, a huge amount of variation possibilities, and thus three-dimensional shapes, exist. The adapted shape of the complex molecules allows for the exposure of functional surfaces influencing the physicochemical properties.
11
1.2.2.1.1 Arabinoxylan
AX is the main cell wall polysaccharide, however, not only the relative content but also the structure varies
with the tissue type (Fig. 1.3). The general structure of xylans found in cereals is a linear backbone consisting of (1 4)-linked 𝛽-D-xylopyranosyl residues with a diversity of side chains substituting the xylose units
at position O2, O3, or both. The substitutions along the xylan backbone are characterised according to their
position, resulting in the denotations mXyl2, mXyl3, and dXyl describing a substitution on the O2, O3, or
both. While the most frequent substitutions remain single units of 𝛼-L-arabinofuranose, 𝛼-D-glucuronic acid,
and its methyl ether 4-0-methyl-glururonic acid, some 2-3 units side chains with xylopyranosyl and galactopyranosyl residues associated with the arabinofuranosyl residues are also found. A frequent ratio used to explain the structure of AX is the arabinose/xylose (A/X) ratio describing the relative amount of arabinose
units per xylose unit and thus creating a rough estimate of the degree of arabinose substitution (DAS). The
distribution of substitutions along the xylan backbone is not random, but probably the result of a biosynthetic
mechanism favouring di-substitutions as well as contiguous blocks of unsubstituted xypyranosyl residues
(Dervilly-Pinel et al., 2004).
Although the xylan chain has previously been considered a rigid structure, later studies have confirmed that
AX indeed is a dynamic flexible conformation. Contrary to cellulose, which adopts a twofold rigid conformation due to the presence of two static hydrogen bonds connecting adjacent glucose residues in the backbone, the xylan adapts three-fold screw-symmetry. This conformation is supported by the presence of only
one hydrogen-bond between residues in the xylan backbone, dynamically shifting to coordinating water, and
thus forming water bridges instead (Almond and Sheehan, 2003). It is further indicated, that the presence of
arabinose side chains does not appear to influence the conformation of the xylan backbone (Ordaz-Ortiz et
al., 2004). In correlation with AX acetic acid and the hydroxycinnamic acids; ferulic and p-coumaric acid are
also found as esters. Ferulic and p-coumaric acids are specifically found linked to the O5 of the arabinofuranosyl units (Saulnier et al., 2007a). Dependent on the permutation of the constituent units, the molecular
weight, as well as the overall three-dimensional organisation of the molecules, the AX can either occur as
water-extractable (WE-AX) or water-unextractable (WU-AX).
Theoretically, the presence and concentration of side chains substituting the xylose backbone would render
the AX more soluble, due to the hindrance of polymer interaction, and thus aggregation and precipitation.
However, due to the complexity of AX, the presence of side chains also supports the formation of cross-links
between cell wall polymers through diferulic bridges, possibly cancelling out the effect (Saulnier et al.,
2007b). This is seen in both endosperm and pericarp tissues, where the WU-AX displays a higher A/X ratio,
and thus a higher DAS.
In general there is a tendency towards WE-AX having a lower molecular weight than the WU-AX regardless
of the fraction (Gruppen et al., 1991; Izydorczyk and Biliaderis, 1995). However, due to a high dispersity
index owing to differences in mass and structure, a definite comparable mass is difficult to obtain (DervillyPinel et al., 2001).
12
Figure 1.3. The main structural features of AX from the endosperm A) and maternal tissues B) of wheat grain. Components are arabinose (A), xylose (X), galactose (G), glucuronic acid (Ga), ferulic acid (F), unsubstituted xylose
(uX), di-substituted xylose (dX), O3 mono-substituted xylose (mX3), O2 mono-substituted xylose (mX2). Adapted
from Saulnier et al. (2012).
1.2.2.1.2 Cellulose
The abundance of cellulose in cereals is largely dependent on cell wall properties, and thus location and tissue influences the concentration. Cellulose is composed of linear homopolymers of (1
4)-linked 𝛽-D-
glycopyranosyl residues, with each glucose residue rotated 180° from the adjacent residue. The structure of
the linear cellulose polymer allows for static intra-molecular hydrogen-bonding, stabilising a rigid two-fold
symmetry of the molecule (Almond and Sheehan, 2003). In addition, inter-molecular hydrogen bonds and
van der Waals forces stabilise interchain cellulose associations. In the cell wall cellulose is found in microfibrillar structures of tightly associated cellulose molecules, forming a highly ordered crystalline ribbon interspersed with less organised, amorphous regions. The nature of the structure is hydrophobic, and the composition makes it relatively resistant towards enzymatic attack (Taiz and Zeiger, 2010). In contrast to dicotyledons, where cellulose is the main cell wall polysaccharide, the abundance of cellulose in monocotyledons
like cereals is low. However, cellulose is present in higher amounts in the pericarp and decreasing in
amounts approaching the endosperm. The low cellulose content in the endosperm tissues of the grain is consistent with the role of these tissues. They have no load-bearing functions, and must be quickly degradable
during germination and embryo growth (Rhodes et al., 2002).
1.2.2.1.3 𝛽-glucan
Mixed-linked-𝛽-glucan is composed of homopolymers of predominately (1
residues (70%) occasionally interrupted by (1
4)-linked-𝛽-D-glycopyranosyl
3)-linked-𝛽-D-glycopyranosyl residues (30%). The 𝛽-(1 3)
linkages are present singly, interrupting the chain in most cases at every 3 rd or 4th 𝛽-(1 4)-linkage. This
gives rise to stretches of cellulose-like sequences of cellotriosyl (DP3) and cellotetraosyl (DP4) (Burton and
Fincher, 2009; Wood, 2007). The distribution of DP3 and DP4 occurs randomly (Staudte et al., 1983). The
ratio of 𝛽-(1 4) / 𝛽-(1 3) linkages reflected in DP3/DP4 ratio varies, and influences the physical proper-
13
ties of the molecules. High and low ratios render the molecules more uniform, and hence more capable of
aligning into insoluble aggregates (Burton et al., 2010; Papageorgiou et al., 2005). The relatively high
DP3/DP4 ratio of wheat (ranging 3.0-4.5) compared to other cereals (1.8-3.5 for barley, 1.9-3.0 for rye, and
1.5-2.3 for oat), could be the reason for the low solubility of 𝛽-glucan here (Cui et al., 2000; Lazaridou and
Biliaderis, 2007).
1.2.2.1.5 Pectins
Pectins are very complex multi-domain polysaccharides. The main structural element is a backbone of
(1 4)-linked-𝛼-D-galacturonic acid residues, that, dependent on side-chain and backbone substitutions, is
identified as homogalacturonan, rhamnogalacturonan type I/II, xylogalacturonan, and arabinogalactan type
I/II. Pectins are present in the primary cell wall, and a major component of the middle lamella in many
plants. It functions in cell cohesion and cell wall elasticity especially during growth and expansion. Their
presence in wheat has, however, only recently been confirmed. Chateigner-Boutin et al. (2014) found small
but significant amounts of pectic molecules in the pericarp and endosperm tissues during grain development.
Their presence probably induced elasticity during tissue expansion. In mature grain pectic molecules were
found in the subcuticle layer of the testa in limited amounts.
1.2.2.1.6 Xyloglucan
Xyloglucan consists of a backbone of (1 4)-linked-𝛽-D-glycosyl residues heavily branched with xylose and
𝛽-galactose and minor amounts of fucose and arabinose attached to xylose units. Xyloglucan is the major
cell wall component of dicotyledons, but is present only in minor amounts in wheat (Burton and Fincher,
2014)
1.2.2.1.7 Mannans
Minor amounts of mannans, gluco-, and galactomannas are present in wheat. Their significance in cell wall
structure is however not known (Burton and Fincher, 2014).
1.2.2.2 Additional cell wall components
1.2.2.1.4 Lignin
Although lignin is not a carbohydrate, it is considered a DF due to its resistance to digestion and association
with other DF influencing their resistance to digestion (AACC, 2001). Lignin is formed by polymerisation of
three different hydroxycinnamyl alcohols or monolignols; p-coumaryl, coniferyl, and sinapyl alcohols. The
three units form an irregular, three-dimensional pattern of ether and carbon-carbon bonds. The covalent interaction between lignin and other cell wall polysaccharides might directly or indirectly appear through esterified ferulic acids (Iiyama et al., 1994). Lignin works in strengthening of the plant cell wall, especially
through deposition during secondary cell wall formation. Here, it increases the cell wall matrix rigidity by
cementing or anchoring the main polysaccharides, and hence rendering it resistant to degradation by pathogens, as well as digestion by human gastrointestinal enzymes (Bach Knudsen, 2014).
14
1.2.2.2.1 Phenolic acids
Phenolic acids are aromatic acids, consisting in the sim-
a)
plest form of a phenolic ring and an organic carboxylic
acid. Additionally, hydroxyl groups might exist on the aromatic ring, and more rings might be coupled to form
complex polymers. The complexity of the molecule affects
the bioavailability adversely. In cereals phenolic acids are
found in three forms: soluble free, soluble conjugated (esterified to sugars and other low molecular weight compounds), and insoluble bound (linked to cell wall polysaccharides, proteins, lignin, cutin and suberin) (Naczk and
b)
Shahidi, 2004). The insoluble fraction is most abundant in
wheat, accounting for 77%, whereas the soluble conjugated form account for 22% (Li et al., 2008). The main phenolic acids found in wheat are the hydroxycinnamic acids
ferulic acid and p-coumaric acid. In cereals hydroxycinnamic acids are ester-linked at the O5 position on
the 𝛼-L-arabinosyl residues of AX, promoting cross-links
between these polymers through homo-dimerization with
other hydroxycinnamic acids (Fig. 1.4). Additionally, they
act as cross-linking agents between polysaccharides an
Figure 1.4. a) Cross-linking of arabinoxylan
through dehydro-diferulates, b) chemical
structure of arabinoxylan with a xylan backbone (Xyl) substituted with arabinose residues (Ara) with ester-linked ferulic acid
(FA).
Adapted from Brouns et al. (2012)
other cell wall components such as lignin and proteins
(Iiyama et al., 1994) (Fig. 1.6). Collectively, this contributes to cell wall assembly, tissue cohesion, mechanical properties, as well as restricts expansion (Antoine et al., 2003; Iiyama et al., 1994). The cross-linking
between polysaccharides is confirmed by the presence of dehydrodimers and -trimers (Ralph et al., 1994;
Rouau et al., 2003) probably originating from oxidation by peroxidase (Fry et al., 2000). The amount and
type of phenolic acid is highly variable, both dependent on spatial orientation within the grain, the species
and the variety (Adom et al., 2005), as well as the environment (Heimler et al., 2010). The main phenolic
acid of wheat is ferulic acid, representing 90% of the bound phenolic acids in the aleurone tissue (Saulnier et
al., 2012), and thus the main cross-linking agent, promoting tissue cohesion and mechanical properties
(Iiyama et al., 1994).
1.2.2.2.2 Acetic acid
The presence of acetylations on the xylan backbone of AX is well documented (Saulnier et al., 2007a;
Saulnier et al., 2007b). However, their role in the cell wall is not well understood (Gille et al., 2011). Since
acetylations are most significant in young grains, and decreasing in content during maturation, they could
render the cell walls more flexible and elastic through the restriction of polymer interaction during grain development (Veličković et al., 2014).
15
1.2.3 The cell wall
The plant cell wall constitutes the surface external to the plasma membrane, and functions in protection, support, and cohesion of the cell. In short, the plant cell wall comprises a primary cell wall and a middle lamella.
Once fully matured, the cell might develop a secondary cell wall at the base of the primary one, providing
additional rigidity and strength. The cell wall matrix is highly adapted to the function it serves, why its structure and composition is dependent on location. The general working model of the cell wall matrix proposes
that the non-cellulosic polysaccharides (NCP) and proteins form a gel-like matrix, in which microfibrillar
cellulose and associated glucomannans are embedded (Stone, 2006). In wheat the cellulosic microfibrils are
embedded in a tightly coordinated network of AX and smaller amounts of 𝛽-glucan, lignin, proteins, phenolic acids, and additional minor compounds. The inner grain tissues contain high amounts of water within their
cell walls; preceding outwards, this is replaced with lignin encrusting the matrix, while covalently associating with the polymers present (McCann and Roberts, 1991). The relative amount of each component is not
only dependent on the cereal species, but also greatly influenced by the variety. AX being the major nonstarch polysaccharide (NSP) of wheat grain thus contributes to a great variation in the general fibre fraction,
both with regard to content and physicochemical properties (Saulnier et al., 1995).
1.2.3.1 The tissues
Roughly, the grain constitutes three overall separable compartments – the starchy endosperm, the aleurone,
and the maternal tissues. Although the starchy endosperm and the aleurone layer botanically both form part
of the endosperm, they are morphologically very different. In addition, when subjecting the grain to conventional milling procedures, the aleurone layer is separated along with the maternal tissues, collectively termed
the bran, from the starchy endosperm, and now regarded as white flour. Table 1.1 shows the distribution of
tissues and individual components within the wheat grain as found in previous studies.
1.2.3.1.1 The starchy endosperm
The cell walls constitute 2-7% of this tissue. They typically contain thin, hydrophilic primary cell walls and
are absent in secondary cell walls. The two main polysaccharides of the wheat starchy endosperm cell walls
are AX and 𝛽-glucan. The distribution is greatly dependent on species, with AX being the dominant polymer
of wheat (Comino et al., 2013). The ratio of 𝛽-glucan to AX is found to a mean of 22/78 g/g in starchy endosperm (Mares and Stone, 1973a, b), although some spatial variation exists. Characteristic to cereals, these
cell walls are essentially free of pectin and pectic substances, while a low, but though significant, content of
cellulose (2-4 % of the cell wall), glucomannan, and phenolic acids exists (Bacic and Stone, 1981; Mares and
Stone, 1973a).
Within the endosperm tissue, the only carbohydrate units constituting the AX are arabinose and xylose
(Saulnier et al., 2007a) (Fig. 1.3A). This renders the AX of this tissue predominantly neutral, due to the absence of glucuronic and 4-0-methyl-glucuronic acids (Selvendran, 1984). In this tissue the average level of
uXyl backbone residues is 50-60%, and substitutions mainly occur as single side-chain units occupying the
mXyl3 (21%) or dXyl (13%) positions. The presence of few short arabinose side chains has, however, been
suggested (Gruppen et al., 1992; Izydorczyk and Biliaderis, 1993)
16
Within the wheat endosperm tissue approximately one third of the AX is WE-AX (Ordaz-Ortiz and Saulnier,
2005). Most WU-AX is solubilised under alkaline conditions, indicating the nature of WU-AX arise from the
presence of phenolic ester cross-linkages (Gruppen et al., 1992; Selvendran, 1984). Although the amount of
the main phenolic acid, ferulic acid, is low in both WE-AX and WU-AX (0.2-0.4 and 0.6-0.9% of the AX) it
is consistently higher in WU-AX (Bonnin et al., 2000; Dervilly-Pinel et al., 2001). In addition, the amount of
dehydrodiferulic acid relative to monomeric ferulic acid is higher in wheat endosperm WU-AX than WE-AX
(Lempereur et al., 1998). Additionally, other molecular differences aside from phenolic cross-linking also
exist. The study of WU-AX after alkaline extraction has showed that it displays a slightly higher molecular
weight and A/X ratio than WE-AX (Izydorczyk and Biliaderis, 1995). The higher A/X ratio of WU-AX does
not explain its solubility, as the presence of side chains should prevent chain-chain interactions between AX,
and thus aggregation into insoluble complexes. Hence, it is thought that the solubility of AX in the endosperm is dictated by the proportion of cross-links through diferulic bridges (Saulnier et al., 2012).
The area constituting the starchy endosperm displays a large spatial diversity regarding cell wall composition
and polysaccharide morphology. An example is 𝛽-glucan, which concentration depends on cell wall thickness and thus increases towards the site of the germ. AX exhibits different structural characteristics according to location with regard to general degree of substitution as well as level of dXyl (Saulnier et al., 2009).
Large structural variation in AX have also been found in different wheat cultivars (Gebruers et al., 2008;
Saulnier et al., 2007b; Toole et al., 2011)
1.2.3.1.2 The aleurone tissue
Aleurone cells form a continuous single layer peripherally to the starchy endosperm. However, in the crease
region the aleurone cells are modified and appear as transfer cells. Due to their distinct function in solute
uptake, they exhibit different composition and structure than the aleurone cell. Although botanically a part of
the endosperm, the aleurone cells morphologically display strikingly differences from the starchy endosperm
cells, especially with regard to their cell walls. The aleurone cell wall is thick and bilayered. The cell walls of
aleurone cells are mainly composed of AX and 𝛽-glucan. The relative amount of 𝛽-glucan is increased relative to the starchy endosperm (Antoine et al., 2003; Mares and Stone, 1973a, b). Cellulose is present in higher concentrations (Barron et al., 2007), as well as lignin (Antoine et al., 2003). Although phenolic acids are
present in higher concentrations than in the starchy endosperm, the presence of dimers are still low relative
to the outer tissues (Antoine et al., 2003; Parker et al., 2005). During germination this tissue is enzymatically
degraded, proposing a possible explanation for the less excessive cross-linking.
The bulk of the AX found in the aleurone cell wall is WU-AX. It, however, displays a lower A/X ratio than
the starchy endosperm AX (Antoine et al., 2003; Bacic and Stone, 1981; Saulnier et al., 2007a). In accordance with the nature of the AX in the aleurone layer being WU-AX, it is further esterified and contain higher
amounts of ferulic and diferulic acid than the AX of the starchy endosperm. The dehydrodiferulic acid can
act as a cross-linking agent between cell wall polysaccharides, whereas esterified monomeric ferulic acid
may participate in cross-linking of polysaccharides to cell wall proteins and lignin (Fig. 1.6). Indeed, proteins have been identified in this tissue associated with cellulosic glucan, glucomannan, and highly substituted AX (Rhodes et al., 2002; Rhodes and Stone, 2002). The interactions was suggested to occur through tyro-
17
sine-hydroxycinnamic acid dimerization (Rhodes and Stone, 2002). The distribution of ferulic acid is asymmetrical, with the anticlinal cell walls existing between adjacent aleurone cells showing twice the abundance
compared to the periclinal cell walls bordering the starchy endosperm on one site and the seed coat on the
other. The increased concentration in these walls are suggested to increase the resistance to collapse following germination, allowing prolonged secretion of hydrolases to the starchy endosperm (Rhodes et al., 2002).
In addition, some acetyl residues is found substituting the xylose backbone of the AX as well as minor
amounts of p-coumaric acid (Rhodes et al., 2002). The presence of acetyl residues as substituents on the xylans of plant cell walls is well known (Timmel, 1964). They are known to render the conformation and solubility of the cell wall AX (Kenji et al., 1994; Northcote, 1972).
1.2.3.1.3 The maternal tissues
The maternal tissues comprise the outer parts of the grain; the seed coat and pericarp. The major role of the
outer layers is protection, and thus the cell walls are thick, hydrophobic, and rigid. The main compounds
comprising this tissue are cellulose and hemicelluloses in the form of complex, highly substituted xylans,
while lignin, phenolic esters, and proteins are also present (DuPont and Selvendran, 1987; Parker et al.,
2005; Ring and Selvendran, 1980). The maternal tissues hold the largest concentration of cellulose, with the
content being highest in the pericarp, and decreasing towards the middle of the grain, consistent with less
load bearing function and higher degradability during germination. Lignification supports the formation of a
secondary cell wall, which also is present in these tissues tissue (Northcote, 1963; Selvendran, 1984). The
presence of esterified phenolic acids form the basis of covalent cross-links within the matrix, further
strengthening the network and resistance towards the action of cell wall degrading enzymes from pathogenic
organisms (Parker and Waldron, 1995). The presence of cross-links is confirmed in the relative abundance of
dehydrodiferulic acid compared to its monomeric form (Antoine et al., 2003; Barron et al., 2007) (Table
1.1).
The diversity of the AX originating from different tissues are reflected in the large variations in A/X ratio
found as low as 0.13 in nucellar epidermis and testa (Parker et al., 2005), intermediate in the inner pericarp
(Antoine et al., 2003), and as high as 1.14 in the outer pericarp (Antoine et al., 2003). Contrary to the endosperm, the maternal tissues contain glucuronic and 4-0-methyl-glucuronic acids usually linked directly to the
O2 of the xylose unit, and thus rendering the AX more acidic (Fig. 1.3B). In addition, galactose, in the form
of galactopyranose residues, is also found associated with the AX of maternal tissues, most often as a part of
a 2-3 units side chain. Due to the diversity of monomers comprising the AX of these tissues, it is often described as glucuronoarabinoxylan or heteroxylan. Here, it will, however, continuously be referred to as AX
in order to avoid any confusion.
Both WE-AX and WU-AX exist in the maternal tissues. However, the WE-AX content is significantly lower
than in the starchy endosperm, only accounting for about 10 % of the total AX (Maes and Delcour, 2002).
The nature of the WE-AX is less substituted than the WE-AX of the endosperm, due to a higher proportion
of uXyl and lower proportion of dXyl residues (Delcour et al., 1999). Significant differences exist between
WE-AX and WU-AX. The WE-AX of maternal tissues shows a low DAS (A/X ratio of 0.45) and two populations of low molecular weight (20 kDa and 5 kDa) when viewing the molecular weight distribution ana-
18
lysed with gel permeation chromatography (Maes and Delcour, 2002). The WU-AX of maternal tissues
shows a significant higher DAS (A/X ratio of 0.82) and two populations with a molecular weight of 100-120
kDa and 5-10 kDa. Common to both AX fractions, the molecular weight of the soluble portion decreases
when increasing the ethanol concentration (Maes and Delcour, 2002).
The resistance of AX to degradation by endo-xylanases within the different maternal tissues illustrates well
the variations. Ordaz-Ortiz et al. (2004) found that the endosperm layers were almost completely degradable
by endo-xylanases, whereas the pericarp was completely resistant. The AX of the pericarp adapts a highly
substituted and complex structure with poor degradability (Saulnier et al., 2007a; Schooneveld-Bergmans et
al., 1999a). In the outer pericarp, the level of substitution reaches 80% and the dXyl 40% rendering the A/X
ratio close to 1 (Brillouet and Joseleau, 1987). The presence of high levels of hydroxycinnamic acids supports the nature of the highly resistant pericarp fraction. Both feruloylated AX (Ng et al., 1997;
Schooneveld-Bergmans et al., 1999b) and large amounts of dehydrodiferulic acids (Antoine et al., 2003;
Parker et al., 2005) have been isolated from this fraction. Although the total amount of ferulic acids is relatively constant within the maternal tissues, the proportion of dimerization is significantly higher in the pericarp fraction than in the inner bran layers. The proportion is 12-16 % in the aleurone and testa, whereas it
exceeds 50 % in the pericarp (Antoine et al., 2003). Like the aleurone cell wall, the bran also contains acetyl
residues substituting the xylan backbone (Mandalari et al., 2005).
Table 1.1. Contents of different wheat grain fractions. The part each fraction represents of the grain, cell wall, nonstarch polysaccharides (NSP), arabinoxylan (AX), water-extractable arabinoxylan portion (WE-AX/AX), ferulic acid,
dimerization of ferulic acid, lignin, cellulose, and uronic acids.
Bran
Pericarp
Outer periInterAleurone
Starchy
Whole
carp
mediate
endosperm grain
layera
1,4
5,1
Part of grain, % 12-18
3.5-4.0
3.2-3.85
5-81,5
835
6
Cell wall, %
40
2-77
2
3
5
3
3
NSP, %
43
73
73-78
56
35
3.52
132
2
1,3
5,11
3,11
1,8
AX, %
23
37-43
45-49
38-40
17.61.6-2
7.3-7.41,2
1,3,5,11
31
WE-AX / AX, % 8-102,4
05
3310
2
3,11
3
3,5
0.58
1.13-1.26
0.36
0.38-0.47
0.5-0.619
0.622
A/X
2
3
1
2.4
2.8-9.3
11.5
1.02
𝜷-glucan, %
1,5
3
5
5
1
5
Ferulic acid, %
0.5-0.6
0.3-0.4
0.2-0.5
0.5-0.6
0.6-0.8
0.005
0.04-0.091
Dimerization of
ferulic acid, %
14-551,5
~503
37-533,5
153
4-141,3
05
2-371
2,4
Lignin, %
7-11
1.82
2
3
1
1
Cellulose, %
7
~23
5
0.1
2.81
4
11
11
Uronic acids, %
3-6
2
1
a
Composed of hyaline, testa, and inner pericarp.
1
Brouns et al. (2012), 2Bach Knudsen (2014), 3Antoine et al. (2003), 4Maes and Delcour (2002) , 5Barron et al. (2007),
6
Hemery et al. (2009b), 7Saulnier et al. (2012), 8Ordaz-Ortiz and Saulnier (2005), 9Saulnier et al. (2007a), 10Selvendran
(1984), 11Benamrouche et al. (2002)
19
1.2.3.2 Architecture and polymer interaction
The main cell wall polymer of wheat grain is the AX. In
the endosperm it occurs along with 𝛽-glucan, whereas it
in the maternal tissues occurs along with cellulose and
variable amounts of lignin.
The cell wall AX exists in a complex interaction with the
remaining cell wall components through hydrogen
bonds, covalent bonds, or mechanical entanglement
(Saulnier et al., 2007b).
In the endosperm the main interacting polymer is 𝛽glucan. Fig. 1.5 illustrates the association of the primary
polymers of the aleurone cell walls. It is evident that the
cell wall consists of different compartments of both isolated or interacting AX and 𝛽-glucan. The AX is most
abundant in cell corners and near the interface between
adjacent cells, while the 𝛽-glucan is most abundant lining the inner periphery of the cell wall. Intermediary
space seems to be occupied by both polymers in close
interaction. Similar distribution of polymers is observed
Figure 1.5. Double labeling of AX and betaglucans in aleurone cell walls from wheat grain. A
polyclonal anti-xylan and a monoclonal antibetaglucan antibodies were used with a second
stage goat anti-rabbit (orangered fluorescence) and
a second stage goat anti-mouse (green fluorescence) antibodies, respectively. Yellow fluorescence indicates the presence of both beta-glucans
and AX. Adapted from Saulnier et al. (2007a).
in the starchy endosperm cell walls (Saulnier et al.,
2007b).
Limited information exists regarding the molecular interaction of AX and 𝛽-glucan and its significance to
cell wall properties. However it was suggested that hydrogen bonding could form the basis of polymer interaction, provided that a certain length of uninterrupted 𝛽-(1 4)-glucan strand and unsubstituted AX are present (Izydorczyk and MacGregor, 2000). A study on the interaction of AX and 𝛽-glucan using composite
films supported this theory, suggesting a possible side of interaction being the aleurone cell walls due to the
presence of low substituted AX (Ying et al., 2015).
Similar interactions should theoretically occur between cellulose, consisting solely of 𝛽-(1 4)-linked glucose residues and unsibstituted strands of AX. This was confirmed by Köhnke et al. (2011) and Selig et al.
(2015), who found that the adsorption of AX onto cellulose depended both on degree of substitutions as well
as substitution pattern. However the association is very weak and therefore not considered of great significance (Paananen et al., 2004)
Phenolic acids acting as cross-linking agents can promote the interaction of the individual AX polysaccharides, as well as AX and lignin or protein (Iiyama et al., 1994; Rhodes et al., 2002; Rhodes and Stone, 2002).
This contributes to cell wall assembly, tissue cohesion, and retardation of growth and expansion. The mechanism of cross-linking is probably oxidative and mediated by peroxidase present in different concentrations
in the plant cell wall tissues, dependent on function (Fry et al., 2000). It has been shown that the oxidation in
young tissues is peroxidase limited, whereas it in mature tissues is hydrogenperoxide limited (Fry et al.,
2000). Proposed examples of cross-linking in the cell wall are depicted in Fig. 1.6, from where it is evident
20
that the interactions of cell wall components occur through mono- or dimeric hydroxycinnamic acids, additional reactive side groups, as well as individual amino acids constituting the cell wall proteins. Polysaccharides might be inter-molecularly linked through uronic acids and hydroxyl groups present as a part of their
structure, through the dehydrogenative coupling between homo- or hetero dihydroxycinnamic acids. The
presence of monomeric hydroxycinnamic acid might form basis for the interaction with either tyrosine or
cysteine residues of cell wall proteins. The coordination possibilities of polysaccharides and lignin are many,
and can happen through direct ester or ether linkages. The presence of hydroxycinnamic acids however allow
for both ferulic and dehydrodiferulic acid bridge formation between one or two polysaccharides and lignin
(Iiyama et al., 1994).
1)
2)
Figure 1.6. Proposed covalent cross-links and mechanisms of interaction between polysaccharides and polysaccharides
and lignins or proteins within the plant cell wall. 1) Polysaccharide-polysaccharide cross-link: (a) ester-linkage carboxyl
groups of uronic acids on one polysaccharides and hydroxyl groups of another polysaccharide; (b) dehydrogenative coupling between two ferulic acids to form dehydrodiferulic acid (DDFA); (c) dehydrogenative coupling between a 𝜌coumaric acid and a ferulic acid. Protein-protein cross-link: (d) isodityrosine intra- or intermolecular cross-linking. Polysaccharide-protein: (e-g) different proposals to interactions between hydroxycinnamic acids esterified to a polysaccharide
and tyrosines or cysteines of cell wall proteins (Bacic et al., 1988; Rhodes and Stone, 2002). 2) (a) direct ester linkage
between a uronic acids on a polysaccharide and hydroxyl group son the lignin surface; (b) direct ether linkage between
polysaccharides and lignins; (c) hydroxycinnamic acid esterified to polysaccharides; (d) hydroxycinnamic acids directly
esterified to lignin surface; (e) hydroxycinnamic acid directly etherified to lignin surface; (f) covalent ester-ether bridge
between polysaccharide and lignin (or between lignins); (g) dehydrodiferulic acid in diester linkage between polysaccharides (h) etherified to lignin. (Reprinted from: (Iiyama et al., 1994))
21
1.3 NUTRITIONAL PROPERTIES OF ARABINOXYLAN AND ARABINOXYLAN OLIGOSACCHARIDES
DF is not digested by endogenous enzymes due to lack of the necessary enzymes in the human
gastrointestinal tract. Instead, it is fermented by microorganisms primarily in the colon. It is well known that
a favorable short chain fatty acid (SCFA) profile promote health effects, reducing the prevalence of a wide
range of lifestyle diseases, such as metabolic syndrome, cardiovascular diseases, and cancer, and thus
counteract the consequences of a typical unhealthy western style diet (Anderson et al., 2009). Such a profile
may be introduced by consumption of variations of DF inducing the production of SCFA.
1.3.1 Dietary fibre definition
The definition of DF was lastly evaluated in the European Regulation in 2011 and is reported as:
,,Fibre means carbohydrate polymers with three or more monomeric units, which are neither digested nor
absorbed in the human small intestine and belong to the following categories:
-
edible carbohydrate polymers naturally occurring in the food as consumed,
-
edible carbohydrate polymers which have been obtained from food raw material by physical, enzymatic or chemical means and which have a beneficial physiological effect demonstrated by generally
accepted scientific evidence,
-
edible synthetic carbohydrate polymers which have a beneficial physiological effect demonstrated by
generally accepted scientific evidence”
(1169/2011/EU, 2011)
1.3.2 Dietary fibre in the gastrointestinal tract
Although DF is not digested by endogenous enzymes in the small intestine, it influences the digestion rate
and extent of other nutrients (Cummings and Englyst, 1995). The presence of soluble DF increases the luminal viscosity (Ellis et al., 1995) and water binding capacity of the digesta (Glitsø et al., 1998). This collectively slows down gastric emptying, and movement of the digesta through the small intestine, and thus reduces the rate of glucose diffusion to the enterocytes (Ellis et al., 1995). After the escape of small intestine,
the DF components enter the large intestine – cecum and colon. The large intestine harbours a great number
and variety of microorganisms, capable of fermenting escaped food components. Dependent on the structure
and physicochemical characteristics of the DF, the rate and extent of fermentation varies. Important determinants are DP as well as type and level of substitutions influencing the extent of matrix coordination (Kabel et
al., 2002). These characteristics affect the solubility differently. While most soluble DF is readily fermentable, insoluble DF might both be fermentable (resistant starch) or non-fermentable (cellulose).
DF fermentation in the large intestine gives rise to the production of unbranched SCFA in the ratio acetate >
propionate ≥ butyrate, gaseous compounds, and additional metabolites, such as lactate and pyruvate (Pomare
et al., 1985; Topping and Clifton, 2001). The relative proportions and the amount synthesised of the unbranched SCFA both depend on the substrate available for fermentation, as well as the bacterial species, and
transit time (Wong et al., 2006).
The production of unbranched SCFA exerts multiple functions in the colon. Their presence causes a decrease
22
in pH (Campbell et al., 1997; Teitelbaum and Walker, 2002; Wong et al., 2006) resulting in an increase in
the bioavailability of the minerals calcium and magnesium (Swennen et al., 2006a; Teitelbaum and Walker,
2002) and inhibition of the growth of pathogenic bacteria (Teitelbaum and Walker, 2002; Wong et al., 2006).
1.3.3 Health related effects of dietary fibres
Epidemiological studies have shown that regular consumption of DF as a part of whole grain cereal products,
associates with reduced risk of all-cause mortality and death from cardiovascular diseases, cancer, diabetes,
respiratory disease, and infections (Huang et al., 2015). As whole grains are rich sources of DF, the suggested beneficial effects may in fact be due to the presence of DF. Physiological effects, which have been directly attributed to the component DF through systematic evidence, includes: laxation, regulation of postprandial
glucose and insulin responses, reduction of serum cholesterol levels, mineral and micronutrient absorption,
and prebiotic effects on the gut microorganisms (Huang et al., 2015). The proposed mechanism of laxation,
glucose-, insulin-, and cholesterol response is in part related to physicochemical properties of the DF. The
increased bulk reaching the large intestine stimulates contraction, and thus increases rate of passage, resulting in laxation (Marlett et al., 2000). The presence of soluble DF in the diet in turn increases the viscous environment of the digesta, prolonging gastric emptying. This reduces transit time through the small intestine,
and slows the rate of carbohydrate digestion and glucose release. Similarly, bile acids are prevented from
reabsorption in the large intestine, due to the viscous appearance of the DF rich digesta. This increases the
need for synthesis of new bile acids from cholesterol, and thus reduces circulating cholesterol levels
(Othman et al., 2011). The stimulation of production of certain SCFA is in addition thought to influence both
postprandial glucose and insulin response, as well as serum cholesterol levels (Delzenne and Roberfroid,
1994). The mechanism of mineral and micronutrient absorption is believed to depend on the production of
SCFA and reduced colonic pH (Tungland and Meyer, 2002). Additionally the presence of DF dilutes other
colonic compounds with potential pathogenic or carcinogenic effects.
Several health effects are thought to depend, at least in part, on the DF acting as prebiotics. That is, substances that influence the growth or activity of microorganisms, beneficially influencing the health of their
host (Gibson et al., 2004). Generally, any compound increasing the bacterial saccharolytic species Lactobacillus and Bifidobacterium is considered prebiotic. The proposed mechanisms of which prebiotics exert their
beneficial effects are many. The stimulation of colonization of beneficial microorganisms might reduce the
number of colonizing pathogens through competitive inhibition, through a shift to unfavourable environmental conditions by reducing pH, and by contributing to a barrier, preventing invasion (Slavin, 2013;
Teitelbaum and Walker, 2002; Wong et al., 2006). Furthermore, the increase in number of beneficial sacchorolytic microorganisms and the presence of DF as a substrate for their fermentation, reduce the level of protein fermentation, which have been shown to result in formation of potential carcinogenic or genotoxic compounds (Slavin, 2013).
Among the unbranched SCFA butyrate is considered the acid with greatest health promoting effects in the
colon. It is the preferred energy source of the colonocytes lining the lumen of the large intestine (Roediger,
1982; Wollowski et al., 2001; Wong et al., 2006). The presence of this unbranched SCFA increases the absorptive capacity of the colonocytes (Topping and Clifton, 2001) and inhibits the growth of colonic carcino-
23
ma cells both in vitro and in vivo (Scheppach et al., 1995; Wollowski et al., 2001; Wong et al., 2006). Once
absorbed over the colonic epithelium, the SCFA are transported to the liver, where the largest part is metabolised (Ingerslev et al., 2014; Kristensen and Wu, 2012). In spite of the high clearance of the liver, the increased production of SCFA causes minor amounts of propionate and butyrate to reach the peripheral circulation. Here butyrate and propionate are thought to exert metabolic effects on peripheral tissues (Bach
Knudsen et al., 2005; Priebe et al., 2010; Robertson et al., 2005). Particularly studies have linked butyrate to
improved peripheral insulin sensitivity (Priebe et al., 2010; Robertson et al., 2003; Robertson et al., 2005).
Prevention of weight gain and obesity have been proposed as a possible mechanisms underlying this effect
(Brahe et al., 2013). Furthermore, butyrate may modulate the secretion of the incretin hormones, glucagonlike peptide 1 and gastric inhibitory polypeptide, affecting insulin sensitivity, although the results are inconsistent (Hooda et al., 2010; Ingerslev et al., 2014; Regmi et al., 2011). Incretin hormones might also be modulated by a combination of butyrate and propionate, whereas acetate show no effect in mice (Lin et al.,
2012). A general increase in peripheral SCFA has been shown to reduce plasma concentrations of nonesterified fatty acids, and thus inhibit lipolysis within adipose tissues (Brighenti et al., 2006; FerchaudRoucher et al., 2005). As a consequence reduced levels of fatty acids are available for ectopic fat accumulation (Britton and Fox, 2011). Ectopic fat storage is associated with multiple lifestyle diseases (Després et al.,
2008; Heilbronn et al., 2004).
Hence, the consumption of DF is associated with multiple health effects; both directly, by influencing the
physicochemical properties of the gastrointestinal environment, and indirectly, by promoting the growth of
beneficial microorganisms, thereby inducing the production of SCFA and reducing the production of potential carcinogenic or genotoxic compounds. Collectively, these effects have been shown to mitigate the prevalence of metabolic syndrome and risk of type 2 diabetes, cardiovascular disease, and cancer.
1.3.3.1 Arabinoxylan as dietary fibre
AX is the main DF component in wheat and other cereal grains (Izydorczyk and Biliaderis, 1995). Several
studies have investigated the mechanism of digestion and fermentation of AX from cereals in general, as
well as from wheat, and wheat fractions in relation to SCFA profile. Multiple physicochemical properties of
AX influence its rate and extent of fermentation. In pigs, it was shown that the fermentation ratio of rye
fractions followed endosperm AX > aleurone AX > pericarp/testa AX, with the last fraction being largely
unfermented This suggests that the fermentation depends on both DAS, extent of cross-linking, and watersolubility. (Glitsø et al., 1999). The same study showed similar faecal DAS irrespective of diet (Glitsø et al.,
1999). Indicating that an A/X ratio of ~1 sets the limit for fermentation. In continuation WU-AX shows
limited breakdown (Damen et al., 2012a; Stevens et al., 1988), allthough some fermentation have been
documented (Damen et al., 2012a; Vardakou et al., 2008; Vardakou et al., 2007). This might be a cause of
the very different structure, and thus physicochemical properties of WU-AX, dependent on type of tissue.
Regarding the SCFA profile, Carneiro et al. (2008) oberved a relative increased butyrate production in the
cecum of pigs fed wheat bran, probably attributable to the fermentation of AX (Bach Knudsen et al., 1993).
This shift in increased butyrate production in response to AX fermentation was also observed for rye AX in
pigs (Bach Knudsen et al., 2005), wheat and rye AX in pigs (Le Gall et al., 2009), a mix of rye flakes and
24
enzyme treated wheat bran in pigs (Nielsen et al., 2014), and wheat AX fractions in rats (Damen et al.,
2011). Corresponding to the increased butyrate production, wheat bran AX was shown to induce the growth
of Roseburia / E. rectale species in pigs (Nielsen et al., 2014) and rats (Damen et al., 2012a). These species
are considered of the most important butyrate producing bacteria in humans (Duncan et al., 2004). Evidence
regarding increased butyrate concentration is however conflicting (Williams et al., 2016), but could possibly
result from an increased total pool size but not concentration (Belobrajdic et al., 2012; Williams et al., 2016).
The SCFA profile of fermented wheat endosperm AX differs remakably from wheat bran AX in rats (Damen
et al., 2012a). A particular raise in acetate was observed, causing a drop in cecum and colon pH.
Concomitantly, supressed protein fermentation probably due to the shift in pH occured (Vince and Burridge,
1980). Furthermore, relative to bran AX, endosperm AX increased the level of Bifidobacteria (Damen et al.,
2012a).
1.3.3.2 Arabinoxylan oligosaccharides as a prebiotic
The resistance to fermentation of certain WU-AX mainly found in certain bran fractions has led to the
investigation of enzyme modified wheat fractions. The main purpose being production of pre-digested AX in
the form of AXOS and xylooligosaccharides (XOS) intended to work as prebiotics by modulating the
colonic microorganisms.
Contrary to intact AX shown in rats to induce none or mediocre bifidogenic effects (Damen et al., 2012a),
XOS showed strong bifidogenic effects during in vitro fermentation with human faeces (Okazaki et al.,
1990). Broekaert et al. (2011) suggested that the lack of endoxylanase in bifidobacteria limitied their ability
to feed properly on intact AX, while they were able to degrade low molecular weight (LMW) AX (AXOS).
The same effects were seen in rats (Damen et al., 2012a).
Vardakou et al. (2007) used an in vitro model of the human colon to investigate the effect of xylanase pretreatment of the WU-AX fraction of wheat starchy endosperm. The enzyme-treated fraction stimulated the
growth of bifidobacteria, while reducing the number of proteolytic species relative to the non-digested WUAX. Both WU-AX and xylanase-treated WU-AX induced bacterial synthesis of extracellular hydrolytic
enzymes, needed for the breakdown of AX and AXOS. However, the induction was signficantly greater in
xylanase-treated WU-AX. The same effects were seen in an in vitro mixed culture fermentation system
(Vardakou et al., 2008).
Another in vitro study using simulators of the human intestinal microbial ecosystem found that AXOS had
no influence on the microbial composition, allthough they induced the synthesis of extracellular AXOSdegrading enzymes (Grootaert et al., 2009). In addition, compared to inulin, AXOS tended to shift the
fermentation to more distal parts of the colon, reflected in the site of expression of AXOS-degrading
enzymes.
Some evidence exists regarding the varying properties of structurally different AXOS. Van Craeyveld et al.
(2008) used rats to demonstrate the fermentation properties of structurally different AXOS. Generally,
AXOS with low average DP (≤5) showed bifidogenic effects regardless of average DAS in rats and chickens
(Courtin et al., 2008; Van Craeyveld et al., 2008). Additionaly AXOS with a low average DP (≤5)
stimulated colonic acetate and butyrate. AXOS with higher average DP (≥5) lowered cecal branched SCFA,
25
and AX with further increased average DP (=61) also lowered colonic branched SCFA, indicative of
supression of protein fermentation. In vitro studies confirmed that average DP negatively correlated with rate
of fermentation, shifting the fermentation site to more distal parts of the colon (Sanchez et al., 2009).
However, the increased average DP (=61) shifted the colonic SCFA content to increased acetate and
propionate (Van Craeyveld et al., 2008). The study failed to show any significant effect of DAS on
fermentation properties, allthough low DAS AXOS (0.27) tended to increase bifidobacteria relative to higher
DAS AXOS (0.69).
Results regarding the prebiotic potentials of AXOS, as well as their metabolic effects, have been conducted
in clinical trials recently. Damen et al. (2012a) investigated the impact of consumption of wheat/rye bread
containing in situ produced AXOS for three weeks on intestinal fermentation and overall gastrointestinal
characteristics. Significant effects of AXOS were found concerning urinary phenol and p-cresol excretion
and stool frequency. Bread containing AXOS reduced the potentially detrimental markers of colonic
bacterial protein fermentation and increased stool frequency. There was a tendency towards bread containing
AXOS increasing the total SCFA pool, mainly caused by an increase in butyrate.
Cloetens et al. (2008) found that AXOS with average DP of 15 and average DAS of 0.26 administration
significantly influenced breath hydrogen and urine and fecal 15N-excretion in healthy volunteers. Elevated
hydrogen excretion indicates increased bacterial fermentation, whereas a shift in urinary to fecal 15Nexcretion suggests stimulation of bacterial growth and metabolic activity. Another study by Cloetens et al.
(2010) investigated the tolerance and prebiotic effects of three weeks orally administered AXOS compared
to placebo. AXOS favorably, significantly influenced the number of bifidobacteria. The stimulatory effect
was most pronounced in subjects with lowest bifidobacteria at baseline. A general rise in the percentage of
bifidobacteria of total fecal microorganisms was observed (François et al., 2012; François et al., 2014; Maki
et al., 2012). In continuation, increased fecal SCFA and reduced fecal pH (François et al., 2012), as well as
reduced branched SCFA (François et al., 2014; Walton et al., 2012) and urinary p-cresol concentrations
(François et al., 2012) were observed, all indicative of reduced protein fermentation, and increased gut
health. Maki et al. (2012) also observed increased postprandial ferulic acid concentrations.
Johansson Boll et al. (2015) investigated AXOS and their metabolic effects in humans. A dose-dependent
decrease in glucose response, and concominant improved insulin sensitivity, were observed with increased
AXOS concentration. The consumed AXOS increased both breath hydrogen and circulating SCFA.
26
Chapter 2
Aim and Experimental Approach
27
2.1 Aims
The investigation of DF rich crops and their potential as health promoting food ingredients is of great relevance. In particular wheat is interesting, as it is the most widely grown cereal worldwide. This study comprised six structurally different wheat grain fractions treated with cell wall degrading enzymes. To evaluate
their potential as health promoting ingredients the following aims were formulated:
To investigate the response of treatment with cell wall degrading enzymes with emphasis on AX
and AXOS
To identify the fractions with the most potential for an efficient production of a beneficial
AX/AXOS profile
To explore additional alterations induced by the enzyme treatment
In view of the previously presented literature, following hypotheses were formulated:
The wheat grain fractions with the least arabinose substituted and cross-linked AX will show the
highest susceptibility to the enzyme treatment. Therefore AX of the starchy endosperm and aleurone
tissues will display the greatest response, whereas the pericarp will display the least
The aleurone tissue will provide the largest quantity of liberated AXOS, as this tissues contains substantially larger amount of AX than starchy endosperm
The modifications of the cell wall matrix will cause some liberation of additional metabolites.
2.2 Experimental approach
To induce the greatest potential release of soluble AX and AXOS, a mixture of cell wall degrading enzymes
was applied to the isolated wheat fractions. The purpose of this was to cause a modification of the cell wall
matrix, and thus trigger an increased liberation of AX. Dupont Industrial Biosciences ApS performed this
preparation prior to the commencement of this study.
To test the presented hypotheses, the following experiments were conducted. To evaluate the specific alterations incurred by the enzyme treatment, the untreated raw fractions were embedded in the study. The enzyme
treatment was performed by the addition of buffer to fractions, and thus fractions only added buffer were
additionally embedded.
The identification of structural and compositional properties of the individual wheat fractions was initially
obtained by analysing the monosaccharide composition following acid hydrolysis. This analysis was conducted using gas liquid chromatography (GLC) following derivatization of monomers to alditol acetates.
Klason lignin and uronic acids were additionally determined by a gravimetric and colorimetric method respectively.
To reveal the individual distribution of XOS with regard to DP, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was performed. This analysis was able to identify the concentration of XOS ranging from 2-6 units along with a range of other mono- and oligosaccharides.
28
To obtain a measure of the extraction yield incurred by the treatment, the poly- and oligosaccharide analyses
were additionally performed on the soluble residues obtained by isolating the supernatants of all buffertreated fractions. The residual pellets were analysed to further emphasise this point.
The liberation of additional non-carbohydrate metabolites in response to the enzyme treatment was finally
examined using non-targeted liquid chromatography-mass spectrometry (LC-MS). This analysis was able to
reveal additional changes upon treatment or differences between fractions, potentially contributing to any
health related properties of the fractions.
Fig. 2.1 provides an overview of experimental events.
Figure 2.1. Flowchart of treatments and analyses of wheat grain fractions.
29
Chapter 3
Materials and Methods
30
3.1 WHEAT FRACTIONS
In this study a total of six different wheat grain fractions from the common hard winter wheat cultivar, Tiger,
was used. The fractions originate from the European Union Framework Programme 6, HEALTHGRAIN
(HEALTHGRAIN). The wheat grain fractions comprised isolated bran, pericarp, aleurone with different purities (1 and 2), starchy endosperm, as well as a whole grain fraction. Three variants of each fraction was collected; a dry raw fraction, a blank buffer-treated fraction, and an enzyme buffer-treated fraction. Buffertreated fractions with or without enzymes were embedded in the study as whole fractions, or separated fractions, where the content was separated into supernatant and pellet. Prior to analysis all fractions, except the
supernatants, which were analysed wet, were freeze-dried and ground to pass a 0.5 mm sieve.
3.2 PRODUCTION OF WHEAT FRACTIONS
The isolated grain fractions were provided by Bühler A.G. Uzwill, Switzerland. The bran and flour were obtained by conventional milling procedures, where the grains underwent cleansing and subsequent tempering.
During tempering the grains were allowed to absorb water until fully soaked. This toughens the bran, and
causes the seed coat and aleurone layer to fuse, while it softens the starchy endosperm allowing optimal
grinding of the starchy endosperm. The soaking causes the bran to maintain some structure, and allows it
easily to be separated from the flour by sieving. Whole grain fractions were obtained by uniting the bran and
flour fractions in the original ratio after milling. The pericarp fraction was obtained by debranning by friction, also called peeling, according to the published Bühler A.G. patent applications (Eugster and
Gerschwiler, 2003). Tempering proceeded for a reduced amount of time, compared to conventional milling,
in order to limit water penetration to the outermost regions of the grain. The peripheral layers, that is the pericarp, were removed by rubbing the grains against each other. This reduced the grain weight with 3-3.5%.
The aleurone fractions were obtained by Bühler using physical dry processing according to the published
Bühler A.G. patent applications (Bohm et al., 2011; Bohm and Kratzer, 2010): A bran fraction was enriched
with aleurone cells using grinding, air-classification, and sieving (Aleurone 1). The aleurone 1 fraction was
then further purified to give high purity fraction (Aleurone 2) using electrostatic separation. The separation
of fractions was additionally described by Hemery et al. (2009a).
3.3 PRETREATMENT OF ISOLATED WHEAT FRACTIONS
3.3.1 Enzyme treatment
The isolated wheat grain fractions were enzymatically treated by DuPont Industrial Biosciences, DuPont
Aps. The fractions were suspended in buffer with or without added enzyme-mix reaching a final concentration of 0.1 g/mL. The enzyme mix consisted of commercially relevant dosages of three cell wall degrading
enzymes: Xylanase from Bacillus subtilis and Glucanase and Cellulase from Trichoderma reesei. The xylanase belonged to the glycoside hydrolase (GH) 11 family, and showed specificity towards WU-AX. Twenty-five mL double distilled H2O (10% w/w), or 14 mL double distilled H2O (10% w/w) and 11 mL enzymemix, was added to 2.5 g isolated wheat grain fractions in a container with closed lid. Fractions were placed in
31
a rotormixer at 50°C and left to equilibrate with regard to temperature (30min). Reaction was continued at
50°C for 3h. Subsequently, fractions were cooled and frozen (-20°C).
3.3.2 Sample preparation
Prior to analysis a set of buffer-treated wheat fractions were separated into supernatant and pellet. The fractions were transferred to centrifuge tubes and centrifuged (10,600 x g, 20 min, 4°C). The supernatant was
isolated and progressed to further analysis in wet form. The pellet was freeze-dried and ground to pass a 0.5
mm sieve. Whole fractions containing wheat fraction and buffer were freeze-dried collectively. Following
grounding they also proceeded to further analyses. Dry-matter was determined by placing the samples in a
vacuum oven at 50°C for 20 h. All analyses were performed in duplicates on the prepared freeze-dried or wet
fractions.
3.4 STARCH
Starch was measured by a colorimetric method described by Bach Knudsen (1997): 150 mg of fractions were
weighed into 50 mL flat bottom centrifuge tubes with screw caps. Acetate buffer (0.1M, pH 5), containing
(glacial) acetic acid (Merck 63) and sodium acetate (Merck 6267), and thermostable 𝛼-amylase (EC 3.2.1.1
120 000 U, Megazyme Ltd., Cat. No. E-BLAAM., 53,7 U/mg., Ireland) were added and fractions were incubated on a water bath for 1h at 100°C with occasional mixing (at least 3 times) in order to hydrolyse starch
polymers to maltodextrins or monomeric glucose. Released oligosaccharides were completely hydrolysed to
monomeric glucose by incubating with amyloglucosidase from Aspergillus niger (EC 3.2.1.3, Megazyme
Ltd, Cat. No. E-AMGDF, Ireland) for 2 h at 60°C. This enzyme caused the release of terminally linked glucose residues. The tubes were centrifuged (2200 x g, 10 min) and glucose monomers released to the supernatant were quantified spectrophotometrically with a glucose oxidase kit (Megazyme Ltd., Cat. No. K-GLUC).
This reagent contains glucose oxidase (≥12.000 U) and peroxidase (≥650 U) in the presence phydroxybenzoic acid and 4-aminoantipyrine. Glucose oxidase catalyzed the reaction of glucose with water
and oxygen gas yielding gluconate and hydrogen peroxide. Hydrogen peroxide then reacted with phydroxybenzoic acid and 4-aminoantipyrine in a reaction catalyzed by peroxidase. The product of this reaction, quinoneimine, is pink and absorbs light at 515 nm. The absorbance at this wavelength is proportional to
the glucose concentration. By including a standard series with known glucose concentrations in the assay,
the sample concentration was obtained.
3.5 POLYSACCHARIDES AND LIGNIN
The concentration of NSP, total carbohydrates (CHO), and Klason lignin were measured using a modification of the method previously described previously by Bach Knudsen (1997); the total CHO, soluble-, and
insoluble NSP were acid-hydrolysed to their constituent monomeric sugars. These were determined as alditol
acetates following derivatization by GLC for neutral sugars and by a colorimetric method for uronic acids
(UA), while Klason lignin was determined gravimetrically. Four parallel runs were performed (A, B, C, and
32
D) representing total NSP, total NCP, insoluble NSP, and total CHO respectively (Fig. 3.1). The samples
(125-250 mg depending on DF content) were weighed into 50 mL flat bottom centrifuge tubes with screw
caps.
Sample
as A
Removal of starch
Precipitation of
soluble NSP
as A
Extraction of
soluble NSP
Swelling, 12 M
H2SO4
Add 2 M H2SO4
Swelling, 12 M
H2SO4
Hydrolysis, 2M H2SO4
as A
as A
as A
Procedure B,
Total NCP
Procedure C,
Insoluble NSP
Procedure D,
Total CHO
Portion to
uronic acid
Reduction and
derivatization
Determination GC
Procedure A,
Total NSP
Figure 3.1. Analytical procedure for determination of non-starch polysaccharides (NSP),
non-cellulosic polysaccharides (NCP), insoluble NSP, and total carbohydrates (CHO). The
low molecular weight (LMW) compounds were determined as the difference between total
NSP and total CHO.
33
3.5.1 Enzymatic hydrolysis of starch
Samples for run A, B, and C were added 9.8 mL of acetate-buffer with CaCl2 (0.1M/20mM, pH 5), containing (glacial) acetic acid (Merck 63), sodium acetate (Merck 6267), CaCl2 (Merck 2382), and 100 𝜇L thermostable 𝛼-amylase (EC 3.2.1.1, Megazyme Ltd., Cat. No. E-Blaam) and incubated for 1h at 100°C with occasional mixing (three times) in order to hydrolyse starch polymers to maltodextrins or monomeric glucose.
Afterwards samples were cooled on ice until about 40°C and incubated with 100 𝜇L amyloglucosidase from
Aspergillus niger (EC 3.2.1.3, Megazyme, Cat. No. EMGDF) for 2h at 60°C to hydrolyse the released oligosaccharides to glucose monomers. Here the samples split, and soluble NSP (run A and B) were precipitated
by adding 40 mL 99% ethanol reaching a concentration of 80% ethanol and placed on ice bath for 1h. This
caused the soluble polysaccharides (>DP 10) to precipitate. Afterwards the samples were centrifuged (2200 x
g, 10 min), the supernatant discarded, and the pellet washed twice with 85% ethanol and once with acetone
in order to remove any LMW components. Samples were left to dry in a fume hood with max ventilation
until completely dry. Insoluble NSP (run C) were instead added 40 mL phosphate buffer (0.2 M, pH 7), containing dihydrogen phosphate monohydrate (Merck 6346) and disodium hydrogenophosphate (Merck 6586),
and incubated for 1h at 100°C. Afterwards samples were centrifuged (2200 x g, 10 min) and the supernatant
containing the soluble NSP was discarded. Pellets were washed once more in phosphate buffer to ensure
elimination of soluble NSP and afterwards washed once in 85% ethanol and acetone respectively. Like run A
and B, samples in run C were left in a fume hood until completely dry.
3.5.2 Acid hydrolysis of starch free residues and direct samples
Once dry, run A, C, and D were subjected to 12 M sulphuric acid (Merck 731) for 1h at 35°C with occasional mixing in order to sulfonate any cellulose present. The sulfonation of cellulose prevented hydrogenbonding between cellulose fibres, and thus prevented the tight interaction of cellulose microfibrils. Afterwards samples were immediately diluted to a 2 M sulphuric acid concentration. Run B was also added 2 M
sulphuric acid and all samples (run A, B, C, and D) were incubated for 1h at 100°C with occasional mixing
in order to hydrolyse all carbohydrates to monosaccharides including to the now sulfonated cellulose (run A
and C). Samples from run B and D were directly filtered (Whatman no. 5) and ready for derivatization. Supernatants were subjected only to 1 M sulphuric acid and 30 min incubation at 100°C.
3.5.3 Production of alditol acetate derivates for gas liquid chromatography
An aliquot (3 mL) of filtered hydrolysate from run A, B, C, and D was added internal standard (allose, Sigma A-6390) and the monosaccharides present were reduced to their alditol form with potassium borohydride
under alkaline conditions. This was achieved by adding 100 𝜇L potassium borohydride (Merck 820747) and
400 𝜇L 12 M ammonia (Merck 5432) to the samples, and incubating them at 45°C for 1h. This reduction
ensured that no sugars present in their ring form were acetylated, as this would further complicate the chromatogram. Adding 1-methylimidazole (Merck 805852) under acidic conditions catalysed the further acetylation of the hydroxyl groups present on the alditols when adding acetic anhydride. This acetylation prevented
hydrogen bonding between monomers and interactions with additional components present in the sample,
and thus resulted in alditol acetates sufficiently volatile to be detected in the GLC. This last step was
34
achieved by adding 200 𝜇L (glacial) acetic acid (Merck 63) to the samples, and subsequently isolating 500
𝜇L of this solution and adding 500 𝜇L 1-methylimidazole. Finally samples were added 5 mL acetic anhydride, 1 mL 99% ethanol, 5 mL milliQ water, and 5 mL 7.5M potassium hydroxide (Merck 5033) with 5-10
min intervals between each. The alditol acetates were isolated in the upper phase. GLC analysis of constituent alditol acetates was performed on a Perkin Elmer Autosystem XL GC fitted with a flame-ionisation detector. A 30m x 0.32 i.d. narrow-bore capillary column (Supelco SP 2380, Cat. No. 2-4116) was used with a
temperature of 210°C. The injector and detector temperature were of 260°C and helium was used as a carrier
gas. The principle of the detection is that the compound eluting from the column passes through the flame,
causing the production of ions and electrons. Positive ions are attracted to the cathode, where they pick up
electrons and are neutralised, whereas negative ions and electrons are attracted to the anode in an opposite
manner. The loss of electrons from the cathode and gain of electrons to the anode will cause a flow of electrons, manifested in an electric current. This current translates into peaks reflecting the concentration of a
compound.
3.5.3.1 Data processing
TotalChrom Navigator – GC was used for the data analysis. The concentration of each compound was determined the use of standards containing known concentrations of each monosaccharide and relating these to
the internally added standard. All constituent sugars were converted to equivalent polysaccharides using
convertion factors 0.89 for rhamnose and fucose, 0.88 for arabinose and xylose, 0.90 for mannose, galactose,
and glucose, and 0.915 for uronic acids.
3.5.4 Colorimetric determination of uronic acids
Uronic acids were measured by a colorimetric method described by (Scott, 1979). This was performed by the
use of a colorimetric reagent selectively reacting with a chromogen formed from the uronic acids galacturonic and glucuronic acid in concentrated sulphuric acid at 70 °C. Half a mL of hydrolysate and standards (0,
12.5, 50.0, 75.0, 100.0 𝜇g/mL galacturonic acid) was added 0.5 mL sodium-/boric acid solution (Merck
6404/165). This solution minimised the interference of neutral sugars, and thus improved the sensitivity of
the reaction. Six mL concentrated sulphuric acid was added, the samples mixed, and immediately placed in a
fume hood for 40 min. at 70°C and subsequently cooled to room temperature. This step caused the formation
of 5-formyl-2-furancarboxylic acid from the uronic acids. Afterwards 0.2 mL 3,5-dimethylphenol (Merck
821221) (0.1 g in 100 mL glacial acetic acid) was added, the samples mixed, and measured spectrophotometrically at 400 and 450 nm after minimum 10 and maximum 20 min. 3,5-dimethylphenol reacts selectively
with the formed chromogen. By substracting the absorbance at 400 nm from the absorbance at 450 nm, the
interference of neutral sugars are corrected for. In addition, the measurements were performed within 20 min
after the addition of colorimetric agent minimising the significance of side reactions.
3.5.5 Klason lignin
Klason lignin was measured gravimetrically as the residue resistant to 12 M sulphuric acid; samples from run
A and C functioned as double determination of Klason lignin and were filtered through a fritted glass cruci-
35
ble on a filter instrument. Flow through was collected and ready for derivatization along with run B and D.
Fritted crucibles were washed thoroughly with water in order to remove any sulphuric acid residues. Washed
crucibles were dried at 103°C for 20h, weighed, ashed (3.5h at 520 °C), and weighed again. The difference in
mass allowed for detection of Klason lignin.
3.5.6 𝜷-glucan
Total mixed linked 𝛽-glucan was measured by a colorimetric method described by McCleary and GlennieHolmes (1985): 𝛽-glucan was quantified by hydrolysis to 𝛽-glucooligosaccharides by lichenase (EC
3.2.1.73, Megazyme Ltd., Cat. No. E-Lichn) and further to glucose monomers by 𝛽-glucosidase (EC
3.2.1.21, Megazyme Ltd., Cat. No. E-BGluc). The released monomers were finally quantified spectrophotometrically by the glucose oxidase reagent (as previously described). One hundred fifty mg of samples were
prepared in a centrifuge tube and added 0.5 mL 50% ethanol. Subsequently, 2.5 mL sodium phosphate buffer
was added (20 mM, pH 6.5), containing dihydrogen phosphate monohydrate (Merck 6346), and samples
were incubated for 5 min in a boiling water bath. Samples were cooled and added 100 𝜇L lichenase and incubated at 40°C for 1h. Samples were then diluted with 12 mL milliQ water and centrifuged (2200 x g, 10
min). Twenty 𝜇L of supernatants were placed on a microtiter plate and each sample was added 𝛽-glucosidase
or acetate buffer (50 mM, pH 4, Merck 6268). 𝛽-glucan concentration was determined as the difference in
glucose concentration between the sample added 𝛽-glucosidase and buffer.
3.6 OLIGOSACCHARIDES
The concentration of oligosaccharides and in particular XOS were analysed by HPAEC-PAD (Dionex,
Sunnyvale, CA) as described by Lærke et al. (2015). The separation utilises the fact that carbohydrates are
weak acids that at high pH become partially ionised and form oxyanions. This allows them to be separated
by anion-exchange mechanisms on highly efficient anion-exchange columns. To 100 mg sample 2.00 mL
50% (v/v) ethanol was added and samples were following vortex mixing placed on water bath at 65°C for 60
min. for extraction with occasional mixing. This ethanol concentration allows for the extraction of far the
majority of LMW compounds, while still eliminating some polysaccharides. This is important in order to
obtain a chromatogram with separable peaks. Afterwards samples were centrifuged (2200 x g, 10 min) and
supernatants were diluted reaching a final dilution of 10 and 100 or 15 and 150 in blank and enzymatically
treated samples respectively. To dilutions were added internal standard (lactose) with a final concentration of
12.5 mg/mL. Diluted supernatants were filtered through a 22 𝜇m nylon filter (0.20 𝜇m, 13 mm, VWR no.
514-4030). The oligosaccharides were eluted from a Carbo-Pac PA100 anion-exchange column by a gradient
of milliQ water (eluent A), 0.225 M NaOH (Fluka 71686) (eluent B), and 0.5 M Na acetate (Sigma S3272)
(eluent C). This column is packed with polymeric pellicular resin beads with a positively charged surface
providing the anion-exchange effect. An elution program constituting the following flow of eluents was used
to separate the XOS: flow rate of 0.6 mL/min with 92.5% eluent A and 7.5% eluent B until 17 min where the
flow rate was increased to 1.0 mL/min, and the proportion of eluent B was increased to 11.5%. After 27 min,
eluent B was increased to 25% until 47 min, and simultaneously, 0.5% eluent C was added until 37 min and
36
further increased to 1.5% until 47 min at the expense of eluent A. Subsequently, the proportions of eluents B
and C were kept at 50% and 7%, respectively, until 55 min, where eluent C was increased to 11% while eluent B remained unchanged. Finally, at 60 min, eluent composition was changed to 50% eluent B and 50%
eluent C until 66 min, after which eluent composition returned to the initial concentrations at a flow rate of
0.7 mL/min and continued for 10 min. An extended elution programme was used to perform the separation
of cello- and glucooligosaccharides. This programme was similar to the previous until 37 min except that the
flow was maintained at 0.6 mL/min until 27 min and 0.7 mL/min from 27 to 37 min. After 37 min the eluent
C was increased to 1.5% at the expense of eluent A and the flow to 0.75 mL/min, this remained until 57 min.
Here the flow was increased to 0.8 mL/min and eluent B increased to 41.6% and eluent C to 7.0%. This continued for 10 min where the flow was again raised to 0.9 mL/min while eluent B was increased to 50% and
eluent C to 11%. This continued until 70 min where eluent B and C both were increased to 50% and the flow
increased to 1.0 mL/min. After 8 min the flow and eluent concentrations returned to the initial settings and
continued for 10 min. The elution principle is based on gradient elution. By increasing the ionic strength of
the mobile phase, the bound carbohydrates are displaced by ions competing for binding sites. This causes the
elution of increasingly charged carbohydrates and thus carbohydrates of increasing length.
A gold electrode employed a repeating sequence of three potentials was used to detect the eluted carbohydrates. The electrical current generated by their oxidation at the surface of the electrode translated into a signal reflecting the quantity. Following carbohydrate oxidation, two additional potentials were applied; the
first to clean the gold electrode by oxidation and the second to reduce the electrode back to gold, permitting
detection of the next compound.
Quantification of the carbohydrates was performed by external standards of xylose (Merck KGaA, Darmstadt, Germany), xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose (Megazyme International Ireland, Wick- low, Ireland) using mixtures in concentrations ranging from 2 to 20 mg/L.
3.7 NON-TARGETED LC-MS METABOLOMICS
3.7.1 Ultra high performance liquid chromatography-mass spectrometry
To 50 mg of sample was added 500 𝜇L 10% acetonitrile (VWR, West Chester, Pennsylvania, USA) with
glycocholic acid (Sigma, MO, USA) as internal standard. The final concentration were 100 mg sample/mL
and 0,01 mg internal standard/mL. Following 1 min of thoroughly mixing, samples were subjected to sonification for 30 min in order to release any compounds bound to the matrix. Samples were placed in 4°C for 20
min. to allow some protein precipitation and afterwards centrifuged (15 min. at 4°C, 20,000 x g). Two hundred fifty 𝜇L of the supernatants were transferred to Nanosep 10K Omega filters (Pall Life Sciencesm Ann
Arbor, MI, USA) and centrifuged (20,000 x g, 15 min, 4°C). Filtered supernatants were analysed. An ultraperformance liquid chromatography Ultimate 3000 (UHPLC, Dionex, Sunnyvale, CA, USA) system coupled to a MicrOTOF-Q II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) was used for the
UHPLC analysis. The analytical column was a HSS T3 column (100 x 2.1 mm, 1.8 𝜇m; Waters, Ireland)
held at 30°C. Electrospray ionisation (ESI) in both negative and positive mode was used for introducing the
sample to the mass spectrometer. This system separates metabolites by elution on a reversed phase column
37
and subsequent ionization using ESI, introducing the sample into the mass spectrometer consisting of a tandem quadrupole coupled to a TOF. Capillary voltage was 4500 V with a scan rate of 1 Hz. Nitrogen functioned as nebulizer and drying gas with a gas pressure of 1.8 bar. Drying gas temperature was 200°C and
flow were 8.0 L/min. Spectra were obtained over a scan range of 50-1000 m/z. 5 mM lithium formate solution (water/isopropanol/formic acid; 50:50:0.2 v/v/v ratio) was injected prior to each chromatographic run as
an external calibrate. The injection volume was 3.2 𝜇L, and the flow rate was 400 𝜇L/min. The mobile phases consisted of (A) milliQ water with 0.1 % formic acid (Fluka, Sigma-Aldrich, St Louis, MO, USA) and (B)
acetonitrile with 0.1 % formic acid. The column was equilibrated for 2 min before injection with 5% (B),
followed by a linear increase to 90% (B) over 11.89 min. Isocratic conditions were kept for 18 s before returning to 5% (B) in 12 sec. To check for potential cross-contamination from samples, control blank samples
(10% acetonitrile) were reinjected after every four samples. Bruker Daltonics software packages MicrOTOF
control v.2.3, HyStar v.3.2, and Data Analysis v.4.0 were used for MS control and data calibration.
3.7.2 Data pre-processing and multivariate data-analysis
Acquired mass spectra were calibrated and peak detection was performed using the “Find Molecular Features” option in Compass DataAnalysis Version 4.2 (Bruker Daltonics GmbH). The spectra were exported to
Bruker Compass ProfileAnalysis 2.1 for initial statistical evaluation. A matrix was generated with retention
time, m/z, and respective intensities. This matrix was exported to LatentiX 2.10 (Latent5 Aps.). Prior to principal component analysis (PCA) data were pareto scaled. Compounds were tentatively identified based on
queries in the METLIN (http://metlin.scripps.edu/) and Human Metabolome Database
(http://www.hmdb.ca/) online databases. Only compounds present in at least 50% of the fractions were included in the PCA.
3.8 CALCULATIONS
Cellulose was calculated as:
𝐶𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 = 𝑁𝑆𝑃𝐺𝑙𝑢𝑐𝑜𝑠𝑒 (12𝑀) − 𝑁𝑆𝑃𝑔𝑙𝑢𝑐𝑜𝑠𝑒 (2𝑀)
NCP was calculated as:
𝑁𝐶𝑃 = 𝑟ℎ𝑎𝑚𝑛𝑜𝑠𝑒 + 𝑓𝑢𝑐𝑜𝑠𝑒 + 𝑎𝑟𝑎𝑏𝑖𝑛𝑜𝑠𝑒 + 𝑥𝑦𝑙𝑜𝑠𝑒 + 𝑚𝑎𝑛𝑛𝑜𝑠𝑒 + 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 𝑁𝐶𝑃𝑔𝑙𝑢𝑐𝑜𝑠𝑒
+ 𝑢𝑟𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑𝑠 + 𝑐𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒
Soluble NCP was calculated as:
𝑁𝐶𝑃𝑆𝑜𝑙𝑢𝑏𝑙𝑒 = 𝑁𝐶𝑃𝑇𝑜𝑡𝑎𝑙 − 𝑁𝐶𝑃𝐼𝑛𝑠𝑜𝑙𝑢𝑏𝑙𝑒
Total NDC was calculated as:
𝑇𝑜𝑡𝑎𝑙 𝑁𝐷𝐶 = 𝐶𝐻𝑂𝑇𝑜𝑡𝑎𝑙 − 𝑆𝑡𝑎𝑟𝑐ℎ
LMW-NDC as:
𝑁𝐷𝐶𝐿𝑀𝑊 = 𝐶𝐻𝑂𝑇𝑜𝑡𝑎𝑙 − 𝑁𝑆𝑃𝑇𝑜𝑡𝑎𝑙 − 𝑆𝑡𝑎𝑟𝑐ℎ
AXOS was calculated as the difference between total AX and high molecular weight (HMW) AX. Where the
total AX was the sum of anhydrous arabinose and xylose in the direct hydrolysis procedure without ethanol
38
precipitation and the HMW-AX was the sum of anhydrous arabinose and xylose after precipitation with 80%
ethanol in the NSP procedure:
𝐴𝑋𝑂𝑆 = 𝐶𝐻𝑂𝐴𝑟𝑎𝑏𝑖𝑛𝑜𝑠𝑒+𝑋𝑦𝑙𝑜𝑠𝑒 − 𝑁𝑆𝑃𝑇𝑜𝑡𝑎𝑙 (𝐴𝑟𝑎𝑏𝑖𝑛𝑜𝑠𝑒+𝑋𝑦𝑙𝑜𝑠𝑒)
The A/X ratio was calculated as the amount of arabinose per xylose residue. This was true in all procedures.
For the calculation of NCP average values for the individual sugar residue from run A and B were used. For
the calculation of uronic acids average values from run A and C were used. Direct hydrolysis values were
obtained on the basis of double determinations.
39
Chapter 4
Results
40
4.1 POLYSACCHARIDES AND LIGNIN
In this analysis the NSP and CHO were identified according to their resistance to different concentrations of
acid hydrolysis and subsequently classified according to the monosaccharaides present. This procedure allowed the distinction of soluble and insoluble HMW compounds as well as identification of LMW compounds. The LMW compounds were identified as the compounds not precipitating with 80% ethanol.
4.1.1 Whole fractions
The chemical characterisation of raw, blank-buffer, and enzyme-buffer treated wheat fractions was performed. This was done in order to be able to characterise the specific changes induced by pure buffer and
enzyme treatment respectively.
The analysis of starch in each fraction was done in order to differentiate the origin of the glucose monomers.
The white flour showed the highest content of starch and aleurone1 the lowest (Table 4.1). The raw peeling
fraction had the highest content of NSP present as HMW-AX, cellulose, and lignin. The raw aleurone fractions had the highest content of 𝛽-glucan. The uronic acid content also depended on fraction, and was most
abundant in the peeling fraction, decreasing in content towards the middle of the grain. The quantitative
highest content of soluble NSP in the raw fractions was seen in bran (Table 4.1). The same was true for soluble AX (Table 4.2). The A/X ratio of the raw fractions was in the range of 0.5-0.6 for all fractions except
the peeling fraction, which had an A/X ratio of 1.0 (Table 4.2).
In response to treatments, total NSP decreased from raw to addition of blank-buffer and further from blankbuffer to enzyme-buffer. In particular, this was evident for HMW-AX that decreased remarkably in all fractions. The highest loss was seen in aleurone2 in response to enzyme-buffer (Fig. 4.1), whereas high losses
also were observed in response to blank-buffer in aleurone1, whole grain, and white flour. 𝛽-glucan also decreased in all but the peeling fraction in response to both blank-buffer and enzyme-buffer. Neither blankbuffer, nor enzyme-buffer affected the soluble-UA/total-UA ratio. Cellulose and Klason lignin varied in response and did not show any systematic changes (Table 4.1).
AXOS increased in response to enzyme-buffer. The most pronounced increase was seen in the aleurone2
fraction, followed by the aleurone1 fraction (Table 4.2). The loss of HMW-AX corresponded approximately
to the increase in AXOS, demonstrating redistribution the AX present in the fractions (Fig. 4.1).
An overall A/X ratio of each fraction was evident from the analysis of total NDC. The lowest general ratio
was found in the aleurone2 fraction, and the highest in the peeling fraction. The A/X ratio found in the total
NSP fraction increased in response to enzyme-buffer in all fractions to a level of ~1. Notably no change was
found from raw to blank-buffer treated fractions (Table 4.2).
For all, but the white flour and whole grain fractions, the increase in AXOS in response to the average A/X
ratio showed an inverse linear relationship (r2=0.96) (Fig. 4.2).
41
Table 4.1. Distribution of carbohydrates (CHO), starch, total non-starch polysaccharides (NSP), high molecular weight arabinoxylan (HMW-AX), cellulose, Klason lignin, and 𝛽glucan in raw, blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions.
Sample
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
+
Enzyme-mix
Raw Raw
+
Raw
+
Raw
+
Raw
+
Raw
+
Digestible CHO, %
Starch1, %
Total NSP2, %
Soluble NSP, %
HMW-AX, %
𝜷-glucan1, %
Cellulose3, %
Klason lignin4, %
Total UA1, %
Soluble-UA/Total-UA
5.5
60.6
6.8
33.7
2.4
11.4
7.2
2.0
0.2
6.0
56.7
1.8
23.9
1.6
15.2
9.1
1.8
0.2
7.7
43.9
4.2
16.9
0.4
14.5
7.9
1.7
0.2
6.8
84.4
2.6
40.9
0.4
26.7
8.8
3.5
0.2
5.5
75.5
2.2
41.7
0.3
19.2
8.4
3.1
0.1
7.0
75.3
6.3
40.3
0.4
20.4
8.9
3.1
0.2
1.9
60.0
3.9
34.3
3.4
10.6
6.3
1.9
0.2
3.2
55.1
5.6
18.4
2.9
17.9
7.1
1.6
0.2
4.8
40.9
3.0
12.2
0.8
17.6
6.1
1.5
0.2
2.2
49.2
5.5
29.2
4.5
6.0
4.7
1.1
0.3
3.2
42.9
2.9
21.2
3.9
6.6
5.0
0.9
0.2
5.1
26.8
4.7
8.2
1.3
9.9
4.7
0.9
0.3
69.6
13.0
2.3
7.2
0.6
1.9
1.5
0.4
0.3
62.5
12.5
2.8
3.7
0.6
3.5
2.2
0.4
0.3
64.3
9.0
1.7
2.0
0.2
3.2
2.1
0.3
0.4
83.2
5.1
1.7
2.1
0.2
0.2
1.7
0.1
0.8
75.4
2.9
0.9
1.2
0.3
-0.3
0.7
0.0
0.8
75.4
2.1
-0.2
0.2
0.2
0.2
0.9
0.0
1.0
1
Determined by a colorimetric method
Determined by precipitation in 80% ethanol in the NSP Procedure
3
Calculated as the difference in glucose when swelling with 2 or 12 M sulphuric acid and precipitating in 80% ethanol in the NSP Procedure
4
Determined by a gravimetric method
2
42
Table 4.2. Distribution of total arabinoxylan (AX), high molecular weight arabinoxylan (HMW-AX), arabinoxylan oligosaccharides (AXOS), and arabinose/xylose ratio (A/X) in
total non-starch polysaccharides (NSP) and total non-digestible carbohydrates (NDC) in raw, blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions.
Sample
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
Enzyme-mix
Raw
+
Raw
+
Raw
+
Raw
+
Raw
+
Raw
+
Total AX1
HMW-AX2
SolubleAX
InsolubleAX
AXOS3
A/X ratio
Total
NSP2
Total
NDC1
AXOS3
33.7
4.6
28.5
23.9
0.5
27.8
16.9
3.4
40.9
0.8
39.7
41.7
3.6
39.2
40.3
5.1
34.3
2.5
21.9
18.4
2.8
21.1
12.2
2.5
29.2
3.6
25.8
21.2
2.4
23.6
8.2
2.9
7.2
1.5
6.1
3.7
1.1
5.7
2.0
0.6
2.1
1.1
1.6
1.2
0.8
1.7
0.2
0.1
29.1
23.4
13.5
40.1
38.1
35.2
31.9
15.6
9.7
25.7
18.9
5.3
5.6
2.6
1.4
1.0
0.4
0.1
4.6
10.9
0.0
0.0
3.5
8.9
4.6
15.5
2.4
3.8
0.4
1.5
0.6
0.9
1.1
1.2
0.6
1.0
0.5
0.8
0.6
1.0
0.7
1.5
0.7
0.7
1.1
1.1
0.6
0.7
0.5
0.6
0.7
0.7
0.8
0.8
1.5
0.5
0.0
0.0
0.8
0.4
0.9
0.5
0.9
0.6
1.4
0.8
0.6
1.0
0.6
0.5
0.6
0.6
1
Determined by direct acid hydrolysis without ethanol precipitation
2
Determined by precipitation in 80% ethanol in the NSP Procedure
3
Calculated as the difference between arabinoxylan determined by direct acid hydrolysis and determined by precipitation in 80% ethanol in the NSP-procedure
43
45
40
35
% of DM
30
25
AXOS
20
HMW-AX
15
10
5
0
-
+
-
Bran
+
Peeling
-
+
-
Aleurone1
+
Aleurone2
-
+
Whole Grain
-
+
White flour
Figure 4.1. The distributiom of high molecular weight arabinoxylan (HMW-AX) and arabinoxylan
oligosaccharides (AXOS). Fractions were treated with blank-buffer (-) or enzyme-buffer (+).
300
White flour
% increase in AXOS with enzyme
250
Aleurone2
200
Aleurone1
150
Bran
100
Whole grain
50
0
0
0.2
0.4
0.6
0.8
1
Peeling
1.2
1.4
Av. A/X ratio (NDC)
Figure 4.2. The relative increase in arabinoxylan oligosaccharides (AXOS) in relation to the
average arabinose/xylose (av. A/X) ratio found in the analysis of non-digestible carbohydrates
(NDC). R2=0.96 for aleurone1, aleurone2, bran, and peeling fraction.
44
4.1.2 Separated fractions
The release of components from pellet to supernatant was investigated by separating the buffer-treated samples in supernatant and pellet by centrifugation. Supernatant and pellet were related by expressing the
amount of NDC totally present in supernatant and the amount of insoluble NSP totally present in pellet. This
was done with the assumption that no LMW and soluble residues (the difference between insoluble NSP and
NDC) was present in the pellet. This allowed a more direct illustration of the differences in soluble carbohydrates between the different fractions and upon enzyme treatment within the individual fraction. Fig. 4.3
demonstrates how the total soluble amount of NDC depends on both fraction and enzyme treatment. The aleurone2 fraction showed the highest quantitative increase in amount of soluble NDC, whereas the bran fraction showed the highest relative increase in soluble NDC in response to enzymatic treatment (Fig. 4.3).
The white flour showed a remarkable high relative amount of NDC present in the supernatant. This was
caused by the relatively low amount of NDC present in this fraction. However, in quantitative terms the
amount was low.
100
90
% NDC in supernatant
80
70
60
50
40
30
20
10
0
-
+
Bran
-
+
Peeling
-
+
Aleurone1
-
+
Aleurone2
-
+
Whole Grain
-
+
White flour
Figure 4.3. The relative amount of non-digestible carbohydrates (NDC) present in the supernatant
when separating the fraction in supernatant and pellet. Amount in supernatant compared relatively to
the total quantitative amount of NDC present in supernatant and insoluble NSP present in pellet.
Fractions were treated with blank-buffer (-) or enzyme-buffer (+).
45
4.1.2.1 Supernatants
There was a clear response to enzyme treatment in the supernatant of the wheat fractions reflected in the
drastic increase in NDC (Table 4.3). The greatest relative increase was seen in the aleurone2 fraction, which
also quantitatively showed the highest concentration of NDC. Aleurone1 was second highest followed by
bran. The white flour showed the least increase in NDC, followed by the peeling fraction. The drastic raise in
NDC was largely due to a raise in total AX present as AXOS (Fig. 4.4).
Enzyme treated aleurone2 held the highest content of total AX, HMW-AX and AXOS. This fraction also
showed the highest relative increase in response to treatment with regard to the mentioned. The second highest content was observed in aleurone1 followed by bran (Table 4.3). The A/X ratio greatly changed in response to enzyme treatment. The total AX A/X ratio was greatly reduced, as was the A/X of AXOS, which it
essentially consisted of. On the contrary the A/X ratio of HMW-AX increased in all fractions in response to
treatment.
Some response to enzyme treatment was also observed for UA, which showed a limited increase in all fractions, and most pronounced in the aleurone2 fraction.
46
Table 4.3. Distribution of soluble non-digestible carbohydrates (NDC), soluble arabinoxylan (AX), arabinoxylan oligosaccharides (AXOS), and uronic acids (UA) in supernatants of
blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions.
SampleID
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
+
+
+
+
+
+
Enzyme-mix
NDC (mg/mL)1
7.4
21.3
4.3
6.8
9.2
24.8
10.1
30.0
9.3
13.5
0.9
11.9
0.5
1.5
1.5
13.9
1.5
16.4
1.0
3.8
Total AX
(mg/mL)
0.9
0.3
1.2
0.6
0.8
0.3
0.8
0.3
0.8
0.5
A/X
HMW-AX
(mg/mL)
0.4
1.5
0.1
0.3
1.0
1.9
1.3
2.7
0.7
0.4
0.6
0.8
1.2
1.2
0.6
0.8
0.7
0.8
0.7
1.3
A/X
AXOS (mg/mL)2
0.4
10.3
0.4
1.3
0.5
12.0
0.2
13.7
0.2
3.3
1.3
0.2
1.1
0.5
1.2
0.2
2.7
0.2
1.6
0.4
A/X
UA (mg/mL)
0.1
0.2
0.1
0.2
0.1
0.2
0.1
0.3
0.0
0.1
1
Determined by direct acid hydrolysis without ethanol precipitation.
2
Calculated as the difference between AX determined by direct acid hydrolysis and determined by precipitation in 80% ethanol in the NSP-procedure.
9.2
0.8
10.7
2.0
0.8
0.7
0.7
0.7
0.2
1.3
0.0
0.2
2.2
1.8
0.6
0.1
47
35
30
mg/mL
25
20
Soluble NDC
15
AXOS
10
5
0
-
+
Bran
Peeling
+
-
+
Aleurone1
-
+
Aleurone2
-
+
Whole Grain
-
+
White flour
Figure 4.4. The relative distribution in supernatant of soluble non-digestible carbohydrates (NDC)
and arabinoxylanoligosaccharides (AXOS). Fractions were treated with blank-buffer (-) or enzymebuffer (+).
4.1.2.2 Pellets
The pellets were analysed with regard to the content of insoluble NSP. Any presence of soluble components
indicates supernatant not removed from the pellet. Indeed, it was impossible to remove all buffer-solution
when separating the fractions in supernatant and pellet (data not shown). To be able to visualise the loss of
components to supernatant, the total quantitative amount of each component in the remaining pellet was calculated and related to the original sample mass before the separation.
The fractions subjected to enzymatic treatment showed a loss of total NSP essentially due to a loss in insoluble NSP relative to the corresponding blank-buffer treated fractions (Table 4.4). The loss was largely due to
a loss in insoluble HMW-AX (Fig. 4.5). The most pronounced relative decrease in both insoluble NSP and
HMW-AX from the pellet was seen in aleurone2, whereas the highest quantitative loss was observed in the
bran fraction.
The A/X ratio increased in all fractions in response to enzyme treatment, and resulted in a relatively constant
level in the enzyme treated fractions (~1) (Table 4.4).
The amount of cellulose in the fractions varied significantly, with the peeling fraction displaying the highest
concentration, followed by aleurone1, bran, aleurone2, whole grain, and white flour.
The amount of insoluble UA was highest in the peeling fraction, and generally all fractions showed limited
decrease in UA in response to enzymatic treatment.
48
Table 4.4. Distribution of total non-starch polysaccharides (NSP), arabinoxylan (AX), cellulose, and uronic acids (UA) pellets of blank-buffer (-) or enzyme-buffer (+) treated wheat
grain fractions.
SampleID
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
Enzyme-mix
+
+
+
+
+
+
Total NSP
%TotalDM1
53.2
41.4
78.3
74.6
49.3
36.9
39.6
22.5
10.6
Insoluble
NSP
%TotalDM1
50.6
39.5
74.0
72.7
46.0
36.0
35.1
20.2
10.2
Insoluble
AX %TotalDM1
28.1
18.1
37.8
38.0
20.4
13.1
16.4
7.1
4.0
0.7
1.0
1.2
1.2
0.6
1.1
0.5
0.9
0.6
A/X
Cellulose
%TotalDM2
10.2
10.0
23.7
22.1
13.8
14.1
8.0
7.8
2.6
Insoluble
UA
%TotalDM1
0.7
0.6
2.5
2.4
1.3
1.2
0.7
0.5
0.3
1
Determined by precipitation in 80% ethanol in the NSP-procedure
2
Calculated as the difference in glucose when swelling with 2 or 12 M sulphuric acid and precipitating in 80% ethanol in the NSP Procedure
7.1
2.2
1.1
6.4
1.7
0.9
1.8
1.1
0.5
0.6
0.0
1.1
2.1
0.4
0.2
0.1
0.0
0.0
49
80
70
60
% of DM
50
40
Insoluble NSP
Insoluble HMW-AX
30
20
10
0
-
+
Bran
-
+
Peeling
-
+
Aleurone1
-
+
Aleurone2
-
+
Whole Grain
-
+
White flour
Figure 5.5. The relative distribution in pellets of total non-starch polysaccharides (NSP) and high
molecular weight arabinoxylan (HMW-AX). Fractions were treated with blank-buffer (-) or enzymebuffer (+).
50
4.2 OLIGOSACCHARIDES
5.2.1 Whole fractions
In the HPAEC-PAD analysis multiple oligosaccharides were identified using a standard-mix of oligosaccharides typical for wheat, and quantified using a standard curve by relating the height to an internally added
standard. Analysis of whole fractions revealed a significant difference in oligosaccharides both between fractions and in response to enzyme treatment. It was possible to identify XOS of varying DP ranging from 2-6.
Fig. 4.6 & 4.7 show a fingerprint chromatogram of each fraction in response to treatment. From the fingerprint it is evident that the basis of each fraction was significantly different, and that the treatment induced
different responses. The raw peeling, whole grain, and white flour fractions were generally less rich in oligosaccharides than bran, aleurone1, and aleurone2. The peeling fraction showed very limited response to the
treatments, whereas bran, aleurone1, and aleurone2 showed large fluctuations in oligosaccharides. Although
bran, aleurone1, and aleurone2 showed the largest fluctuations in XOS, whole grain and white flour showed
remarkable fluctuations in other oligosaccharides (Fig. 4.9).
A PCA analysis revealed which components resulted in the largest variances across fractions, and within
each fraction, in response to treatment (Fig. 4.8). Raw fractions were placed in close proximity although
peeling/whole grain/white flour seemed to cluster closer than bran/aleurone1/aleurone2. Of the last cluster,
bran and aleurone 2 differed the most. The peeling fraction was largely resistant to any treatment, whereas
whole grain and white flour were pulled in the direction of maltodextrins and bran/aleurone1/aleurone2 together were pulled in the direction of the XOS in response to enzymatic treatment.
Fig. 4.9 shows the distribution of identified components in the fractions by the use of standards. Evidently,
the most effect in general was seen when applying the enzyme-buffer treatment regarding the total content of
analysed components, whereas the most susceptible fractions were bran, aleurone1, and aleurone2.
Regarding the individual components, the raw fractions showed high contents of sucrose, raffinose, maltose,
and maltotriose especially in bran, aleurone1, and aleurone2 (Fig. 4.9). This was remarkably different from
blank-buffer treated samples, where the same fractions showed high contents of glucose and fructose. The
whole grain and white flour both showed very high levels of maltose in response to blank-buffer treatment,
while their glucose content increased in response to enzyme-buffer compared to blank-buffer. Glucose also
increased along with arabinose, raffinose, and to a lesser extent galacturonic acid in response to enzymebuffer in bran, aleurone1, and aleurone2.
51
a)
X1
X2
X3
X4 X5 X6
b)
X1
X2
X3
X4 X5 X6
c)
X1
X2
X3
X4 X5 X6
Figure 4.6. Fingerprint chromatograms of bran a), peeling b), and aleurone1 c). In chromatograms raw fractions are shown in black ( ), blank-buffer in blue ( ), and enzyme-buffer in magenta ( ). Xylooligosaccharides are indicated with X1 (xylose), X2 (xylobiose), X3 (xylotriose),
X4 (xylotetraose), X5 (xylopentaose), and X6 (xylohexaose).
52
a)
X1
X2
X3
X4
X5 X6
b)
X1
X2
X3
X2
X3
X4 X5 X6
c)
X1
X4 X5 X6
Figure 4.7. Fingerprint chromatogram of aleurone2 a), whole grain b), and white flour c). In
chromatograms raw fractions are shown in black ( ), blank-buffer in blue ( ), and enzyme-buffer
in magenta ( ). Xylooligosaccharides are indicated with X1 (xylose), X2 (xylobiose), X3 (xylotriose), X4 (xylotetraose), X5 (xylopentaose), and X6 (xylohexaose).
53
a)
b)
1,0
Maltoheptaose
Component 2 (19,3 % )
0,5
Maltotetraose
Galacturonsyre
D-Xylose
0,0
D-Fukose
-0,5
Maltohexaose
Sukrose
Rafinose
-1,0
-1,0
-0,5
0,0
0,5
1,0
Component 1 (51,3 % )
Figure 4.8. PCA score a) and loading plot b). Bran/aleurone1/aleurone2 raw fractions are indicated with a black circle ( ). Bran/aleurone1/aleurone2 blank-buffer and enzymebuffer are indicated with a blue ( ) and magenta ( ) circle respectively.
54
50
45
D-Fukose
Arbinose
40
Galactose
Glukose
D-mannose
35
D-Xylose
Fruktose
30
Sukrose
g/kg
Xylobiose
Rafinose
25
Xylotriose
Maltose
20
Xylotetraose
Xylopentaose
Maltotriose
15
Xylohexaose
Maltotetraose
10
Galacturonsyre
Maltopentaose
Maltohexaose
5
Maltoheptaose
0
Raw
Bran
+
Raw
Peeling
+
Raw
Aleurone1
+
Raw
Aleurone2
+
Raw
Whole Grain
+
Raw
-
+
White flour
Figure 4.9. The distribution of mono-and oligosaccharides identified in raw, blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions.
55
The raw samples did not show any appreciable content of XOS (Table 4.5). Of the enzyme-buffer
treated fractions the most pronounced difference in the XOS was seen in xylotriose, which also was
present in the highest concentration (Table 4.5). After xylotriose, xylobiose and then xylotetraose
reached second- and third highest concentrations. Bran contained the highest quantitative amount of
XOS followed by aleurone1 and aleurone2. Very high contents of monomeric xylose were in addition
identified in bran, aleurone1, and aleurone2. This was not the case in raw and blank-buffer treated
samples.
Hydrolysis products of cellulases and glucanases on cellulose and 𝛽-glucan respectively were in addition identified (Table 4.5). Bran, aleurone1, and aleurone2 responded to the enzymatic treatment by
an increase in these hydrolysis products.
56
Table 4.5. Distribution of xylooligosaccharides (XOS), glucooligosaccharides (GOS), and cellooligosaccharides (COS) with different degree of polymerisation in raw, blank-buffer
(-) or enzyme-buffer (+) treated wheat grain fractions.
Sample
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
Enzyme-mix
Raw
+
Raw
+
Raw
+
Raw
+
Raw
+
Raw
+
D-xylose, g/kg
Xylobiose, g/kg
Xylotriose, g/kg
Xylotetraose, g/kg
Xylopentaose, g/kg
Xylohexaose, g/kg
0.02
nd
nd
0.00
nd
nd
0.21
0.01
0.01
0.05
0.01
0.01
14.54
4.87
5.78
2.57
2.19
0.98
0.08
nd
nd
0.01
nd
nd
0.51
0.00
0.01
0.00
0.00
0.04
3.61
0.45
0.18
0.06
0.04
0.06
0.01
nd
nd
0.02
nd
nd
0.23
0.01
0.00
0.04
0.01
0.02
15.69
4.27
4.59
1.65
1.69
0.63
0.01
nd
nd
0.01
nd
nd
0.11
0.00
0.00
0.04
0.00
0.02
15.51
3.79
4.04
2.15
1.72
0.74
0.01
nd
0.01
nd
nd
nd
0.04
0.01
0.01
0.01
0.00
0.01
3.89
1.74
1.18
0.75
0.34
0.01
0.00
nd
nd
nd
nd
nd
0.00
0.01
0.03
0.01
0.00
0.00
1.38
0.50
0.32
0.28
0.04
0.00
Total XOScorr1, g/kg
0.17
0.08
16.39
0.01
0.05
0.79
0.02
0.07
12.83
0.01
0.07
12.44
0.01
0.03
4.02
0.00
0.05
1.15
Cellotriose, g/kg
1,3:1,4-ßGlucotriose, g/kg
Cellotetraose, g/kg
1,3:1,4-ß
Glucotetraose, g/kg
Cellopentaose, g/kg
Cellohexaose, g/kg
nd
0.00
0.00
nd
0.00
0.00
nd
0.05
0.00
nd
0.00
0.00
nd
0.03
0.00
nd
0.00
0.00
nd
nd
0.19
0.00
0.10
0.66
nd
nd
0.01
0.00
0.00
0.00
nd
nd
0.21
0.00
0.15
0.29
nd
nd
0.45
0.00
0.28
0.42
nd
nd
0.11
0.00
0.00
0.00
nd
nd
0.00
0.00
0.00
0.00
nd
nd
nd
0.08
0.02
0.19
3.19
0.01
3.02
nd
nd
nd
0.01
0.00
0.02
0.00
0.00
0.05
nd
nd
nd
0.09
0.00
0.19
0.98
0.00
5.03
nd
nd
nd
0.00
0.00
0.24
1.22
0.10
6.78
nd
nd
nd
0.28
0.02
0.23
0.00
0.00
1.34
nd
nd
nd
0.16
0.00
0.17
0.00
0.00
0.52
0.05
nd
0.55
6.45
nd
0.69
8.79
nd
0.67
1.34
nd
0.33
0.52
Total GOS+COS
g/kg
nd
0.48
6.98
nd
0.04
1Value corrected for the presence of monomeric xylose
Not detected (nd)
57
5.2.2 Supernatants
The oligosaccharide analysis of the supernatants of the fractions showed the somewhat same pattern as
the analysis of the whole fractions. There was an effect of enzyme treatment, and the effect was most
pronounced in the bran, aleurone1, and aleurone2 fractions. The most prominent difference in the
XOS was seen in xylobiose, which also was present in the highest concentration (Table 4.6). After
xylobiose, xylotriose and then xylopentaose followed in bran, aleurone1, and aleurone2, whereas xylotriose and then xylotetraose followed in the peeling, whole grain, and white flour fractions. However, the differences were small. Aleurone2, followed by bran and aleurone1 contained the highest total
concentration of the analysed XOS.
Hydrolysis products of cellulases and glucanases on cellulose and 𝛽-glucan respectively were in addition identified (Table 4.6). Although the concentrations were very low, there were some differences
from blank-buffer to enzyme treated fractions. The largest differences were seen in aleurone2, aleurone1, and bran in decreasing order.
Fig. 4.10 shows the distribution of identified components in the fractions by the use of standards. The
most effect in general was seen when applying the enzyme-buffer treatment regarding the total content
of analysed components, whereas the most susceptible fractions were bran, aleurone1, and aleurone2.
Regarding differences between the individual fractions in response to the enzyme treatment, the same
pattern was seen as in the whole fractions.
58
Table 4.6. Distribution of xylooligosaccharides (XOS), glucooligosaccharides (GOS), and cellooligosaccharides (COS) with different degree of polymerisation in blankbuffer (-) or enzyme-buffer (+) treated wheat grain fractions.
Sample
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
Enzyme-mix
+
+
+
+
+
+
D-xylose, mg/mL
Xylobiose, mg/mL
Xylotriose, mg/mL
Xylotetraose, mg/mL
Xylopentaose, mg/mL
Xylohexaose, mg/mL
0.056
0.001
0.001
0.005
0.001
0.002
1.906
0.565
0.195
0.072
0.121
0.086
0.154
0.004
0.002
0.001
0.001
0.007
0.446
0.031
0.014
0.004
0.002
0.012
0.060
0.000
0.000
0.003
0.001
0.002
2.304
0.549
0.166
0.094
0.160
0.062
0.026
0.000
0.000
0.003
0.001
0.002
2.150
0.700
0.192
0.089
0.127
0.071
0.005
0.000
0.000
0.001
0.000
0.001
0.491
0.155
0.089
0.043
0.024
0.000
0.000
0.000
0.001
0.001
0.000
0.001
0.171
0.056
0.032
0.029
0.004
0.000
Total XOScorr1, mg/mL
0.010
1.039
0.015
0.063
0.007
1.032
0.006
1.179
0.002
0.312
0.003
0.121
Cellotriose, mg/mL
1,3:1,4-ß-Glucotriose,
mg/mL
Cellotetraose, mg/mL
1,3:1,4-ßGlucotetraose, mg/mL
Cellopentaose, mg/mL
Cellohexaose, mg/mL
0.000
0.000
0.000
0.000
0.003
0.154
0.000
0.293
0.000
0.000
0.000
0.041
0.018
0.000
0.008
0.024
0.000
0.000
0.000
0.000
0.027
0.000
0.012
0.037
0.036
0.000
0.022
0.036
0.009
0.002
0.009
0.000
0.005
0.000
0.004
0.000
0.016
0.003
0.023
0.075
0.000
0.117
0.000
0.000
0.000
0.000
0.000
0.000
0.006
0.003
0.020
0.091
0.000
0.205
0.009
0.000
0.028
0.125
0.000
0.320
0.021
0.002
0.022
0.021
0.000
0.014
0.022
0.003
0.017
0.022
0.000
0.041
0.000
0.060
0.499
0.073
0.796
0.055
0.043
0.047
0.108
Total GOS+COS,
mg/mL
0.061
0.225
0.000
1
Value corrected for the presence of monomeric xylose
Not detected (nd)
59
5
4.5
D-Fukose
Arbinose
4
Galactose
Glukose
3.5
D-mannose
D-Xylose
Fruktose
3
Sukrose
mg/mL
Xylobiose
Rafinose
2.5
Xylotriose
Maltose
2
Xylotetraose
Xylopentaose
Maltotriose
1.5
Xylohexaose
Maltotetraose
Galacturonsyre
1
Maltopentaose
Maltohexaose
0.5
Maltoheptaose
0
-
+
Bran
-
+
Peeling
-
+
Aleurone1
-
+
Aleurone2
-
+
Whole Grain
-
+
White flour
Figure 4.10. The distribution of mono- and oligosaccharides identified in supernatants of blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions.
60
4.3 NON-TARGETED LC-MS METABOLOMICS
Whole fractions were analysed with non-targeted LC-MS to identify non-carbohydrate metabolites present in
an extraction solvent of 10 % acetonitrile. Metabolites were detected in both negative and positive ion mode
as the metabolites does not respond equally well to negative or positive ionization charge. Emphasis was on
phenolic compounds, and compounds differing significantly between fractions when plotting the data in a
PCA score plot. Metabolite content was evaluated as related relative to the ferulic acid score in the peeling
blank-buffer fraction.
The results of the PCA revealed a general clustering of bran/aleurone1/aleurone2 and peeling/whole
grain/white flour roughly separating these two groups. The first principal component accounted for most of
the separation of these groups, whereas both the first and second principal components contributed to minor
separation within the groups. Both in negative and positive ion mode the enzyme treatment generally did not
cause enough variation to show in any of the principal components in the cluster of peeling/whole
grain/white flour. Within this group the first principal component accounted for the separation of white flour
and peeling/whole grain, whereas the second principal component accounted for the separation of peeling
and whole grain/white flour (Fig. 4.11). The cluster of bran/aleurone1/aleurone2 differed dependent of ion
mode. In negative ion mode the enzyme treatment caused a small separation in the second principal component direction. The first principal component accounted for a small separation of bran from aleurone1/aleurone2. The second principal component contributed to a separation of bran, aleurone1, and aleurone2. Aleurone1 and aleurone2 were less separated than bran and aleurone1, and bran and aleurone2 were
most separated (Fig. 4.11a & 4.11b). In positive ion mode the enzyme treatment caused a larger separation
in both the direction of first and second principal components. While the second principal component increased in response to enzyme treatment, the first principal component decreased. Within the cluster of
bran/aleurone1/aleurone2 the enzyme treated fractions were slightly separated as accounted by the first principal component, with aleurone2 being the most different. The same was the case for the untreated fractions,
although these were generally more evenly separated (Fig. 4.11c & 4.11d).
Of the identified metabolites ferulic acid was essentially the metabolite causing the greatest variation in response to enzyme treatment. It greatly increased in all fractions, except in the white flour fraction in negative
ion mode, and whole grain/white flour fractions in positive ion mode (Table 4.7 & 4.8).
In negative ion mode citric acid also positively correlated with enzyme treatment in all fractions, though
most pronounced in white flour, whole grain, and peeling. Azelaic acid did not differ in response to enzyme
treatment, but instead largely depended on fraction, with the highest content being found in bran, aleurone1,
and aleurone2, and the lowest in white flour. The same was true for suberic acid and ethyl 2hydroxyisovalerate, although here bran alone showed high contents. Whole grain and white flour both
showed increased contents of malic acid in relation to all other fractions.
In positive ion mode L-tryptophan was identified in very high contents in bran, aleurone1, and aleurone2,
and responding negatively to enzymatic treatment. On the contrary, this compound increased in response to
enzyme treatment in whole grain and white flour. Betaine was identified in high contents in bran, aleurone1,
and aleurone2, and to a lesser extent in the peeling fraction; again responding negatively to enzyme treatment. The opposite was seen in whole grain and white flour, which showed increased betaine content in re-
61
sponse to enzyme treatment. Adenosine and deoxy-adenosine was identified in higher concentrations in bran,
aleurone1, and aleurone2, responding negatively to enzyme treatment. Both L-phenylalanine and L-leucin/Lisoleucine were lower in white flour than the remaining fractions. In aleurone1 and aleurone2 the contents
decreased in response to enzyme treatment, while it increased in white flour
62
a)
c)
PC 2
PC 2
PC 2
PC 2
0.2
0.2
0.5
1.0
0.1
0.1
0.5
0.0
0.0
0.0
0.0
-0.5
-0.5
-0.1
-1.0
-0.1
-0.2
-1.0
-1.5
-0.3
-0.2
-1.5
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
PC 1
-0.15
-0.10
b)
-0.05 -2
0.00
-1
0.05
00.15
0.10
0.20
1
0.25
PC 12
PC 1
d)
PC 2
PC 2
PC 2
0.2
NI (11)
0.2
NI (8)
NI (9)
NI (14)
0.5
0.1
NI (18)
0.0
L-Tryptophan
NI (19)
-0.5
Malic acid
Pantothenic acid
NI (3)
-0.1
NI (15)
NI (12)
Gluconic acid
-0.10
-0.05 -2 0.00
0.05-1
NI (6)
NI (7)
-0.2
Phenylalanine
Suberic acid
-0.3
Azelaic acid
-0.15
NI (3)
Betaine
NI (16)
-0.2
PC 1
NI(1)
Deoxyadenosine Adenosine
0.0
NI (1)
-1.5
2.0
NI (2)
Citric acid
NI (5)
-1.0
-0.1
L-Tryptophan
NI (10)
NI (13)
NI (17)
0.0
0.1
NI (12)
0.10
0.15
0
0.20
1 0.25
PC 1 2
PC 1
Leucine/Isoleucine
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
PC 1
Figure 4.11. PCA score and loading plots of whole fractions and metabolites in negative a), b) and positive c), d) ion mode. Score plot a) and c) show fractions; bran (▽), peeling
(○), aleurone1 (△), aleurone2 (×), whole grain (+), and white flour (◇) with buffer blank ( ) or buffer-enzyme ( ). Loading plot b) and d) show metabolites.
63
-0.10
Table 4.7. Metabolites identified with non-targeted LC-MS in negative ion mode in blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions. Values are relative to
ferulic acid in the peeling blank-buffer fraction.
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
Enzyme-mix
+
+
+
+
+
+
Metabolites negative
ion mode
0
61
1
10
1
59
5
48
0
12
0
0
Ferulic acid
114
112
39
45
69
66
58
53
34
41
3
4
Azelaic acid
11
10
4
4
7
5
4
3
4
4
0
0
Sebacic acid
31
28
5
6
33
27
40
31
15
15
7
8
L-Tryptophan
3
2
2
2
2
2
3
3
2
2
2
2
Salicylic acid
31
29
5
6
11
9
4
3
5
7
0
0
Suberic acid
28
39
16
29
25
31
26
33
18
30
4
29
Citric acid
10
9
1
2
9
8
12
11
3
3
1
2
Pantothenic acid
Ethyl 218
17
5
6
7
6
3
2
5
6
0
0
hydroxyisovalerate
22
17
19
20
16
13
15
12
35
27
36
31
Malic acid
18
14
20
19
13
12
10
11
6
6
7
7
Gluconic acid
NI(1), 193.051 m/z
29
23
15
14
19
18
16
17
13
14
3
3
NI(2), 243.124 m/z
24
26
8
5
13
11
5
4
5
6
0
0
NI(3), 241.083 m/z
9
13
8
8
13
14
15
15
6
8
2
2
NI(4), 217.108 m/z
16
17
5
5
7
6
3
3
3
4
0
0
NI (5), 133.014 m/z
0
0
1
2
0
0
0
0
3
1
2
2
NI(6), 223.046 m/z
6
4
10
10
4
3
2
2
2
2
1
1
NI(7), 175.097 m/z
9
8
4
5
3
2
1
1
2
2
0
0
NI(8), 169.087 m/z
8
5
4
5
2
1
1
0
5
5
0
0
NI(9), 253.108 m/z
6
6
8
8
4
4
3
2
1
1
0
0
NI(10), 165.040 m/z
3
3
5
5
2
2
1
1
1
1
1
1
NI(11), 329.233 m/z
167
165
53
59
156
149
165
161
111
109
90
97
NI(12), 313.238 m/z
68
53
16
14
44
68
88
72
17
6
2
2
NI1(3), 329.233 m/z
68
56
15
17
53
48
71
70
27
25
11
12
NI1(4), 327.218 m/z
42
39
6
7
52
44
63
58
19
19
12
14
NI1(5), 311.223 m/z
42
35
8
7
27
23
26
21
8
5
0
0
NI(16), 311.223 m/z
34
31
7
8
18
14
17
14
6
5
0
0
NI(17), 659.474 m/z
32
29
4
4
29
26
31
28
13
12
8
9
NI(18), 377.085 m/z
0
0
2
3
0
0
1
1
9
10
25
27
NI(19), 341.109 m/z
0
0
1
1
1
1
2
2
7
7
20
13
Not identified (NI)
Values are means (n 2)
64
Table 4.8. Metabolites identified with non-targeted LC-MS in positive ion mode in blank-buffer (-) or enzyme-buffer (+) treated wheat grain fractions. Values are relative to
ferulic acid in the peeling blank-buffer fraction.
Bran
Peeling
Aleurone1
Aleurone2
Whole Grain
White flour
Enzyme-mix
+
+
+
+
+
+
Metabolites positive ion
mode
22
49
1
11
1
58
3
45
22
8
22
22
Ferulic acid
153
146
29
32
186
165
213
182
63
67
14
26
L-Tryptophan
2
2
3
3
3
2
3
2
1
1
1
1
Trans-cinammic acid
148
129
93
84
163
131
177
138
41
66
26
39
Betaine
67
64
43
39
95
67
113
91
49
40
13
17
Adenosine
31
31
15
13
48
31
56
40
11
13
5
7
Deoxy-adenosine
31
29
35
39
42
34
41
31
15
17
4
10
L-Phenylalanine
46
44
53
58
62
52
62
47
22
25
6
15
L-Leucine/L-Isoleucine
19
17
23
18
16
13
10
9
10
10
2
4
Cytosine
NI(1), 160.133 m/z
38
39
6
5
55
41
58
55
11
10
1
2
NI(2), 296.227 m/z
46
43
10
11
46
40
46
42
18
19
9
15
NI(3), 152.057 m/z
50
46
41
34
53
47
73
59
30
29
8
11
NI(4), 279.233 m/z
29
20
6
4
34
28
36
31
6
4
9
9
NI(5), 146.093 m/z
25
23
7
7
30
23
33
30
10
10
2
3
NI(6), 72.081 m/z
10
7
17
17
13
7
11
7
7
8
2
9
NI(7), 144.102 m/z
5
4
18
15
4
3
2
1
2
1
0
0
NI(8), 177.055 m/z
22
49
1
11
1
58
3
45
22
8
22
22
NI(9), 685.241 m/z
0
10
1
2
0
10
10
10
12
12
25
26
NI(10), 325.113 m/z
3
3
6
7
3
3
4
3
12
12
14
16
Not identified (NI)
Values are means (n 2)
65
Chapter 5
Discussion
66
This thesis investigated the response of treatment with cell wall degrading enzymes on different fractions
from the common hard winter wheat cultivar Tiger. The main aim of this setup was to investigate the response of treatment with emphasis on AX and AXOS. Additional aims were to identify the fractions most
susceptible to treatment, and hence most efficiently providing a beneficial AXOS profile, and explore additional compositional changes upon enzyme treatment.
5.1 RESPONSE TO ENZYME TREATMENT
The enzyme treatment caused a decrease in NSP, which was mainly caused by a loss of HMW-AX. The loss
occurred concomitantly with increase in AXOS of similar magnitude. The susceptibility of the fractions to
enzyme treatment appeared to depend on the A/X ratio (Fig. 4.2). This was demonstrated as an inverse linear
relationship between the average DAS (expressed as average A/X) and relative increase in AXOS in the fractions with the highest NSP content (Fig. 4.2). It was further confirmed when studying the A/X ratio found in
the HMW-AX fraction (Table 4.2). All fractions demonstrated an increase in this ratio, indicating a loss of
lower substituted AX to the LMW fraction.
The relative distribution of tissues in each
fraction was previously described by Hemery
100
et al. (2009a) and Hemery et al. (2009b), who
Starchy
endosperm
Aleurone
layer
Intermediate
layer
Outer pericarp
wheat grain fractions from the cultivar Tiger
%
investigated the proportions in comparable
(Fig. 5.1). This distribution may explain some
of the properties displayed by the fractions.
Different xylanases from different GH families and organisms show different substrate
specificity (Berrin and Juge, 2008). GH family
10 and 11 are the only xylanases hydrolysing
AX (Kormelink et al., 1993a; Kormelink et al.,
0
Bran
Peeling
Aleurone
Figure 5.1. Proportions (%) of the different tissues of
wheat grain in the fractions. The starchy endosperm, the
aleurone layer, the intermediate layer (inner pericarp, testa,
and nucellar epidermis), and the outer pericarp.
Results from Hemery et al. (2009b)
1993b). Of these, GH11 xylanases prefer unsubstituted regions of AX (Berrin et al., 2007; Biely et al., 1997; Jänis et al., 2005). The results are consistent
with the used xylanase belonging to the GH11 family. Furthermore, the majority of the XOS produced in
response to enzyme treatment were present as xylobiose and xylotriose. These have previously been described as the main products of GH11 xylanase (Berrin et al., 2007). As expected, the enzyme treatment displayed the highest specificity towards WU-AX, producing mainly AXOS, but also to some extent rendering
the WU-AX more soluble as expressed in a rise in WE-AX in some fractions.
The least effect of HMW-AX degradation and AXOS production was seen in the peeling fraction. This fraction essentially constituted the pericarp of the grain, which is initially more highly substituted than the remaining grain tissues (Fig. 5.1). This tissue also shows a higher degree of cross-linking through the presence
of dehydrodiferulic acids, and thus provides steric hindrance towards hydrolysis (Table 1.1). Additional
cross-linking is also possible between polysaccharides through glucuronic acid, and between polysaccharides
67
and lignin (Iiyama et al., 1994), both which were abundant in this fraction (Table 4.1). Beaugrand et al.
(2004a) reached the same conclusion regarding the resistance of the pericarp tissue.
Overall the A/X ratio appeared to be the major factor setting the limit for further AX degradation when ~1.
This was also previously concluded by Benamrouche et al. (2002).
PCA score and loading plot of the fractions in relation to the identified oligosaccharides, revealed the major
components causing the variation across the fractions, and within each fraction in response to treatment (Fig.
4.8). The greatest variance was caused by the production of XOS in the NSP rich fractions with the lowest
A/X ratio. The highly cross-linked and rigid pericarp, comprising much of the peeling fraction, rendered it
largely resistant to any alterations. The white flour, consisting of high levels of starch, was pulled in the direction of starch hydrolysis products, whereas whole grain constituting all fractions, but dominated by the
white flour fraction, pulled in both directions.
The analysis of XOS was not able to account for a substantial amount of AXOS (Table 4.5). The amount of
arabinose substituted XOS (AXOS) presents an unknown factor, and must necessarily explain some of the
difference. The presence of longer XOS with a DP >6 could in addition contribute to the observed discrepancies between the two analyses. For the extraction of LMW-compounds, 50% ethanol was used; it is possible
that some precipitation of oligosaccharides could arise from this concentration. Swennen et al. (2006b),
however, showed that the precipitation of XOS was extremely low below 80% ethanol.
In the supernatants there was a shift in the most dominant XOS from xylotriose to xylobiose in the aleurone
containing fractions, along with a relative increase in monomeric xylose in all fractions. This could suggest
than some further hydrolysis of XOS had occurred in the supernatants. Prior to analysis, supernatants were
stored as frozen; however, the isolation process involved thawing and handling during a few hours. This was
distinct from the treatment of the fractions analysed as whole, which was subjected to freeze-drying directly.
It is likely, that this difference in preparation induced the additional hydrolysis of some polymers.
Substantial amounts of monomeric xylose were found in the fractions in response to enzyme treatment. Previously Van Craeyveld et al. (2010) investigated the action of different GH8, 10, and 11 xylanases on wheat
bran AX, and found release of high levels of monomeric arabinose and xylose in response to all enzymes.
Subsequently, they identified the action of several AX degrading endogenous enzymes (arabinofuranosidases
and xylosidases). They concluded that the release of monomeric hydrolysis products could be ascribed to the
activity of endogenous enzymes, the hydrolytic properties of the exogenous added enzymes, and the presence of xylosidases as contaminations in the exogenous added enzymes in decreasing order of significance.
The enzyme treatment in this study did not involve inactivation of endogenous enzymes. This probably
caused the large release of monomeric xylose.
Although the fermentation of monomeric xylose has been sparsely investigated in vitro using gut bacterial
species (Geraylou et al., 2014; Palframan et al., 2003), the fermentation profile might not be of great significance in vivo if ingested as such. Xylose is, when present in monomeric form, absorbed passively in the
small intestine and enters into the portal vein. Previous studies, investigating the absorption of monomeric
xylose from the small intestine, showed that the absorption was moderate (57%) in rats and complete in
roosters (Schutte et al., 1991; Yuasa et al., 1997). The complete degradation of AX to its monomeric prod-
68
ucts seem therefore not to be beneficial, if the purpose is to enhance the prebiotic potential of the DF fraction.
It cannot be excluded, that additionally to the structure and physicochemical properties of the AX in the individual fractions, protein inhibitors also play some part in the susceptibility of AX to exogenously added
xylanase. The best characterised xylanase inhibitors present in wheat are XIP (xylanase inhibitor protein)
and TAXI (Triticum aestivum xylanase inhibitor) (Juge and Delcour, 2006). However, bacterial xylanases
have not been found sensitive to the inhibition by XIP (Berrin and Juge, 2008). The apparent xylanase inhibition activity of TAXI is quite stable, and found mainly to be affected by genotype (Mendis et al., 2013).
Croes et al. (2009) investigated the internal location within the grain of the xylanase inhibitors in the wheat
cultivar Tiger, and found the largest concentration in the aleurone fraction, followed by the bran, whole
grain, white flour, and peeling fraction in decreasing order of magnitude.
The response of blank-buffer was investigated in order to differentiate between alterations induced by endogenous enzymes and exogenously added enzymes. It is likely that the addition of buffer alone during incubation at 50 °C caused some activation of endogenous enzymes present in the fractions. The setting could
provoke events naturally occurring during germination, where the starchy endosperm and aleurone layer are
mobilised to support the growth of the embryo. It is well established that the hydration of seeds induces germination, and thus the activation of endogenous enzymes (Hegarty, 1978). Several studies have confirmed
the presence of endogenous enzymes in cereals (Boskov Hansen et al., 2002; Vatandoust et al., 2012). In particular proteinases (Jones and Lookhart, 2005), starch degrading-, and cell wall polysaccharide degrading
enzymes (Poutanen, 1997). A study investigating the activity of endogenous phytase in wheat bran, showed
that incubation with distilled water at 55°C for a minimum of 80 min was able to increase the activity fourfold. The conditions of the treatment used in this study were quite similar to that, confirming that endogenous enzymes probably induced alterations as well. The distribution of endogenous enzymes is, however, not
uniform, and the outer parts of the grain such as the aleurone and bran layers as well as the germ have been
found to present the highest concentrations of enzymes (Poutanen, 1997). In particular AX degrading enzymes have been characterised in both wheat flour (Cleemput et al., 1995) and bran (Beldman et al., 1996).
The treatment with blank-buffer caused a decrease in NSP mainly in the form of HMW-AX in all but the
peeling fraction (Table 4.2). The presence of XOS and monomeric xylose were, however, not affected.
Hence, the observed decrease in HMW-AX in response to buffer treatment seemed not to be caused by the
formation of XOS with a maximum DP of 6 (Table 4.2; Fig. 4.6 & 4.7). The fact that the A/X ratio additionally did not change, could suggest that the decrease in HMW-AX was caused by the release of longer
oligosaccharides (>6 units), allowing for the release of higher DAS AX, and thus not affecting the HMWAX A/X ratio substantially.
69
5.2 PROPERTIES AND EXTRACTION YIELD OF ARABINOXYLAN AND ARABINOXYLAN
OLIGOSACCHARIDES
Separating the samples into supernatant, representing the soluble fraction, and pellet, representing the insoluble fraction, illustrated the direct effects of the enzyme treatment (Fig. 4.3). It further allowed for exact determination of the extraction yield upon treatment. The enzyme treatment induced a soluble fibre fraction,
and the response was greatest in the fractions containing aleurone tissue (the aleurone1, aleurone2, and bran).
The increase in soluble NDC largely resulted from an increase in AXOS (Fig. 4.4). The enzyme treatment
also induced some increase in soluble HMW-AX, except in the whole grain and white flour fractions (Table
4.3). The extraction yield of AX was 16.4, 13.9, and 11.9% in the aleurone2, aleurone1, and bran respectively. The extraction yield of bran was slightly higher than previously observed using endo-xylanases from either GH10 or GH11 families (~10%) (Van Craeyveld et al., 2010). It is likely that the use of additional cellwall degrading enzymes in this study worked synergistically producing an increased yield. Indeed Van
Craeyveld et al. (2010) illustrated that the aleurone cell layer remained largely intact after enzyme treatment
when staining the 𝛽-glucan in microscopic analysis, while this study showed a degradation of 𝛽-glucan.
The A/X ratio of total AX drastically decreased in all fractions, confirming that the previous observed increase in HMW-AX A/X ratio indeed resulted from the loss of lower substituted LMW-AX (AXOS) (Table
4.2). This was further confirmed when regarding the remaining WU-AX found in the pellet, where there was
a concomitant increase in the A/X ratio in response to enzyme treatment (Table 4.4). All fractions displayed
an A/X ratio ~1 in response to enzyme treatment, indicating that this degree of substitution set the limit for
further hydrolysis. The influence of arabinose substitution on the activity of xylanases was confirmed in a
previous study by Bonnin et al. (2006). They showed that, regardless of GH family and origin, the xylanase
activity was affected by arabinose content in a manner linearly related to the A/X ratio. Beaugrand et al.
(2004b) further reported an A/X ratio of ~1 in residual wheat bran fractions following xylanase action.
The aleurone-containing fractions also showed the highest concentrations of XOS. Although the aleurone2
fraction was shown to display the largest content of AXOS (Table 4.2; Fig. 4.1), the bran was shown to contain the largest concentration of XOS. It is possible that some of the oligosaccharides of the aleurone2 fractions were additionally degraded to monomeric products, due to the action of endogenous enzymes, and thus
resulting in the loss of XOS. However, the increased amount of monomeric xylose in the aleurone fractions
was not able to explain the entire difference.
In agreement with previous studies, the AX susceptibility to enzyme degradation and AXOS production differed significantly between fractions, owing to structural and physicochemical properties (Beaugrand et al.,
2004b; Benamrouche et al., 2002; Bonnin et al., 2006; Van Craeyveld et al., 2010). This study showed that
the most readily degradable AX was found in the bran and aleurone fractions. Additionally, to aleurone tissue, the bran fraction contained a relative higher content of intermediate tissue (Fig 5.1), previously shown
to display low A/X ratio (0.36) (Antoine et al., 2003). This probably counterbalanced the lower content of
aleurone tissue, explaining the comparable effects on AXOS release observed in the bran and aleurone fractions. Indeed Benamrouche et al. (2002) showed through histological analysis that the aleurone and nucellar
epidermis were completely degraded in response to treatment with endo-xylanase. Additionally, they showed
that the pericarp was completely resistant to histological changes, as also confirmed in this study.
70
Collectively this study revealed that the fractions initially were significantly different with regard to NSP
content and relative distribution. When regarding the raw fractions in relation to the buffer treated samples, it
was evident that some changes were induced in response to the activation of endogenous enzymes, probably
as a result of germination-like conditions. It was also evident that the fractions responded differently to the
enzyme treatment. The susceptibility of the AX to enzyme degradation seemed to depend on the content of
aleurone tissue in the fraction, as the highest yield of AXOS and general extraction yield (supernatants) were
found in the aleurone2 fraction. The different structural features of the AX such as the DAS and the extent of
cross-linking in the cell wall probably influenced the susceptibility to xylanase treatment, as also previously
found (Beaugrand et al., 2004b; Bonnin et al., 2006; Van Craeyveld et al., 2010). Additionally, the results
indicated that the added xylanase might display specificity towards WU-AX with the main conversion product being LMW-AX. However, due to the initially relatively low amount of WE-AX, it is difficult draw any
final conclusions to this statement. Generally the whole grain and white flour fraction showed somewhat inconsistent results. This was attributed to their relatively low initial NSP and in particular AX content, and
thus small quantitative variations could cause substantial relative variations within these samples.
5.3 FURTHER ALTERATIONS
In addition to xylanase, cellulase and 𝛽-glucanase were also embedded in the enzyme treatment in order to
induce AX susceptibility by modifying the general matrix structure. Whereas generally no effect of cellulase
on cellulose content was seen, 𝛽-glucan decreased in response to enzyme treatment. Due to the cellulose-like
stretches of 𝛽-glucan both 𝛽-glucanase and cellulase were contributing to the hydrolysis of this polysaccharide. The enzyme treated samples displayed higher background absorption during 𝛽-glucan analysis (data not
shown), indicating a high amount of free glucose present, some probably originating from the hydrolysis of
𝛽-glucan.
The intact hydrolysis products of cellulose and 𝛽-glucan were also analysed (Table 4.5). Again the most affected fractions by the enzyme treatment were the aleurone-containing fractions. Contrary to the observed
decrease in 𝛽-glucan in response to enzyme treatment (Table 4.1), the hydrolysis products of 𝛽-glucan were
limited. The dominating product was cellohexaose, expected to originate from cellulose, as the consecutive
stretches of 𝛽-(1 4)-linked glycosyl residues are limited to predominantly 3 or 4 in 𝛽-glucan. It is, however, possible that the hydrolysis of 𝛽-glucan results in some longer stretches of uninterrupted 𝛽-(1 4)-linked
glycosyl residues as Wood et al. (1994) found that ~8% of the 𝛽-glucan contained 4-15 consecutive 𝛽(1 4)-linked units. As virtually no loss of cellulose was found in the polysaccharide analysis, these products
might indeed originate from 𝛽-glucan.
The analysis of the pellet remaining after isolation of the supernatant confirmed that the cellulose content
was not affected by the enzyme treatment. The cellulose might be embedded in too dense a matrix structure
to become extensively hydrolysed. The uronic acid content was greatest in the peeling fraction in accordance
with the AX of the pericarp tissue being highly substituted with glucuronic acids.
71
The PCA plots obtained from the LC-MS metabolomics analysis, showed same separation pattern as the previous PCA plot from the oligosaccharide analysis. However, while the separation in the PCA plot of the oligosaccharides mainly was caused by the enzyme treatment, the separation was mainly caused by the fraction
in the PCA plot of the LC-MS metabolomics analysis.
The only metabolite identified increasing in response to the enzyme treatment was ferulic acid (Table 4.7;
4.8). In the cell wall ferulic acid is known to occur in association with AX (Fig. 1.4). Consistent with the
greatest degradation of AX in the aleurone containing fractions, these also showed the largest liberation of
ferulic acid. Although the aleurone cell walls contain a great deal of ferulic acid, previous studies showed
that it is mainly present in its monomeric form (Antoine et al., 2003; Brouns et al., 2012). This may limit its
ability to promote intermolecular cross-linking between cell wall polymers. Iiyama et al. (1994) previously
suggested that monomeric ferulic acid is able to promote cross-linking between polymers and lignin. The
presence of lignin in the aleurone cell walls was relatively limited. This is probably why AX degradation was
extensive, and liberation of ferulic acid was observed in response to the enzyme treatment in these fractions.
This is in agreement with previous results from Benamrouche et al. (2002) and Lequart et al. (1999) who
showed that endo-xylanase was able to release carbohydrates containing ferulic acid from non-lignified cell
walls. This effect could provide an additional dimension to the health promoting effect of enzyme treated
wheat bran fractions. The liberation of ferulic acid in an enzyme treated aleurone fraction of wheat, previously was shown to provide a combinational beneficial health effect in obese mice (Pekkinen et al., 2014).
Generally a higher concentration of the dicarboxylic acid, azelaic acid, was found in the bran fraction. This
metabolite has previously been proposed as a biomarker for whole grain consumption due to its discriminative presence in bran but not endosperm (Coulomb et al., 2015). Due to its beneficial antibacterial and anticancer activities, it is potentially contributing to the beneficial effects ascribed to whole grain intake
(Coulomb et al., 2015).
Altogether higher amino acid contents (phenylalanine, tryptophan, and leucine/isoleucine) were observed in
the outer grain fractions (bran, peeling, and aleurone). Also nucleosides (adenosine and cytosine) and a deoxyribonucleoside (deoxyadenosine) were identified in essentially all but the white flour fraction. Citric acid
and malic acid are both metabolites associated with the citric acid cycle and central carbon metabolism, and
were also identified in varying amount. Both amino acids, nucleosides, and citric acid cycle metabolites may
indicate metabolic activity in the grain (Coulomb et al., 2015; Gillissen et al., 2000).
The aromatic amino acid, phenylalanine, may influence metabolic activity by serving as a precursor for a
range of secondary metabolites important for plant growth and human nutrition and health (Tzin and Galili,
2010).
Consistent with previous studies, betaine was identified in high amounts in the outer grain layers (Bruce et
al., 2010; Gillissen et al., 2000). Betaine functions as a methyl donor in the methionine cycle and is an important nutrient for the prevention of chronic disease (Craig, 2004).
Finally, it is important to note that the extraction of metabolites were induced by sonification, which disrupts
the cell wall matrix and improves the extractability of cell wall associated components (Hielscher, 2016). It
is therefore possible that some components here regarded as liberated, would physiologically not have been
liberated during digestion in the gastrointestinal tract.
72
Changes broad upon by the activation of endogenous enzymes were also observed. 𝛽-glucan was found to
decrease in response to the action of endogenous enzymes in the fractions with the highest initial concentration. This is in agreement with previous studies, showing that endogenous 𝛽-glucanases were present mainly
in the bran of ungerminated wheat. During germination it spreads throughout the entire kernel (Vatandoust et
al., 2012).
The activation of endogenous enzymes caused cellulose to decrease in the peeling fraction exclusively.
Wheat grain have been shown to contain cellulases, responsible for the hydrolysis of cellulose (Schmitz et
al., 1974), however the site of production is not known. One could speculate that the cellulase content would
be largest in the tissue holding the greatest cellulose content, that is the pericarp. This would provide a plausible explanation for the observed drop in cellulose specifically restricted to the peeling fraction. The remaining fractions showed an increase in cellulose in response to treatments. Possibly, a loss of NSP mainly in the
form of HMW-AX, caused the relative amount of cellulose in the fraction to increase.
The identification of oligosaccharides revealed a complex pattern of responses to buffer and enzyme treatment. Generally, the raw fractions had high sucrose contents, which in response to buffer treatment resulted
in an increase of its hydrolysis products glucose and fructose. The fractions holding the largest starch content
responded to treatments with increasingly maltose concentration, which is a hydrolysis product of starch.
Along with this, a concomitant decrease in starch content was observed (Table 4.1). These events were ascribed to the action of endogenous enzymes, responsible for the mobilisation of energy, and thus breakdown
of glucose and fructose containing polymers.
The raw aleurone fractions held initially higher maltodextrin levels than the remaining fractions. It is possible that the additional processing of these raw fractions, due to the higher complexity of the isolation process, caused an activation of some endogenous enzymes. The maltodextrins decreased in response to buffer
treatment concomitant with an increase in their hydrolysis product, glucose. Again this was likely the action
of additionally activated endogenous enzymes.
73
Chapter 6
Conclusion
74
This master’s thesis investigated the response of enzyme treatment with emphasis on AX and AXOS in
structurally different wheat grain fractions. Incubation with an enzyme-mix containing the cell wall degrading enzymes primarily resulted in the conversion of HMW-AX to AXOS and to a much lesser extent the
conversion of WU-AX to WE-AX.
In general, the susceptibility of the fractions to enzyme modifications were highly dependent on DAS, manifested in an inverse relationship between relative increase in AXOS and A/X ratio. Ultimately an A/X ratio
of ~1 set the limit for further enzyme degradation of AX. The most pronounced effect of enzyme modification was seen in the most pure aleurone2 fraction, owing to the initially most susceptible AX profile. This
fraction showed an AX extraction yield of 16.4%. The peeling fraction showed generally resistant to any
modifications, as explained by its high DAS and ability to form extensive cross-links.
Investigation of the XOS produced in response to enzyme treatment showed that a substantial amount was
present as monomeric xylose. This was expected to originate from the action of endogenous xylosidases. It is
likely, that inactivation of endogenous enzymes by heat treatment prior to incubation could limit the complete degradation of XOS to monomeric xylose, and thus provide additional health promoting effects.
The only identified non-carbohydrate metabolite liberated by the enzyme treatment was ferulic acid. This
liberation was greatest in the fractions also most susceptible to enzyme modifications regarding the AX.
The collected modifications observed in the aleurone containing fractions, regarding the solubilisation of
NSP and production of AXOS along with a liberation of ferulic acid, could improve the nutritional and
health promoting profile, and thus potentially form a basis for the use of otherwise regarded low-value cereal
co-products.
75
Chapter 7
Future Perspectives
76
This study was able to identify a decrease in HMW-AX along with a concomitant increase in LMW-AX in
response to enzyme treatment. However, it was not able to identify all products of degradation, due to the
limitations of the HPAEC-PAD analysis. These limitations consisted in the constrained identification of unsubstituted XOS, and the restricted length of the identified oligosaccharides due to the absence of XOS
standards with a DP>6. Further knowledge regarding the DP of AX liberated to the supernatant in enzyme
treated fractions could be acquired by determination of the reducing end sugar residues, and thus obtaining a
measure of the average DP. This could potentially be executed by a method similar to the one used in this
study to determine the monosaccharide composition after hydrolysis. However, the reduction should be performed prior to hydrolysis, and thus only cause acetylation, and thereby detection of the reducing end sugar
monomer (Courtin et al., 2000).
The oligosaccharide analysis revealed, that a substantial amount of monomeric xylose was produced in response to the enzyme treatment. The purpose of producing oligosaccharides with a prebiotic potential is
greatly compromised if the products are further hydrolysed to monomers. The monomeric xylose is mostly
absorbed in the small intestine, and thus does not exert any potential as a prebiotic. Van Craeyveld et al.
(2010) showed that it was possible to reduce the production of monomeric AX degradation products to a
minimum during xylanase treatment by implementing an additional step of heat treatment initially. This
could provide a future perspective to a more efficient production of prebiotics in the form of AXOS.
The health promoting effects of AX and AXOS might be added a further dimension if the enzyme modification of the cell wall matrix promote the liberation of additional compounds with beneficial bioactive effects.
This study showed that a liberation of ferulic acid occurred concomitant with a release of AXOS. A recent
study by Pekkinen et al. (2014), observed a synergistic effect of liberated ferulic acid when present in an enzyme modified aleurone preparation fed to obese mice. These effects were suggested to originate from the
liberation of additional phytochemicals with bioactive effects, and the induction of prebiotic effects due to an
increase of AXOS. Thus, it is likely that there exist beneficial effects of the enzyme modification of cell wall
polysaccharides beyond an improved fermentation profile. This could provide a further perspective for the
development of a further modified enzyme profile.
The incorporation of xylanase-modified AX and AXOS into palatable and high quality foods is an area that
deserves great attention. It has previously been shown that the presence of AXOS negatively affects the
bread making and general bread quality, due to loss of water holding capacity (Courtin and Delcour, 2002).
Recently in situ production of AXOS with thermophile xylanase in breads has been proposed as a method to
avoid some of the undesirable effects on dough properties (Damen et al., 2012b; Dornez et al., 2011). The
yield is however quite low (~2%). Additionally, AXOS has been proposed as a sucrose replacer in cookies.
Further options of enriching different foodstuff with AXOS are interesting in order to raise the AXOS concentration and should be studied.
77
The wheat grain relative and quantitative NSP composition, structural features, and physicochemical properties are greatly dependent on cultivar, and to a lesser extent environment (Saulnier et al., 2007b). There exists thus a potential for improving the AX quality by manipulating the biosynthesis, and thus influence these
parameters. This could potentially increase the general AX content in the grain as well as increase the fraction of AX susceptible to enzyme degradation. Recent studies have investigated key genes in the biosynthesis of AX in wheat endosperm using RNAi techniques (Lovegrove et al., 2013; Pellny et al., 2012). However, the manipulation of AX expression has mainly been focused on decreasing the relative viscosity of the
endosperm by decreasing AX concentration, as beneficial in alcohol production and feeding of monogastric
livestock (Freeman et al., 2016). It is possible that the knowledge gained in these studies regarding the AX
synthesis could be used to develop wheat cultivars with optimal AX properties with regard to human nutrition and health.
78
REFERENCES
1169/2011/EU, R. (2011). REGULATION (EU) No 1169/2011 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL. Vol. 2016, pp. ANNEX I, EUR - Lex Access to European Union law.
Adom, K. K., Sorrells, M. E., and Liu, R. H. (2005). Phytochemicals and antioxidant activity of milled fractions of
different wheat varieties. Journal of Agricultural and Food Chemistry 53, 2297-2306.
Almond, A., and Sheehan, J. K. (2003). Predicting the molecular shape of polysaccharides from dynamic interactions
with water. Glycobiology 13, 255-264.
Amrein, T. M., Gränicher, P., Arrigoni, E., and Amadò, R. (2003). In vitro digestibility and colonic fermentability of
aleurone isolated from wheat bran. LWT-Food Science and Technology 36, 451-460.
Anderson, J. W., Baird, P., Davis, R. H., Ferreri, S., Knudtson, M., Koraym, A., Waters, V., and Williams, C. L. (2009).
Health benefits of dietary fiber. Nutrition reviews 67, 188-205.
Anderson, J. W., Randles, K. M., Kendall, C. W., and Jenkins, D. J. (2004). Carbohydrate and fiber recommendations
for individuals with diabetes: a quantitative assessment and meta-analysis of the evidence. Journal of the
American College of Nutrition 23, 5-17.
Antoine, C., Peyron, S., Mabille, F., Lapierre, C., Bouchet, B., Abecassis, J., and Rouau, X. (2003). Individual
contribution of grain outer layers and their cell wall structure to the mechanical properties of wheat bran.
Journal of Agricultural and Food Chemistry 51, 2026-2033.
Awika, J. (2011). Major cereal grains production and use around the world. In "Advances in cereal science:
implications to food processing and health promotion", Vol. 1089, pp. 1-13. American Chemical Society
Atlantic City, NJ.
Bach Knudsen, K., Jensen, B. B., and Hansen, I. (1993). Oat bran but not a beta-glucan-enriched oat fraction enhances
butyrate production in the large intestine of pigs. The Journal of nutrition 123, 1235-1247.
Bach Knudsen, K. E. (1997). Carbohydrate and lignin contents of plant materials used in animal feeding. Animal Feed
Science and Technology 67, 319-338.
Bach Knudsen, K. E. (2014). Fiber and nonstarch polysaccharide content and variation in common crops used in broiler
diets. Poultry science, PS3902.
Bach Knudsen, K. E., Serena, A., Kjær, A. K. B., Jørgensen, H., and Engberg, R. (2005). Rye bread enhances the
production and plasma concentration of butyrate but not the plasma concentrations of glucose and insulin in
pigs. The Journal of nutrition 135, 1696-1704.
Bacic, A., Harris, P. J., and Stone, B. A. (1988). Structure and function of plant cell walls. The biochemistry of plants
14, 297-371.
Bacic, A., and Stone, B. (1981). Chemistry and organization of aleurone cell wall components from wheat and barley.
Functional Plant Biology 8, 475-495.
Barron, C., Surget, A., and Rouau, X. (2007). Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain
and their carbohydrate and phenolic acid composition. Journal of Cereal Science 45, 88-96.
Bass, E. J. (1988). "Wheat flour milling. In Wheat: Chemistry and Technology," American Association of Cereal
Chemists.
Beaugrand, J., Chambat, G., Wong, V. W., Goubet, F., Remond, C., Paes, G., Benamrouche, S., Debeire, P.,
O'Donohue, M., and Chabbert, B. (2004a). Impact and efficiency of GH10 and GH11 thermostable
endoxylanases on wheat bran and alkali-extractable arabinoxylans. Carbohydr Res 339, 2529-40.
Beaugrand, J., Crônier, D., Debeire, P., and Chabbert, B. (2004b). Arabinoxylan and hydroxycinnamate content of
wheat bran in relation to endoxylanase susceptibility. Journal of Cereal Science 40, 223-230.
Bechtel, D. B., Zayas, I., Kaleikau, L., and Pomeranz, Y. (1990). Size-distribution of wheat starch granules during
endosperm development. Cereal Chemistry 67, 59-63.
Beldman, G., Osuga, D., and Whitaker, J. (1996). Some characteristics of β-d-xylopyranosidases, α-larabinofuranosidases and an arabinoxylan α-l-arabinofuranohydrolase from wheat bran and germinated wheat.
Journal of Cereal Science 23, 169-180.
Belobrajdic, D. P., Bird, A. R., Conlon, M. A., Williams, B. A., Kang, S., McSweeney, C. S., Zhang, D., Bryden, W. L.,
Gidley, M. J., and Topping, D. L. (2012). An arabinoxylan-rich fraction from wheat enhances caecal
fermentation and protects colonocyte DNA against diet-induced damage in pigs. British Journal of Nutrition
107, 1274-1282.
Benamrouche, S., Crônier, D., Debeire, P., and Chabbert, B. (2002). A chemical and histological study on the effect of
(1→ 4)-β-endo-xylanase treatment on wheat bran. Journal of Cereal Science 36, 253-260.
Bennett, D., and Parry, D. W. (1981). Electron-probe Microanalysis Studies of Silicon in the Epicarp Hairs of the
Caryopses of Hordeum sativum Jess., Avena sativa L., Secale cereale L. and Triticum aestivum L. Annals of
Botany 48, 645-654.
79
Berrin, J.-G., Ajandouz, E. H., Georis, J., Arnaut, F., and Juge, N. (2007). Substrate and product hydrolysis specificity
in family 11 glycoside hydrolases: an analysis of Penicillium funiculosum and Penicillium griseofulvum
xylanases. Applied microbiology and biotechnology 74, 1001-1010.
Berrin, J.-G., and Juge, N. (2008). Factors affecting xylanase functionality in the degradation of arabinoxylans.
Biotechnology letters 30, 1139-1150.
Biely, P., Vršanská, M., Tenkanen, M., and Kluepfel, D. (1997). Endo-β-1, 4-xylanase families: differences in catalytic
properties. Journal of biotechnology 57, 151-166.
Bohm, A., Bogoni, C., Behrens, R., and Otto, T. (2011). Method for the extraction of aleurone from bran. Google
Patents.
Bohm, A., and Kratzer, A. (2010). Method for isolating aleurone particles. Google Patents.
Bonnin, E., Daviet, S., Sorensen, J. F., Sibbesen, O., Goldson, A., Juge, N., and Saulnier, L. (2006). Behaviour of
family 10 and 11 xylanases towards arabinoxylans with varying structure. Journal of the Science of Food and
Agriculture 86, 1618-1622.
Bonnin, E., Saulnier, L., Vigouroux, J., Dervilly, G., and Thibault, J. (2000). Potentialities of endogenous peroxidases
and feruloyl esterases in wheat for modifications of arabinoxylans. In "VTT SYMPOSIUM", Vol. 207, pp. 8190. VTT; 1999.
Boskov Hansen, H., Andreasen, M., Nielsen, M., Larsen, L., Knudsen, B. K., Meyer, A., Christensen, L., and Hansen,
Å. (2002). Changes in dietary fibre, phenolic acids and activity of endogenous enzymes during rye breadmaking. European Food Research and Technology 214, 33-42.
Brahe, L. K., Astrup, A., and Larsen, L. H. (2013). Is butyrate the link between diet, intestinal microbiota and obesity‐
related metabolic diseases? Obesity Reviews 14, 950-959.
Bray, G. A., Nielsen, S. J., and Popkin, B. M. (2004). Consumption of high-fructose corn syrup in beverages may play a
role in the epidemic of obesity. The American journal of clinical nutrition 79, 537-543.
Brighenti, F., Benini, L., Del Rio, D., Casiraghi, C., Pellegrini, N., Scazzina, F., Jenkins, D. J., and Vantini, I. (2006).
Colonic fermentation of indigestible carbohydrates contributes to the second-meal effect. The American
journal of clinical nutrition 83, 817-822.
Brillouet, J.-M., and Joseleau, J.-P. (1987). Investigation of the structure of a heteroxylan from the outer pericarp
(beeswing bran) of wheat kernel. Carbohydrate Research 159, 109-126.
Britton, K. A., and Fox, C. S. (2011). Ectopic fat depots and cardiovascular disease. Circulation 124, e837-e841.
Broekaert, W. F., Courtin, C. M., Verbeke, K., Van de Wiele, T., Verstraete, W., and Delcour, J. A. (2011). Prebiotic
and other health-related effects of cereal-derived arabinoxylans, arabinoxylan-oligosaccharides, and
xylooligosaccharides. Critical reviews in food science and nutrition 51, 178-194.
Brouns, F., Hemery, Y., Price, R., and Anson, N. M. (2012). Wheat aleurone: separation, composition, health aspects,
and potential food use. Critical reviews in food science and nutrition 52, 553-568.
Brown, L., Rosner, B., Willett, W. W., and Sacks, F. M. (1999). Cholesterol-lowering effects of dietary fiber: a metaanalysis. The American journal of clinical nutrition 69, 30-42.
Bruce, S. J., Guy, P. A., Rezzi, S., and Ross, A. B. (2010). Quantitative measurement of betaine and free choline in
plasma, cereals and cereal products by isotope dilution LC-MS/MS. Journal of agricultural and food chemistry
58, 2055-2061.
Burton, R. A., and Fincher, G. B. (2009). (1, 3; 1, 4)-β-d-glucans in cell walls of the Poaceae, lower plants, and fungi: a
tale of two linkages. Molecular Plant 2, 873-882.
Burton, R. A., and Fincher, G. B. (2014). Evolution and development of cell walls in cereal grains. Front Plant Sci 5,
456.
Burton, R. A., Gidley, M. J., and Fincher, G. B. (2010). Heterogeneity in the chemistry, structure and function of plant
cell walls. Nature chemical biology 6, 724-732.
Bühler, http://www.buhlergroup.com/global/en/home.htm.
Campbell, J. M., Fahey, G. C., and Wolf, B. W. (1997). Selected indigestible oligosaccharides affect large bowel mass,
cecal and fecal short-chain fatty acids, pH and microflora in rats. The Journal of Nutrition 127, 130-136.
Carneiro, M., Lordelo, M., Cunha, L., and Freire, J. (2008). Effects of dietary fibre source and enzyme supplementation
on faecal apparent digestibility, short chain fatty acid production and activity of bacterial enzymes in the gut of
piglets. Animal feed science and technology 146, 124-136.
Carpita, N. C., and Gibeaut, D. M. (1993). Structural models of primary cell walls in flowering plants: consistency of
molecular structure with the physical properties of the walls during growth. The Plant Journal 3, 1-30.
Chateigner-Boutin, A.-L., Bouchet, B., Alvarado, C., Bakan, B., and Guillon, F. (2014). The wheat grain contains pectic
domains exhibiting specific spatial and development-associated distribution. PloS one 9, e89620.
Cleemput, G., Bleukx, W., Van Oort, M., Hessing, M., and Delcour, J. (1995). Evidence for the presence of
arabinoxylan hydrolysing enzymes in European wheat flours. Journal of Cereal Science 22, 139-145.
Cloetens, L., Broekaert, W. F., Delaedt, Y., Ollevier, F., Courtin, C. M., Delcour, J. A., Rutgeerts, P., and Verbeke, K.
(2010). Tolerance of arabinoxylan-oligosaccharides and their prebiotic activity in healthy subjects: a
randomised, placebo-controlled cross-over study. British Journal of Nutrition 103, 703-713.
80
Cloetens, L., De Preter, V., Swennen, K., Broekaert, W. F., Courtin, C. M., Delcour, J. A., Rutgeerts, P., and Verbeke,
K. (2008). Dose-response effect of arabinoxylooligosaccharides on gastrointestinal motility and on colonic
bacterial metabolism in healthy volunteers. Journal of the American College of Nutrition 27, 512-518.
Comino, P., Shelat, K., Collins, H., Lahnstein, J., and Gidley, M. J. (2013). Separation and Purification of Soluble
Polymers and Cell Wall Fractions from Wheat, Rye and Hull less Barley Endosperm Flours for StructureNutrition Studies. Journal of Agricultural and Food Chemistry 61, 12111-12122.
Cornier, M.-A., Dabelea, D., Hernandez, T. L., Lindstrom, R. C., Steig, A. J., Stob, N. R., Van Pelt, R. E., Wang, H.,
and Eckel, R. H. (2008). The metabolic syndrome. Endocrine reviews 29, 777-822.
Coulomb, M., Gombert, A., and Moazzami, A. (2015). Metabolomics study of cereal grains reveals the discriminative
metabolic markers associated with anatomical compartments. Italian Journal of Food Science 27, 142.
Courtin, C., and Delcour, J. A. (2002). Arabinoxylans and endoxylanases in wheat flour bread-making. Journal of
cereal science 35, 225-243.
Courtin, C., Van den Broeck, H., and Delcour, J. (2000). Determination of reducing end sugar residues in oligo-and
polysaccharides by gas–liquid chromatography. Journal of Chromatography A 866, 97-104.
Courtin, C. M., Broekaert, W. F., Swennen, K., Lescroart, O., Onagbesan, O., Buyse, J., Decuypere, E., Van de Wiele,
T., Marzorati, M., and Verstraete, W. (2008). Dietary inclusion of wheat bran arabinoxylooligosaccharides
induces beneficial nutritional effects in chickens. Cereal chemistry 85, 607-613.
Craig, S. A. (2004). Betaine in human nutrition. The American journal of clinical nutrition 80, 539-549.
Croes, E., Gebruers, K., Luyten, N., Delcour, J. A., and Courtin, C. M. (2009). Immunoblot quantification of three
classes of proteinaceous xylanase inhibitors in different wheat (Triticum aestivum) cultivars and milling
fractions. Journal of agricultural and food chemistry 57, 1029-1035.
Cui, W., Wood, P., Blackwell, B., and Nikiforuk, J. (2000). Physicochemical properties and structural characterization
by two-dimensional NMR spectroscopy of wheat β-D-glucan—comparison with other cereal β-D-glucans.
Carbohydrate Polymers 41, 249-258.
Cummings, J. H., and Englyst, H. N. (1995). Gastrointestinal effects of food carbohydrate. The American journal of
clinical nutrition 61, 938S-945S.
Damen, B., Cloetens, L., Broekaert, W. F., François, I., Lescroart, O., Trogh, I., Arnaut, F., Welling, G. W., Wijffels, J.,
and Delcour, J. A. (2012a). Consumption of breads containing in situ–produced arabinoxylan oligosaccharides
alters gastrointestinal effects in healthy volunteers. The Journal of nutrition 142, 470-477.
Damen, B., Pollet, A., Dornez, E., Broekaert, W. F., Haesendonck, I. V., Trogh, I., Arnaut, F., Delcour, J. A., and
Courtin, C. M. (2012b). Xylanase-mediated in situ production of arabinoxylan oligosaccharides with prebiotic
potential in whole meal breads and breads enriched with arabinoxylan rich materials. Food Chemistry 131,
111-118.
Damen, B., Verspreet, J., Pollet, A., Broekaert, W. F., Delcour, J. A., and Courtin, C. M. (2011). Prebiotic effects and
intestinal fermentation of cereal arabinoxylans and arabinoxylan oligosaccharides in rats depend strongly on
their structural properties and joint presence. Molecular nutrition & food research 55, 1862-1874.
Delcour, J. A., Van Win, H., and Grobet, P. J. (1999). Distribution and structural variation of arabinoxylans in common
wheat mill streams. Journal of Agricultural and Food Chemistry 47, 271-275.
Delzenne, N. M., and Roberfroid, M. (1994). Physiological effects of non-digestible oligosaccharides. LWT-Food
Science and Technology 27, 1-6.
Dervilly-Pinel, G., Rimsten, L., Saulnier, L., Andersson, R., and Åman, P. (2001). Water-extractable Arabinoxylan
from Pearled Flours of Wheat, Barley, Rye and Triticale. Evidence for the Presence of Ferulic Acid Dimers
and their Involvement in Gel Formation. Journal of Cereal Science 34, 207-214.
Dervilly-Pinel, G., Tran, V., and Saulnier, L. (2004). Investigation of the distribution of arabinose residues on the xylan
backbone of water-soluble arabinoxylans from wheat flour. Carbohydrate Polymers 55, 171-177.
Després, J.-P., Lemieux, I., Bergeron, J., Pibarot, P., Mathieu, P., Larose, E., Rodés-Cabau, J., Bertrand, O. F., and
Poirier, P. (2008). Abdominal obesity and the metabolic syndrome: contribution to global cardiometabolic risk.
Arteriosclerosis, thrombosis, and vascular biology 28, 1039-1049.
Dornez, E., Verjans, P., Broekaert, W. F., Cappuyns, A. M., Van Impe, J. F., Arnaut, F., Delcour, J. A., and Courtin, C.
M. (2011). In situ production of prebiotic AXOS by hyperthermophilic Xylanase B from Thermotoga maritima
in high-quality bread. Cereal Chemistry 88, 124-129.
Duncan, S. H., Holtrop, G., Lobley, G. E., Calder, A. G., Stewart, C. S., and Flint, H. J. (2004). Contribution of acetate
to butyrate formation by human faecal bacteria. British Journal of Nutrition 91, 915-923.
DuPont, M. S., and Selvendran, R. R. (1987). Hemicellulosic polymers from the cell walls of beeswing wheat bran: part
I, polymers solubilised by alkali at 2. Carbohydrate research 163, 99-113.
Ellis, P., Roberts, F., Low, A., and Morgan, L. (1995). The effect of high-molecular-weight guar gum on net apparent
glucose absorption and net apparent insulin and gastric inhibitory polypeptide production in the growing pig:
relationship to rheological changes in jejunal digesta. British Journal of Nutrition 74, 539-556.
Eugster, W., and Gerschwiler, O. (2003). Method and installation for cleaning cereal. Google Patents.
81
Evers, A., O'Brien, L., and Blakeney, A. (1999). Cereal structure and composition. Crop and Pasture Science 50, 629650.
Evers, T., and Millar, S. (2002). Cereal Grain Structure and Development: Some Implications for Quality. Journal of
Cereal Science 36, 261-284.
Ferchaud-Roucher, V., Pouteau, E., Piloquet, H., Zair, Y., and Krempf, M. (2005). Colonic fermentation from lactulose
inhibits lipolysis in overweight subjects. American Journal of Physiology-Endocrinology and Metabolism 289,
E716-E720.
François, I. E., Lescroart, O., Veraverbeke, W. S., Marzorati, M., Possemiers, S., Evenepoel, P., Hamer, H., Houben, E.,
Windey, K., and Welling, G. W. (2012). Effects of a wheat bran extract containing arabinoxylan
oligosaccharides on gastrointestinal health parameters in healthy adult human volunteers: a double-blind,
randomised, placebo-controlled, cross-over trial. British Journal of Nutrition 108, 2229-2242.
François, I. E., Lescroart, O., Veraverbeke, W. S., Marzorati, M., Possemiers, S., Hamer, H., Windey, K., Welling, G.
W., Delcour, J. A., and Courtin, C. M. (2014). Effects of wheat bran extract containing arabinoxylan
oligosaccharides on gastrointestinal parameters in healthy preadolescent children. Journal of pediatric
gastroenterology and nutrition 58, 647-653.
Freeman, J., Lovegrove, A., Wilkinson, M. D., Saulnier, L., Shewry, P. R., and Mitchell, R. A. C. (2016). Effect of
suppression of arabinoxylan synthetic genes in wheat endosperm on chain length of arabinoxylan and extract
viscosity. Plant biotechnology journal 14, 109-116.
Fry, S. C., Willis, S. C., and Paterson, A. E. (2000). Intraprotoplasmic and wall-localised formation of arabinoxylanbound diferulates and larger ferulate coupling-products in maize cell-suspension cultures. Planta 211, 679692.
Gebruers, K., Dornez, E., Boros, D., Dynkowska, W., Bedő, Z., Rakszegi, M., Delcour, J. A., and Courtin, C. M.
(2008). Variation in the content of dietary fiber and components thereof in wheats in the HEALTHGRAIN
diversity screen. Journal of Agricultural and Food Chemistry 56, 9740-9749.
Geraylou, Z., Rurangwa, E., Wiele, T. v. d., Courtin, C. M., Delcour, J. A., Buyse, J., and Ollevier, F. (2014).
Fermentation of Arabinoxylan-Oligosaccharides, Oligofructose and their monomeric sugars by hindgut
bacteria from Siberian sturgeon and African catfish in batch culture in vitro. Journal of Aquaculture Research
and Development 5, 230.
Gibson, G. R., Probert, H. M., Van Loo, J., Rastall, R. A., and Roberfroid, M. B. (2004). Dietary modulation of the
human colonic microbiota: updating the concept of prebiotics. Nutrition research reviews 17, 259-275.
Gille, S., de Souza, A., Xiong, G., Benz, M., Cheng, K., Schultink, A., Reca, I. B., and Pauly, M. (2011). O-acetylation
of Arabidopsis hemicellulose xyloglucan requires AXY4 or AXY4L, proteins with a TBL and DUF231
domain. Plant Cell 23, 4041-53.
Gillissen, B., Bürkle, L., André, B., Kühn, C., Rentsch, D., Brandl, B., and Frommer, W. B. (2000). A new family of
high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis. The Plant Cell 12, 291300.
Glitsø, L., Brunsgaard, G., Højsgaard, S., Sandström, B., and Knudsen, K. B. (1998). Intestinal degradation in pigs of
rye dietary fibre with different structural characteristics. British journal of nutrition 80, 457-468.
Glitsø, L. V., Gruppen, H., Schols, H. A., Højsgaard, S., Sandström, B., and Bach Knudsen, K. E. (1999). Degradation
of rye arabinoxylans in the large intestine of pigs. Journal of the Science of Food and Agriculture 79, 961-969.
Grootaert, C., Van den Abbeele, P., Marzorati, M., Broekaert, W. F., Courtin, C. M., Delcour, J. A., Verstraete, W., and
Van de Wiele, T. (2009). Comparison of prebiotic effects of arabinoxylan oligosaccharides and inulin in a
simulator of the human intestinal microbial ecosystem. FEMS microbiology ecology 69, 231-242.
Gruppen, H., Hamer, R., and Voragen, A. (1992). Water-unextractable cell wall material from wheat flour. 2.
Fractionation of alkali-extracted polymers and comparison with water-extractable arabinoxylans. Journal of
Cereal Science 16, 53-67.
Gruppen, H., Hamer, R. J., and Voragen, A. G. J. (1991). Barium hydroxide as a tool to extract pure arabinoxylans from
water-insoluble cell wall material of wheat flour. Journal of Cereal Science 13, 275-290.
HEALTHGRAIN, https://www.healthgrain.org.
Hegarty, T. (1978). The physiology of seed hydration and dehydration, and the relation between water stress and the
control of germination: a review. Plant, Cell & Environment 1, 101-119.
Heilbronn, L., Smith, S., and Ravussin, E. (2004). Failure of fat cell proliferation, mitochondrial function and fat
oxidation results in ectopic fat storage, insulin resistance and type II diabetes mellitus. International journal of
obesity 28, S12-S21.
Heimler, D., Vignolini, P., Isolani, L., Arfaioli, P., Ghiselli, L., and Romani, A. (2010). Polyphenol content of modern
and old varieties of Triticum aestivum L. and T. durum Desf. grains in two years of production. Journal of
agricultural and food chemistry 58, 7329-7334.
Hemery, Y., Lullien-Pellerin, V., Rouau, X., Abecassis, J., Samson, M.-F., Åman, P., von Reding, W., Spoerndli, C.,
and Barron, C. (2009a). Biochemical markers: efficient tools for the assessment of wheat grain tissue
proportions in milling fractions. Journal of Cereal Science 49, 55-64.
82
Hemery, Y., Rouau, X., Dragan, C., Bilici, M., Beleca, R., and Dascalescu, L. (2009b). Electrostatic properties of wheat
bran and its constitutive layers: Influence of particle size, composition, and moisture content. Journal of Food
Engineering 93, 114-124.
Hielscher, U. T. (2016). Ultrasonic Extraction and Preservation. Vol. 2016. Hielscher - Ultrasound Technology,
http://www.hielscher.com.
Hooda, S., Matte, J. J., Vasanthan, T., and Zijlstra, R. T. (2010). Dietary oat β-glucan reduces peak net glucose flux and
insulin production and modulates plasma incretin in portal-vein catheterized grower pigs. The Journal of
nutrition 140, 1564-1569.
Huang, T., Xu, M., Lee, A., Cho, S., and Qi, L. (2015). Consumption of whole grains and cereal fiber and total and
cause-specific mortality: prospective analysis of 367,442 individuals. BMC medicine 13, 1.
Iiyama, K., Lam, T., and Stone, B. A. (1994). Covalent Cross-Links in the Cell Wall. Plant Physiol 104, 315-320.
Ingerslev, A. K., Theil, P. K., Hedemann, M. S., Larke, H. N., and Bach Knudsen, K. E. (2014). Resistant starch and
arabinoxylan augment SCFA absorption, but affect postprandial glucose and insulin responses differently. The
British journal of nutrition 111, 1564-76.
Izydorczyk, M. S., and Biliaderis, C. G. (1993). Structural heterogeneity of wheat endosperm arabinoxylans. Cereal
chemistry (USA).
Izydorczyk, M. S., and Biliaderis, C. G. (1995). Cereal arabinoxylans: advances in structure and physicochemical
properties. Carbohydrate Polymers 28, 33-48.
Izydorczyk, M. S., and MacGregor, A. W. (2000). Evidence of intermolecular interactions of β-glucans and
arabinoxylans. Carbohydrate Polymers 41, 417-420.
Johansson Boll, E., Ekström, L. M., Courtin, C. M., Delcour, J. A., Nilsson, A. C., Björck, I. M., and Östman, E. M.
(2015). Effects of wheat bran extract rich in arabinoxylan oligosaccharides and resistant starch on overnight
glucose tolerance and markers of gut fermentation in healthy young adults. European journal of nutrition, 110.
Jones, B. L., and Lookhart, G. L. (2005). Comparison of Endoproteinases of Various Grains. Cereal Chemistry Journal
82, 125-130.
Juge, N., and Delcour, J. A. (2006). Proteinaceous Xylanase Inhibitors: Structure, Function and Evolution. Current
Enzyme Inhibition 2, 29-35.
Jänis, J., Hakanpää, J., Hakulinen, N., Ibatullin, F. M., Hoxha, A., Derrick, P. J., Rouvinen, J., and Vainiotalo, P.
(2005). Determination of thioxylo‐oligosaccharide binding to family 11 xylanases using electrospray
ionization Fourier transform ion cyclotron resonance mass spectrometry and X‐ray crystallography. FEBS
Journal 272, 2317-2333.
Kabel, M. A., Kortenoeven, L., Schols, H. A., and Voragen, A. G. (2002). In vitro fermentability of differently
substituted xylo-oligosaccharides. Journal of agricultural and food chemistry 50, 6205-6210.
Kenji, I., Thi, B. T. L., Natsuki, K., and Stone, B. A. (1994). The International Journal of Plant BiochemistryRapid and
simple determination of O-acetyl groups bound to plant cell walls by acid hydrolysis and 1H NMR
measurement. Phytochemistry 35, 959-961.
Kormelink, F. J., Gruppen, H., Viëtor, R. J., and Voragen, A. G. (1993a). Mode of action of the xylan-degrading
enzymes from Aspergillus awamori on alkali-extractable cereal arabinoxylans. Carbohydrate research 249,
355-367.
Kormelink, F. J., Hoffmann, R. A., Gruppen, H., Voragen, A. G., Kamerling, J. P., and Vliegenthart, J. F. (1993b).
Characterisation by 1 H NMR spectroscopy of oligosaccharides derived from alkali-extractable wheat-flour
arabinoxylan by digestion with endo-(1→ 4)-β-d-xylanase III from Aspergillus awamori. Carbohydrate
research 249, 369-382.
Kristensen, N. B., and Wu, G. (2012). Metabolic functions of the porcine liver. In "Nutritional Physiology of Pigs".
Köhnke, T., Östlund, Å., and Brelid, H. (2011). Adsorption of arabinoxylan on cellulosic surfaces: influence of degree
of substitution and substitution pattern on adsorption characteristics. Biomacromolecules 12, 2633-2641.
Lazaridou, A., and Biliaderis, C. (2007). Molecular aspects of cereal β-glucan functionality: Physical properties,
technological applications and physiological effects. Journal of Cereal Science 46, 101-118.
Le Gall, M., Serena, A., Jørgensen, H., Theil, P. K., and Knudsen, K. E. B. (2009). The role of whole-wheat grain and
wheat and rye ingredients on the digestion and fermentation processes in the gut–a model experiment with
pigs. British journal of nutrition 102, 1590-1600.
Lempereur, I., Surget, A., and Rouau, X. (1998). Variability in dehydrodiferulic acid composition of durum wheat
(Triticum durum Desf.) and distribution in milling fractions. Journal of Cereal Science 28, 251-258.
Lequart, C., Nuzillard, J.-M., Kurek, B., and Debeire, P. (1999). Hydrolysis of wheat bran and straw by an
endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides. Carbohydrate
Research 319, 102-111.
Li, L., Shewry, P. R., and Ward, J. L. (2008). Phenolic acids in wheat varieties in the HEALTHGRAIN diversity
screen. Journal of Agricultural and Food Chemistry 56, 9732-9739.
83
Lin, H. V., Frassetto, A., Kowalik Jr, E. J., Nawrocki, A. R., Lu, M. M., Kosinski, J. R., Hubert, J. A., Szeto, D., Yao,
X., and Forrest, G. (2012). Butyrate and propionate protect against diet-induced obesity and regulate gut
hormones via free fatty acid receptor 3-independent mechanisms. PLoS One 7, e35240.
Lovegrove, A., Wilkinson, M. D., Freeman, J., Pellny, T. K., Tosi, P., Saulnier, L., Shewry, P. R., and Mitchell, R. A.
(2013). RNA interference suppression of genes in glycosyl transferase families 43 and 47 in wheat starchy
endosperm causes large decreases in arabinoxylan content. Plant physiology 163, 95-107.
Lærke, H. N., Arent, S., Dalsgaard, S., and Bach Knudsen, K. (2015). Effect of xylanases on ileal viscosity, intestinal
fiber modification, and apparent ileal fiber and nutrient digestibility of rye and wheat in growing pigs. Journal
of animal science 93, 4323-4335.
Maes, C., and Delcour, J. (2002). Structural characterisation of water-extractable and water-unextractable arabinoxylans
in wheat bran. Journal of Cereal Science 35, 315-326.
Maki, K. C., Gibson, G. R., Dickmann, R. S., Kendall, C. W., Chen, C.-Y. O., Costabile, A., Comelli, E. M., McKay,
D. L., Almeida, N. G., and Jenkins, D. (2012). Digestive and physiologic effects of a wheat bran extract,
arabino-xylan-oligosaccharide, in breakfast cereal. Nutrition 28, 1115-1121.
Mandalari, G., Faulds, C. B., Sancho, A. I., Saija, A., Bisignano, G., LoCurto, R., and Waldron, K. W. (2005).
Fractionation and characterisation of arabinoxylans from brewers' spent grain and wheat bran. Journal of
Cereal Science 42, 205-212.
Mares, D., and Stone, B. (1973a). Studies on wheat endosperm I. Chemical composition and ultrastructure of the cell
walls. Australian Journal of Biological Sciences 26, 793-812.
Mares, D., and Stone, B. (1973b). Studies on wheat endosperm II. Properties of the wall components and studies on
their organization in the wall. Australian Journal of Biological Sciences 26, 813-830.
Marlett, J. A., Kajs, T. M., and Fischer, M. H. (2000). An unfermented gel component of psyllium seed husk promotes
laxation as a lubricant in humans. The American journal of clinical nutrition 72, 784-789.
McCann, M. C., and Roberts, K. (1991). "The cytoskeletal basis of plant growth and form," London, etc.: Academic
Press.
McCleary, B., and Glennie-Holmes, M. (1985). Enzymic quantification of (I1-3),(1.4)-β-D-glucan in barley and malt. J.
Inst. Brew 91, 285-295.
McDougall, G. J., Morrison, I. M., Stewart, D., and Hillman, J. R. (1996). Plant cell walls as dietary fibre: range,
structure, processing and function. Journal of the Science of Food and Agriculture 70, 133-150.
Mendis, M., Ohm, J. B., Delcour, J. A., Gebruers, K., Meinhardt, S., and Simsek, S. (2013). Variability in
Arabinoxylan, Xylanase Activity, and Xylanase Inhibitor Levels in Hard Spring Wheat. Cereal Chemistry 90,
240-248.
Montonen, J., Knekt, P., Järvinen, R., Aromaa, A., and Reunanen, A. (2003). Whole-grain and fiber intake and the
incidence of type 2 diabetes. The American journal of clinical nutrition 77, 622-629.
Naczk, M., and Shahidi, F. (2004). Extraction and analysis of phenolics in food. Journal of Chromatography A 1054,
95-111.
Ng, A., Greenshields, R. N., and Waldron, K. W. (1997). Oxidative cross-linking of corn bran hemicellulose: formation
of ferulic acid dehydrodimers. Carbohydrate Research 303, 459-462.
Nielsen, T. S., Lærke, H. N., Theil, P. K., Sørensen, J. F., Saarinen, M., Forssten, S., and Knudsen, K. E. B. (2014).
Diets high in resistant starch and arabinoxylan modulate digestion processes and SCFA pool size in the large
intestine and faecal microbial composition in pigs. British Journal of Nutrition 112, 1837-1849.
Northcote, D. (1963). Changes in the cell walls of plants during differentiation. In "Symp. soc. exp. biol", pp. 157-74.
Northcote, D. (1972). Chemistry of the plant cell wall. Annual Review of Plant Physiology 23, 113-132.
O'Neill, S., and O'Driscoll, L. (2015). Metabolic syndrome: a closer look at the growing epidemic and its associated
pathologies. obesity reviews 16, 1-12.
Okazaki, M., Fujikawa, S., and Matsumoto, N. (1990). Effect of xylooligosaccharide on the growth of bifidobacteria.
Bifidobacteria and Microflora 9, 77-86.
Ordaz-Ortiz, J. J., Guillon, F., Tranquet, O., Dervilly-Pinel, G., Tran, V., and Saulnier, L. (2004). Specificity of
monoclonal antibodies generated against arabinoxylans of cereal grains. Carbohydrate Polymers 57, 425-433.
Ordaz-Ortiz, J. J., and Saulnier, L. (2005). Structural variability of arabinoxylans from wheat flour. Comparison of
water-extractable and xylanase-extractable arabinoxylans. Journal of Cereal Science 42, 119-125.
Othman, R. A., Moghadasian, M. H., and Jones, P. J. (2011). Cholesterol-lowering effects of oat β-glucan. Nutrition
reviews 69, 299-309.
Palframan, R. J., Gibson, G. R., and Rastall, R. A. (2003). Carbohydrate preferences of Bifidobacterium species isolated
from the human gut. Curr Issues Intest Microbiol 4, 71-5.
Papageorgiou, M., Lakhdara, N., Lazaridou, A., Biliaderis, C., and Izydorczyk, M. (2005). Water extractable (1→ 3,
1→ 4)-β-D-glucans from barley and oats: An intervarietal study on their structural features and rheological
behaviour. Journal of cereal science 42, 213-224.
84
Parker, M. L., Ng, A., and Waldron, K. W. (2005). The phenolic acid and polysaccharide composition of cell walls of
bran layers of mature wheat (Triticum aestivum L. cv. Avalon) grains. Journal of the Science of Food and
Agriculture 85, 2539-2547.
Parker, M. L., and Waldron, K. W. (1995). Texture of Chinese water chestnut: involvement of cell wall phenolics.
Journal of the Science of Food and Agriculture 68, 337-346.
Pekkinen, J., Rosa, N. N., Savolainen, O.-I., Keski-Rahkonen, P., Mykkänen, H., Poutanen, K., Micard, V., and
Hanhineva, K. (2014). Disintegration of wheat aleurone structure has an impact on the bioavailability of
phenolic compounds and other phytochemicals as evidenced by altered urinary metabolite profile of dietinduced obese mice. Nutrition & Metabolism 11, 1-15.
Pellny, T. K., Lovegrove, A., Freeman, J., Tosi, P., Love, C. G., Knox, J. P., Shewry, P. R., and Mitchell, R. A. C.
(2012). Cell Walls of Developing Wheat Starchy Endosperm: Comparison of Composition and RNA-Seq
Transcriptome. Plant Physiology 158, 612-627.
Pomare, E., Branch, W., and Cummings, J. (1985). Carbohydrate fermentation in the human colon and its relation to
acetate concentrations in venous blood. Journal of Clinical Investigation 75, 1448.
Poutanen, K. (1997). Enzymes: An important tool in the improvement of the quality of cereal foods. Trends in Food
Science & Technology 8, 300-306.
Priebe, M. G., Wang, H., Weening, D., Schepers, M., Preston, T., and Vonk, R. J. (2010). Factors related to colonic
fermentation of nondigestible carbohydrates of a previous evening meal increase tissue glucose uptake and
moderate glucose-associated inflammation. The American journal of clinical nutrition 91, 90-97.
Paananen, A., Österberg, B., Rutland, M., Tammelin, T., Saarinen, T., Tappura, K., and Stenius, P. (2004). Interaction
between cellulose and xylan: An atomic force microscope and quartz crystal microbalance study. In "1st
International Symposium on Xylans, Mannans and Other Hemicelluloses Location: ORLANDO, FLORIDA
Date: APR, 2001", Vol. 864, pp. 269-290.
Ralph, J., Quideau, S., Grabber, J. H., and Hatfield, R. D. (1994). Identification and synthesis of new ferulic acid
dehydrodimers present in grass cell walls. Journal of the Chemical Society, Perkin Transactions 1, 3485-3498.
Regmi, P. R., van Kempen, T. A., Matte, J. J., and Zijlstra, R. T. (2011). Starch with high amylose and low in vitro
digestibility increases short-chain fatty acid absorption, reduces peak insulin secretion, and modulates incretin
secretion in pigs. The Journal of nutrition 141, 398-405.
Rhodes, D., Sadek, M., and Stone, B. (2002). Hydroxycinnamic acids in walls of wheat aleurone cells. Journal of
Cereal Science 36, 67-81.
Rhodes, D. I., and Stone, B. A. (2002). Proteins in walls of wheat aleurone cells. Journal of Cereal Science 36, 83-101.
Ring, S. G., and Selvendran, R. R. (1980). Isolation and analysis of cell wall material from beeswing wheat bran
(Triticum aestivum). Phytochemistry 19, 1723-1730.
Robertson, M., Currie, J., Morgan, L., Jewell, D., and Frayn, K. (2003). Prior short-term consumption of resistant starch
enhances postprandial insulin sensitivity in healthy subjects. Diabetologia 46, 659-665.
Robertson, M. D., Bickerton, A. S., Dennis, A. L., Vidal, H., and Frayn, K. N. (2005). Insulin-sensitizing effects of
dietary resistant starch and effects on skeletal muscle and adipose tissue metabolism. The American journal of
clinical nutrition 82, 559-567.
Roediger, W. (1982). Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 83, 424-429.
Rosenfelder, P., Eklund, M., and Mosenthin, R. (2013). Nutritive value of wheat and wheat by-products in pig nutrition:
A review. Animal Feed Science and Technology 185, 107-125.
Rost, T. L., and Lersten, N. R. (1970). Transfer aleurone cells inSetaria lutescens (Gramineae). Protoplasma 71, 403408.
Rouau, X., Cheynier, V., Surget, A., Gloux, D., Barron, C., Meudec, E., Louis-Montero, J., and Criton, M. (2003). A
dehydrotrimer of ferulic acid from maize bran. Phytochemistry 63, 899-903.
Sanchez, J., Marzorati, M., Grootaert, C., Baran, M., Van Craeyveld, V., Courtin, C., Broekaert, W., Delcour, J.,
Verstraete, W., and Van de Wiele, T. (2009). Arabinoxylan‐oligosaccharides (AXOS) affect the
protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human
Intestinal Microbial Ecosystem. Microbial biotechnology 2, 101-113.
Saulnier, L., Guillon, F., and Chateigner-Boutin, A.-L. (2012). Cell wall deposition and metabolism in wheat grain.
Journal of Cereal Science 56, 91-108.
Saulnier, L., Guillon, F., Sado, P.-E., Chateigner-Boutin, A. L., and Rouau, X. (2007a). Plant cell wall polysaccharides
in storage organs: xylans (food applications). Reference Module in Chemistry, Molecular Sciences and
Chemical Engineering, np.
Saulnier, L., Peneau, N., and Thibault, J. F. (1995). Variability in grain extract viscosity and water-soluble arabinoxylan
content in wheat. Journal of Cereal Science 22, 259-264.
Saulnier, L., Robert, P., Grintchenko, M., Jamme, F., Bouchet, B., and Guillon, F. (2009). Wheat endosperm cell walls:
spatial heterogeneity of polysaccharide structure and composition using micro-scale enzymatic fingerprinting
and FT-IR microspectroscopy. Journal of Cereal Science 50, 312-317.
85
Saulnier, L., Sado, P.-E., Branlard, G., Charmet, G., and Guillon, F. (2007b). Wheat arabinoxylans: Exploiting variation
in amount and composition to develop enhanced varieties. Journal of Cereal Science 46, 261-281.
Scheppach, W., Bartram, H., and Richter, F. (1995). Role of short-chain fatty acids in the prevention of colorectal
cancer. European Journal of Cancer 31, 1077-1080.
Schmitz, J. F., McDonald, C. E., and Gilles, K. A. (1974). Arabinoxylanases and cellulases of wheat. Cereal Chemistry
51, 809-821.
Schooneveld-Bergmans, M. E. F., Beldman, G., and Voragen, A. G. J. (1999a). Structural Features of
(Glucurono)Arabinoxylans Extracted from Wheat Bran by Barium Hydroxide. Journal of Cereal Science 29,
63-75.
Schooneveld-Bergmans, M. E. F., Dignum, M. J. W., Grabber, J. H., Beldman, G., and Voragen, A. G. J. (1999b).
Studies on the oxidative cross-linking of feruloylated arabinoxylans from wheat flour and wheat bran.
Carbohydrate Polymers 38, 309-317.
Schutte, J., Van Leeuwen, P., and Lichtendonk, W. (1991). Ileal digestibility and urinary excretion of D-xylose and Larabinose in ileostomized adult roosters. Poultry science 70, 884-891.
Scott, R. W. (1979). Colorimetric determination of hexuronic acids in plant materials. Analytical chemistry 51, 936-941.
Selig, M. J., Thygesen, L. G., Felby, C., and Master, E. R. (2015). Debranching of soluble wheat arabinoxylan
dramatically enhances recalcitrant binding to cellulose. Biotechnology letters 37, 633-641.
Selvendran, R. R. (1984). The plant cell wall as a source of dietary fiber: chemistry and structure. American Journal of
Clinical Nutrition (USA).
Slavin, J. (2013). Fiber and prebiotics: mechanisms and health benefits. Nutrients 5, 1417-1435.
Stanhope, K. L. (2012). Role of fructose-containing sugars in the epidemics of obesity and metabolic syndrome. Annual
review of medicine 63, 329-343.
Staudte, R., Woodward, J., Fincher, G., and Stone, B. (1983). Water-soluble (1→ 3),(1→ 4)-β-D-glucans from barley
(Hordeum vulgare) endosperm. III. Distribution of cellotriosyl and cellotetraosyl residues. Carbohydrate
Polymers 3, 299-312.
Stevens, J., VanSoest, P., Robertson, J., and Levitsky, D. (1988). Comparison of the effects of psyllium and wheat bran
on gastrointestinal transit time and stool characteristics. Journal of the American Dietetic Association 88, 323326.
Stockvault (2016). http://www.freepik.com.
Stone, B. (2006). Cell walls of cereal grains. Cereal foods world 51, 62-65.
Surget, A., and Barron, C. (2005). Histologie du grain de blé (histology of the wheat grain). Ind. Cér. 145, 3-7.
Swennen, K., Courtin, C. M., and Delcour, J. A. (2006a). Non-digestible oligosaccharides with prebiotic properties.
Critical reviews in food science and nutrition 46, 459-471.
Swennen, K., Courtin, C. M., Lindemans, G. C. J. E., and Delcour, J. A. (2006b). Large-scale production and
characterisation of wheat bran arabinoxylooligosaccharides. Journal of the Science of Food and Agriculture
86, 1722-1731.
Taiz, L., and Zeiger, E. (2010). Cell Walls: Structure, Biogenesis, and Expansion. In "Plant Physiology", Vol. 5.
Sinauer.
Teitelbaum, J. E., and Walker, W. A. (2002). Nutritional Impact of Pre-and Probiotics as Protective Gastrointestinal
Organisms*. Annual Review of Nutrition 22, 107-138.
Thompson, R. D., Hueros, G., Becker, H.-A., and Maitz, M. (2001). Development and functions of seed transfer cells.
Plant Science 160, 775-783.
Timmel, T. E. (1964). Wood hemicelluloses: Part 1. In "Advances in carbohydrate chemistry", Vol. 19, pp. 247-302.
Toole, G. A., Le Gall, G., Colquhoun, I. J., Johnson, P., Bedö, Z., Saulnier, L., Shewry, P. R., and Mills, E. C. (2011).
Spectroscopic analysis of diversity of arabinoxylan structures in endosperm cell walls of wheat cultivars
(Triticum aestivum) in the HEALTHGRAIN diversity collection. Journal of agricultural and food chemistry
59, 7075-7082.
Topping, D. L., and Clifton, P. M. (2001). Short-chain fatty acids and human colonic function: roles of resistant starch
and nonstarch polysaccharides. Physiological reviews 81, 1031-1064.
Tungland, B., and Meyer, D. (2002). Nondigestible oligo‐and polysaccharides (Dietary Fiber): their physiology and
role in human health and food. Comprehensive Reviews in food science and food safety 1, 90-109.
Tzin, V., and Galili, G. (2010). The biosynthetic pathways for shikimate and aromatic amino acids in Arabidopsis
thaliana. The Arabidopsis Book, e0132.
Van Craeyveld, V., Dornez, E., Holopainen, U., Selinheimo, E., Poutanen, K., Delcour, J. A., and Courtin, C. M.
(2010). Wheat bran AX properties and choice of xylanase affect enzymic production of wheat bran-derived
arabinoxylan-oligosaccharides. Cereal chemistry 87, 283-291.
Van Craeyveld, V., Swennen, K., Dornez, E., Van de Wiele, T., Marzorati, M., Verstraete, W., Delaedt, Y., Onagbesan,
O., Decuypere, E., and Buyse, J. (2008). Structurally different wheat-derived arabinoxylooligosaccharides
have different prebiotic and fermentation properties in rats. The Journal of nutrition 138, 2348-2355.
86
Vardakou, M., Palop, C. N., Christakopoulos, P., Faulds, C. B., Gasson, M. A., and Narbad, A. (2008). Evaluation of
the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora.
International journal of food microbiology 123, 166-170.
Vardakou, M., Palop, C. N., Gasson, M., Narbad, A., and Christakopoulos, P. (2007). In vitro three-stage continuous
fermentation of wheat arabinoxylan fractions and induction of hydrolase activity by the gut microflora.
International journal of biological macromolecules 41, 584-589.
Vatandoust, A., Ragaee, S., Wood, P. J., Tosh, S. M., and Seetharaman, K. (2012). Detection, localization, and
variability of endogenous β-glucanase in wheat kernels. Cereal Chemistry 89, 59-64.
Veličković, D., Ropartz, D., Guillon, F., Saulnier, L., and Rogniaux, H. (2014). New insights into the structural and
spatial variability of cell-wall polysaccharides during wheat grain development, as revealed through MALDI
mass spectrometry imaging. Journal of experimental botany 65, 2079-2091.
Vince, A. J., and Burridge, S. M. (1980). Ammonia production by intestinal bacteria: the effects of lactose, lactulose
and glucose. Journal of medical microbiology 13, 177-191.
Vincken, J.-P., Schols, H. A., Oomen, R. J., McCann, M. C., Ulvskov, P., Voragen, A. G., and Visser, R. G. (2003). If
homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture. Plant
physiology 132, 1781-1789.
Walton, G. E., Lu, C., Trogh, I., Arnaut, F., and Gibson, G. R. (2012). A randomised, double-blind, placebo controlled
cross-over study to determine the gastrointestinal effects of consumption of arabinoxylan-oligosaccharides
enriched bread in healthy volunteers. Nutrition journal 11, 1.
Wang, H., Offler, C., and Patrick, J. (1994). Nucellar projection transfer cells in the developing wheat grain.
Protoplasma 182, 39-52.
Whelton, S. P., Hyre, A. D., Pedersen, B., Yi, Y., Whelton, P. K., and He, J. (2005). Effect of dietary fiber intake on
blood pressure: a meta-analysis of randomized, controlled clinical trials. Journal of hypertension 23, 475-481.
Williams, B. A., Zhang, D., Lisle, A. T., Mikkelsen, D., McSweeney, C. S., Kang, S., Bryden, W. L., and Gidley, M. J.
(2016). Soluble arabinoxylan enhances large intestinal microbial health biomarkers in pigs fed a red meat–
containing diet. Nutrition 32, 491-497.
Wollowski, I., Rechkemmer, G., and Pool-Zobel, B. L. (2001). Protective role of probiotics and prebiotics in colon
cancer. The American journal of clinical nutrition 73, 451s-455s.
Wong, J. M., De Souza, R., Kendall, C. W., Emam, A., and Jenkins, D. J. (2006). Colonic health: fermentation and
short chain fatty acids. Journal of clinical gastroenterology 40, 235-243.
Wood, P., Weisz, J., and Blackwell, B. (1994). Structural studies of (1→ 3)(1→ 4)-β-D-glucans by 13C-NMR and by
rapid analysis of cellulose-like regions using high-performance anion-exchange chromatography of
oligosaccharides released by lichenase. Cereal Chem 71, 301-307.
Wood, P. J. (2007). Cereal β-glucans in diet and health. Journal of Cereal Science 46, 230-238.
Ying, R., Saulnier, L., Bouchet, B., Barron, C., Ji, S., and Rondeau-Mouro, C. (2015). Multiscale characterization of
arabinoxylan and β-glucan composite films. Carbohydrate Polymers 122, 248-254.
Yuasa, H., Kuni, C., and Watanabe, J. (1997). Comparative Assessment of D‐Xylose Absorption between Small
Intestine and Large Intestine. Journal of pharmacy and pharmacology 49, 26-29.
AACC, D. F. D. C. (2001). Report of the Dietary Fiber Definition Committee to the Board of Directors of the American
Association of Cereal Chemists: The Definition of Dietary Fiber. St Paul, MN: AACC International, 112-126.
87