1. business and industrial
2. energy
3. oil

Dietary Fibres from Flaxseed Kernel: Structural and Conformational
Characterization, and Structure-Function Relationship
By
Huihuang Ding
A Thesis
Presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
in
Food Science
Guelph, Ontario, Canada
© Huihuang Ding, April, 2015
ABSTRACT
DIETARY FIBRES FROM FLAXSEED KERNEL:
STRUCTURAL & CONFORMATIONAL CHARACTERIZATION
AND STRUCTURE-FUNCTION RELATIONSHIP
Huihuang Ding
University of Guleph, 2015
Advisors: Dr. Steve W. Cui
Dr. H. Douglas Goff
Flaxseed (Linum usitatissimum L.), one of the most economically important oilseed
crops, is also rich in soluble and insoluble dietary fibres. Scanning electron microscope
(SEM) images revealed that flaxseed kernel dietary fibres (FKDF) are mostly in the
supporting structure of the cell walls. Five separate FKDF fractions were obtained by a
sequential extraction and fractionation procedure, which were flaxseed kernel (FK)
water-extracted polysaccharides (FK-WP), FK EDTA-extracted polysaccharides (FK-EP),
FK Na2CO3-extracted polysaccharides (FK-NP), FK 1M KOH-extracted polysaccharides
(FK-KPI), and FK 4M KOH-extracted polysaccharides (FK-KPII). The average
molecular weight of FK-WP was 486 kDa, FK-EP 593 kDa, FK-NP 704 kDa, FK-KPI
770 kDa, and FK-KPII 1660 kDa, respectively. All FKDF fractions had the ability to
lower the surface tension of water, among which FK-KPI exhibited the best surface
activity. Rheological properties showed that FKDF fractions had low viscosity, and 2%
(w/v) FKDF water solution exhibited viscoelastic behaviour at 25 °C. FK-KPI, which had
the highest yield and best surface activity, was chosen as a representative to elucidate the
detailed structures of FKDF. The structures of two major fractions of FK-KPI (KPI-ASF
and KPI-EPF) were proposed as RG-I bridge-linked arabinans and xyloglucans as below:
The absolute Mw of KPI-ASF ranged from 550 to 596 kDa with a highly branched and
more sphere-like molecular structure. The absolute Mw of KPI-EPF was calculated to be
1506 kDa by SLS, and the ρ value of KPI-EPF (1.44) also confirmed the highly branched
structure of extracted xyloglucans from flaxseed kernel cell wall. These two fractions
were subjected to in vitro fermentation by pig colonic digesta with psyllium fibre as the
reference. All fibre grown cultures showed a significant increase in total short chain fatty
acids (SCFAs) after 72 h fermentation. Acetic acid was the major SCFAs produced,
followed by propionic acid and butyric acid. Flaxseed dietary fibres were relatively
slower fermentable dietary fibres as compared with psyllium fibre, and they sustained the
production of SCFAs with prolonged fermentation. Enhanced understanding of the
structure-function relationship is hoped to promote the potential application of flaxseed
dietary fibres in related industries.
Acknowledgements
I would like to express my sincerest gratitude to my advisors, Dr. Steve W. Cui and
Dr. H. Douglas Goff for all their support, guidance, and encouragement during my PhD
program. They bring me into the world of polysaccharide science, and provide me the
opportunity of doing research projects on dietary fibres in the University of Guelph, and
Guelph Food Research Centre (GFRC-AAFC). I am deeply thankful for their intellectual
advices, enthusiasm, and patience throughout the project.
I am grateful to my co-supervisor Dr. Jie Chen in Jiangnan University, and my
advisory committee members Dr. Qi Wang and Dr. Massimo F. Marcone for their
guidance. I want to thank Dr. Joshua Gong in GFRC-AAFC, Dr. Alejandro G. Marangoni,
Dr. Arthur R. Hill, Dr. Yoshinori Mine, and Dr. Sheng Chang in the University of Guelph,
Prof. Nongxue Qiu in Shaanxi Normal University, and Dr. Margareta Nyman in Lund
University for their guidance and kind help.
I wish to thank Cathy Wang, Dr. Ying Wu, Dr. Hai Yu, and John Nikiforuk in
AAFC, and Dr. Sandy Smith, Dr. Valerie Robertson, and Dr. Armen Charchoglyan in the
University of Guelph for their technical assistance. I would like to thank Natunola Health
Inc. for providing flaxseed kernel samples.
I want to thank all of my friends and colleagues in the University of Guelph,
GFRC-AAFC, Jiangnan University, and Shaanxi Normal University for their insightful
discussion.
Finally, special thanks to my parents, Ruiming Ding and Xiaolan Zeng, and my wife
Ying Xu for their endless love, support and understanding.
v
Table of Contents
ABSTRACT ....................................................................................................................... ii
Acknowledgements ............................................................................................................v
List of Tables .................................................................................................................... xi
List of Figures ................................................................................................................. xiv
CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ..............................1
1.1. Introduction .................................................................................................................1
1.2. Literature review ........................................................................................................3
1.2.1. Definition and functionality of dietary fibre .........................................................3
1.2.2. Dietary fibres in flaxseed ......................................................................................4
1.2.3. Cell wall polysaccharides ......................................................................................5
1.2.4. Previous research on the structure of flaxseed dietary fibres ..............................12
1.2.5. Application of flaxseed dietary fibres and their potential products ....................13
1.2.6. Structural and conformational characterization of polysaccharide .....................15
1.3. Overall objectives ......................................................................................................21
CHAPTER 2. FLAXSED KERNEL DIETARY FIBRES: EXTRACTION,
FRACTIONATION, AND PARTIAL PHYSICOCHEMICAL PROPERTIES .......23
2.1. Introduction ...............................................................................................................23
2.2. Methodology ..............................................................................................................23
2.2.1. Materials ..............................................................................................................23
2.2.2. Extraction and fractionation procedure ...............................................................24
vi
2.2.3. Determinations of sugar, uronic acid, dietary fibre, starch, protein, ash, and
moisture .........................................................................................................................25
2.2.4. Scanning electron microscope (SEM) .................................................................25
2.2.5. Molecular weight distribution……………………………...…………………..25
2.2.6. Monosaccharide composition analysis ................................................................26
2.2.7. Fourier transform infrared spectroscopy (FTIR) analysis ...................................27
2.2.8. Surface tension analysis ......................................................................................27
2.2.9. Rheological properties determination .................................................................27
2.3. Results and discussion ..............................................................................................28
2.3.1. Extraction, fractionation and chemical composition analysis .............................28
2.3.2. Molecular weight distribution of FKDF fractions...............................................31
2.3.3. Monosaccharide analysis of FKDF fractions ......................................................33
2.3.4. FTIR analysis of FKDF fractions ........................................................................36
2.3.5. Surface activities of FKDF fractions ...................................................................36
2.3.6. Rheological property of FKDF fractions ............................................................38
2.4. Conclusions ................................................................................................................40
CHAPTER
3.
STRUCTURAL
CHARACTERIZATION
OF
RG-I
BRIDGE-LINKED ARABINANS FROM FLAXSEED KERNEL CELL WALL ...42
3.1. Introduction ...............................................................................................................42
3.2. Experimental .............................................................................................................43
3.2.1. Materials ..............................................................................................................43
vii
3.2.2. Further fractionation of FK-KPI fractions ..........................................................43
3.2.3. Total uronic acid analysis, molecular weight distribution, monosaccharide
composition, and total phenol content determination of KPI-ASF ...............................44
3.2.4. Reduction of uronic acids and methylation/ GC-MS analysis ............................44
3.2.5. Nuclear magnetic resonance (NMR) spectroscopy .............................................45
3.2.6. MALDI-TOF-MS analysis ..................................................................................46
3.3. Results and discussion ..............................................................................................47
3.3.1. Fractionation and chemical composition analysis of FK-KPI ............................47
3.3.2. Methylation/ GC-MS analysis of KPI-ASF ........................................................48
3.3.3. NMR analysis of KPI-ASF .................................................................................51
3.3.4. MALDI-TOF-MS analysis of KPI-ASF .............................................................58
3.3.5. Proposed structure of KPI-ASF...........................................................................61
3.4 Conclusion ..................................................................................................................63
CHAPTER 4. STRUCTURAL CHARACTERIZATION OF XYLOGLUCANS
AND PARTIAL FLAXSEED KERNEL CELL WALL MODEL ..............................65
4.1. Introduction ...............................................................................................................65
4.2. Experimental .............................................................................................................65
4.2.1. Materials ..............................................................................................................65
4.2.2. Methylation/GC-MS analysis ..............................................................................66
4.2.3. Nuclear magnetic resonance (NMR) spectroscopy .............................................66
4.2.4. Hydrolysis of KPI-EPF and MALDI-TOF-MS analysis ....................................66
viii
4.3. Results and discussion ..............................................................................................67
4.3.1. Methylation and GC-MS analysis of KPI-EPF ...................................................67
4.3.2 NMR analysis of KPI-EPF ...................................................................................69
4.3.3. MALDI-TOF-MS analysis and proposed structure of KPI-EPF ........................73
4.3.4. Distribution of polysaccharides in flaxseed kernel (FK) cell walls, and partial
FK cell wall model ........................................................................................................77
4.4. Conclusions ................................................................................................................85
CHAPTER 5. CONFORMATION OF RG-I BRIDGE-LINKED ARABINANS AND
XYLOGLUCANS FROM FLAXSEED KERNEL CELL WALL ..............................86
5.1. Introduction ...............................................................................................................86
5.2. Experimental .............................................................................................................86
5.2.1. Materials ..............................................................................................................86
5.2.2. High performance size exclusion chromatography (HPSEC) and intrinsic
viscosity measurement ..................................................................................................86
5.2.3. Light scattering ....................................................................................................87
5.3. Results ........................................................................................................................89
5.3.1. Conformation characterization of KPI-ASF .......................................................89
5.3.2. Conformation characterization of KPI-EPF ........................................................95
5.4. Discussion.................................................................................................................100
5.5. Conclusions ..............................................................................................................101
ix
CHAPTER 6. SHORT-CHAIN FATTY ACID PROFILES OF FLAXSEED
DIETARY FIBRES BY IN VITRO FERMENTATION OF PIG COLONIC
DIGESTA........................................................................................................................102
6.1. Introduction .............................................................................................................102
6.2. Experimental ...........................................................................................................103
6.2.1. Materials ............................................................................................................103
6.2.2. Molecular weight and chemical composition analysis ......................................104
6.2.3. Digesta preparation ...........................................................................................104
6.2.4. Medium preparation ..........................................................................................105
6.2.5. Preparation of in vitro batch culture system......................................................105
6.2.6. Measurement of short-chain fatty acids (SCFAs) .............................................105
6.3. Results and Discussion ............................................................................................108
6.3.1 Chemical composition and monosaccharide ratio of dietary fibres ...................108
6.3.2 Effect of different dietary fibres on the Production of SCFAs ..........................108
6.3.3 Partial structure-function relationship between dietary fibre and fermentation
profiles……………………………………………………………………………….118
6.4. Conclusions ..............................................................................................................121
CHAPTER 7. GENERAL DISCUSSION AND CONCLUSION ..............................123
REFERENCES ...............................................................................................................130
x
List of Tables
Table 1.1. Linkage type of arabinans from different plant sources .....................................9
Table 1.2. Previous research on flaxseed dietary fibres .....................................................12
Table 1.3. Previous research on the application of flaxseed dietary fibres........................13
Table 2.1 Chemical composition of flaxseed kernel (FK) and de-oiled FK ......................30
Table 2.2. Sugar, uronic acid, starch, protein content and yield of FKDF fractions .........31
Table 2.3. The weight average molecular weight (Mw), number average molecular
weight (Mn), intrinsic viscosity ([η]) and polydispersity index (Mw/Mn) of FKDF
fractions ......................................................................................................................33
Table 2.4. Relative neutral monosaccharide composition of FKDF fractions ...................35
Table 3.1. Monosaccharide composition of KPI-EPF, KPI-ASF, KPI-ASF-R, and
KPI-ASF-H .................................................................................................................47
Table 3.2. Linkage type and molar percentage of KPI-ASF and KPI-ASF-R based on
Methylation/ GC-MS analysis ....................................................................................50
Table 3.3. 1H/13C NMR Chemical Shifts (ppm) of sugar resides from KPI-ASF (in D2O at
295 K) .........................................................................................................................55
Table 3.4. Observed Nuclear Overhauser Effect (NOE) contacts from anomeric protons
of KPI-ASF (in D2O at 295 K), and KPI-ASF-H (in DMSO-d6 at 295 K) ................58
Table 3.5. Saccharide series and mass peak of KPI-ASF-H in MALDI-TOF-MS
spectra… .....................................................................................................................60
xi
Table 4.1. Linkage type and molar percentage of KPI-EPF based on Methylation/ GC-MS
analysis .......................................................................................................................68
Table 4.2. 1H/13C NMR Chemical Shifts (ppm) of sugar resides from KPI-EPF (in D2O at
295 K) .........................................................................................................................72
Table 4.3. MALDI-TOF-MS analysis and deduced subunit structures of xyloglucans from
flaxseed kernel cell wall .............................................................................................75
Table 4.4. Linkage type and molar percentage of FK-WPpf based on Methylation/ GC-MS
analysis .......................................................................................................................79
Table 4.5. Linkage type and molar percentage of FK-EPpf based on Methylation/ GC-MS
analysis .......................................................................................................................80
Table 4.6. Linkage type and molar percentage of FK-NPpf-1 based on Methylation/
GC-MS analysis .........................................................................................................81
Table 4.7. Linkage type and molar percentage of FK-NPpf-2 based on Methylation/
GC-MS analysis .........................................................................................................81
Table 4.8. Linkage type and molar percentage of FK-KPIIpf based on Methylation/
GC-MS analysis .........................................................................................................82
Table 5.1. Conformational parameters of KPI-ASF using Static and Dynamic Light
Scattering (SLS & DLS), and High Performance Size Exclusion Chromatography
(HPSEC) .....................................................................................................................93
xii
Table 5.2. Conformational parameters of KPI-EPF using Static and Dynamic Light
Scattering (SLS & DLS), and High Performance Size Exclusion Chromatography
(HPSEC) .....................................................................................................................99
Table 6.1. Composition of anaerobic incubation medium (AIM) in 1 L Milli-Q water...107
Table 6.2. Chemical composition and molecular weight of dietary fibre samples ..........109
Table 6.3. Fitting equation and R2 of SCFA standards in GC analysis ...........................110
Table 6.4. SCFA production of different dietary fibres by in vitro fermentation of pig
colonic digesta ..........................................................................................................112
Table 6.5. Percentage of SCFA production of different dietary fibres by in vitro
fermentation of pig colonic digesta ..........................................................................113
xiii
List of Figures
Figure 1.1. (A) anatomic structure of flaxseed (Vaughan, 1970), and (B) micrographs of
flaxseeds in ruthenium red solution (Naran, Chen, & Carpita, 2008) ..........................4
Figure 2.1. Extraction and fractionation procedure of FKDF fractions .............................24
Figure 2.2. Scanning Electron Microscopy (SEM) images of whole flaxseed (a), bar
represents 100 μm; flaxseed kernel (FK) before oil extraction (b), bar represents 10
μm; and FK after oil extraction (c, d), bar represents 100 μm (c) and 10 μm (d),
respectively. ................................................................................................................29
Figure 2.3. HPSEC elution profiles of FKDF fractions (RI and UV detector) ..................32
Figure 2.4. HPAEC of FKDF fractions (PAD detector) ....................................................34
Figure 2.5. FTIR spectra of FKDF fractions ......................................................................36
Figure 2.6. Surface tension of FKDF fractions with various concentrations at 25 °C ......37
Figure 2.7. Steady shear flow curves of FKDF fractions under different concentrations at
25 °C (a), and dynamic rheology by frequency sweep test of 2% (w/v) FKDF
fractions at 25 °C (b). .................................................................................................39
Figure 2.8. Steady shear flow curves of 1% FKDF fractions at different temperatures ....40
Figure 2.9. Steady shear flow curves of 1% FKDF fractions with different concentrations
of Ca2+ at 25 ⁰C ..........................................................................................................40
Figure 3.1. Fractionation of FK-KPI..................................................................................43
xiv
Figure 3.2. 1H NMR spectra of (a) KPI-ASF (in D2O at 295 K), and (b) KPI-ASF-H (in
DMSO-d6 at 295 K) ....................................................................................................53
Figure 3.3. Parts of (a) COSY and (b) TOCSY spectra of KPI-ASF (in D2O at 295 K) ...54
Figure 3.4. Parts of HSQC spectra of KPI-ASF (in D2O at 295 K) ...................................56
Figure 3.5. Parts of NOESY spectra of (a) KPI-ASF (in D2O at 295 K), and (b)
KPI-ASF-H (in DMSO-d6 at 295 K) ..........................................................................57
Figure 3.6. MALDI-TOF-MS profile of KPI-ASF and KPI-ASF-H .................................59
Figure 3.7. Proposed structure of KPI-ASF as RG-I bridge linked arabinans ...................63
Figure 4.1. 1H spectrum of KPI-EPF (in D2O at 295 K) ...................................................69
Figure 4.2. Parts of COSY (a), and TOCSY spectra (b) of KPI-EPF (in D2O at 295 K) ..71
Figure 4.3. Parts of HSQC spectrum of KPI-EPF (in D2O at 295 K) ................................72
Figure 4.4. MALDI-TOF-MS profile of KPI-EPF ............................................................74
Figure 4.5. Proposed structure of KPI-EPF as xyloglucans...............................................77
Figure 4.6. Fractionation of dietary fibres in flaxseed kernel cell walls ............................78
Figure 4.7. Partial cell wall model from the cotyledons in flaxseed kernel .......................84
Figure 5.1. The molecular size distribution of KPI-ASF (concentration: 0.1 mg/mL,
temperature: 23 ºC) in Milli-Q water (A), in 0.1 M NaNO3 (B), in 6 M NH4OH (C),
and in 0.5 M NaOH (D). ………………………………..………………………..…90
Figure 5.2. HPSEC elution profile of KPI-ASF……………………………………..…..91
xv
Figure 5.3. Stability of KPI-ASF in 6 M NH4OH, (A) HPSEC elution profile of KPI-ASF
dissolving in 6 M NH4OH after 24 h & 48 h, and (B) hydrodynamic radius (Rh) of
KPI-ASF dissolving in 6 M NH4OH after 1 h, 2 h, 4h, 24 h, & 48 h. ……………..92
Figure 5.4. The molecular size distribution of KPI-EPF (concentration: 0.1 mg/mL,
temperature: 23 ºC) in Milli-Q water (A), in 0.1 M NaNO3 (B), and in 0.5 M NaOH
(C)..…………………………………………………………………...…………......95
Figure 5.5. Stability of KPI-EPF in 0.5 M NaOH, (A) HPSEC elution profile of KPI-EPF
dissolving in 0.5 M NaOH after 24 h & 48 h, and (B) hydrodynamic radius (R h) of
KPI-EPF dissolving in 0.5 M NaOH after 1 h, 2 h, 4h, 24 h, & 48 h………...…… 96
Figure 5.6. HPSEC elution profile of KPI-EPF………………………………………….98
Figure 5.7. Schematic conformation of KPI-ASF and KPI-EPF………………….........100
Figure 6.1. Profiles of SCFA standards in GC analysis ...................................................111
Figure 6.2. Acetic acid production by in vitro fermentation of pig colonic digesta ........115
Figure 6.3. Total SCFA production by in vitro fermentation of pig colonic digesta .......116
Figure 6.4. Molecular structures of psyllium arabinoxylans (Yin et al., 2012), KPI-ASF &
KPI-EPF from flaxseed kernel (Chapter 3 & 4), and FG-AFG & FG-NFG from
flaxseed mucilage (Qian, 2014)................................................................................119
xvi
CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW
1.1. Introduction
Flaxseed (Linum usitatissiumum L.), one of the most economically important oilseed crops,
is the third major oilseed crop after canola and soybean in Canada (Statistics Canada, 2011). The
flaxseed production of Canada accounts for 38.6% of world production in 2010, covering 72.3%
of the global flaxseed trade (Statistics Canada, 2011). In 2014/2015 crop year, the total flaxseed
production and exportation of Canada are estimated to be 952 and 700 kilotonnes, increasing by
17% to the highest level since 2010 (Agriculture and Agri-Food Canada, 2014).
The GRAS (generally recognized as safe) status of flaxseed has been confirmed (Food and
Drug Administration, 2009), and it is the richest plant based source of alpha-linolenic acid (ALA)
in the North American diet (Morris, 2001, 2007). Ground flaxseed is a functional supplement for
the people at the risk of cardiovascular diseases, diabetes, and constipation with a
cholesterol-lowering health claim (Health Canada, 2014), as well as an ideal ingredient in
health-oriented, gluten-free, and vegetarian diets due to its high nutritional potential and
relatively low glycemic index (GI) value (Morris, 2007).
Flaxseed is also rich in soluble and insoluble dietary fibres. Compared to the other cereals
and oilseeds, e.g. wheat, barley, oat, soybean, etc. (Dhingra, Michael, Rajput, & Patil, 2012),
flaxseed has extremely high content of dietary fibres (22~28%), lignan (0.6~1.3%) and minerals
(3~4%) (Cui & Mazza, 1996; Johnsson, Kamal-Eldin, Lundgren, & Aman, 2000; Morris, 2007;
Mueller, Eisner, Yoshie-Stark, Nakada, & Kirchhoff, 2010), but low content of starch and sugars
1
(<1.6%) (USDA, 2011). The health benefits of dietary fibres include (but not limited to)
protecting against several chronic diseases (e.g. diabetes, obesity, colon cancer), reducing blood
cholesterol levels, and improving insulin sensitivity (Health Canada, 2012; Elleuch, Bedigian,
Roiseux, Besbes, Blecker, & Attia, 2011). Compared with insoluble dietary fibres, soluble
dietary fibres are easier to incorporate into beverages, dairy products, and processed foods as
thickeners, emulsifiers, stabiliser, and fat replacer (Cui, Wu, & Ding, 2013).
Due to the high nutritional potential and relatively low glycemic index value, flaxseed and
its products have been used for preparing value-added food products, such as muffin, salad
dressing (Stewart & Mazza, 2000), spaghetti (Lee, Manthey, & Hall, 2003) and dairy products
(Hall, Tulbek, & Xu, 2006). When flaxseed is eaten whole, the seed coat will hardly allow the
digestion of the kernel. However, ground flaxseed can be easily oxidized due to the high
percentage of PUFA, thus a patented de-hulling technique was developed to “lock” the PUFA
without destructing the kernel (Cui & Han, 2006; Cui, 2012).
Flaxseed kernel contains two cotyledons and embryo for oil storage, which are also the
major oil storage tissues in some other oilseeds, such as rapeseed/canola, sunflower seed, etc.
(Murphy, Rawsthorne, & Hills, 1993). The content of storage starch in cotyledon, embryo,
and/or endosperm largely decreases during the oilseed maturation, and the carbon skeleton of
cell was mainly composed of dietary fibres (e.g. hemicellulose, pectin, cellulose) (Murphy,
Rawsthorne, & Hills, 1993; Bewley, & Black, 1994).
2
The structure and functionality of flaxseed kernel dietary fibres (FKDF), as well as the
common characteristics between FKDF and the dietary fibres from other oilseeds are important
information to explore their potential for better utilization of the farm wastes and by-products
from oilseed industry, and to produce more value-added products in food, cosmetic, and
pharmaceutical industries.
1.2. Literature review
1.2.1. Definition and functionality of dietary fibre
According to the definition of Health Canada, dietary fibres consist of carbohydrates and
accepted novel fibres with a degree of polymerization (DP) of 3 or more that are not digested and
absorbed by the small intestine (Health Canada, 2012). Generally, their health benefits include
(but not limited to) protecting against several chronic diseases (e.g. diabetes, obesity, colon
cancer), improving laxation, preventing gastrointestinal disorders, reducing blood cholesterol
levels, reducing post-prandial blood glucose, increasing post-prandial satiation, improving
glycemia and insulin sensitivity, and providing energy-yielding metabolites through colonic
fermentation (Health Canada, 2012; Elleuch et al., 2011).
Dietary fibres can be further classified as water-soluble/ well fermented dietary fibres and
water-insoluble/ less fermented ones (Dhingra et al., 2012), involving differences in their
functionalities and physiological effects (Slavin, 1987; Slavin, 2013a; Dhingra et al., 2012).
Soluble dietary fibres, which could increase the viscosity of stomach and intestinal contents,
mainly act in the upper gastrointestinal tract (esophagus, stomach, and duodenum) and jejunum
3
to decrease post-prandial plasma glucose level, and reduce intestinal enzymatic activity (Wood,
Braaten, Scott, Riedel, Wolynetz, & Collins, 1994; Elleuch et al., 2011). However, insoluble
dietary fibres, mainly serving as bulking agents and laxatives, could increase fecal mass and
decrease large intestinal transit time (Phillips & Cui, 2011). They also help prevent constipation,
decrease re-adsorption of bile salts, and lower the risk of cancer in the colon (Verma and
Banerjee, 2010).
1.2.2. Dietary fibres in flaxseed
As mentioned above, whole flaxseed has 27~28% total dietary fibres, among which around
5~8% of soluble ones (flaxseed gum) are from flaxseed mucilage (Qian, Cui, Wu, & Goff, 2012).
The major insoluble fibre fractions are in flaxseed hull, which consist of cellulose, hemicellulose
and lignin (Johnsson et al., 2000).
Figure 1.1. (A) anatomic structure of flaxseed (Vaughan, 1970), and (B) micrographs of
flaxseeds in ruthenium red solution (Naran, Chen, & Carpita, 2008)
4
Fig. 1.1 (A) shows the separate anatomic parts of flaxseed. It is composed of two cotyledons
(~55%), hull (36%-40%), an embryo (~4%), and water soluble mucilage (~2%) (Vaughan, 1970;
Naran, Chen, & Carpita, 2008; Singh et al., 2011). Fig. 1.1B shows the water-soluble mucilage
from the outer layer of flaxseed hull. Flaxseed mucilage exists in two states, one which can be
easily separated from the hull and the other which tightly adheres to the cell walls (Naran, Chen,
& Carpita, 2008). Hull consists of four layers, and the inner hull surface is a thin layer of
endosperm. Flaxseed kernel is composed of two cotyledons and embryo in which the major
portion of oil is located (Vaughan, 1970). After oil extraction and enzymatic hydrolysis of
protein, the supporting structure of the cell walls is mainly composed of dietary fibres (i.e. cell
wall polysaccharides) (Bewley, & Black, 1994; Ding et al., 2014).
1.2.3. Cell wall polysaccharides
1.2.3.1. Cellulose and hemicellulose
Cellulose is a fundamental constituent of plant cell walls, and abundantly exists in
vegetables, fruits, cereals, and legumes (Albersheim, Darvill, O’Neill, Schols, & Voragen, 1996;
Cui, Wu, & Ding, 2013). Cellulose accounts for up to 40% of secondary cell walls (Delmer, &
Amor, 1995), and it is around 4~20% in primary cell wall (Albersheim et al., 1996). Cellulose is
associated to each other in structures as microfibrils, which are composed of around 36 cellulose
chains (Delmer, & Amor, 1995). Cellulose is composed of β-(1,4)-D-glucose, and its molecular
weight ranges from 162 to 2268 kDa (Cui, Wu, & Ding, 2013). Coexisting with cellulose,
5
hemicellulose, pectin and lignin, the microstructure allows enzymatic regulation of cell swelling
and elongating in primary cell wall (Keegstra, Talmadge, Bauer, & Albersheim, 1973; Fry,
1989a).
Hemicelluloses mainly contain xyloglucans, arabinoxylans, β-glucans, and galactomannans
(Cui, Wu, Ding, 2013), which bind to cellulose fibrils through hydrogen bonds (Albersheim et al.,
1996).
Xyloglucans,
the
most
abundant
hemicellulose,
have
linear
backbones
of
β-(1,4)-D-glucopyranose attached with xylopyranosyl branches (Fry, 1989a). The structure of
xyloglucans in various plants, including legumes, corn, olive, tomato, lettuce, carrot, onion,
pepper, liverwort, etc., had been extensively summarized (Fry, 1989a; Hayashi, 1989; Kato,
2001; Vierhuis et al., 2001; Hoffman et al., 2005; Peña, Darvill, Eberhard, York, & O’Neill,
2008). The aforementioned xyloglucans were extracted from the cell walls of the stems, seeds,
shoots, suspension-cultured cells, and/or other edible parts. Different substitution patterns of
each backbone glucosyl residue of xyloglucans were described as a single letter nomenclature
(Fry, York, Albersheim et al., 1993). Most xyloglucans are composed of XXXG and XXGG-type
structure, as well as XXLG and/or XXFG-type units. Among them, the letter “G” is an
unsubstituted β-D-glucopyranosyl unit; the letter “X” represents a terminal α-D-xylopyranosyl
residue (1-6)-linked with G; the letter “L” represents a terminal β-D-galactopyranosyl residue
(1-2)-linked with X, and the letter “F” represents a terminal α-L-fucopyranosyl residue
(1-2)-linked with L (Fry et al., 1993). The side-chain distributions of xyloglucans are both
species and tissue specific, and the structure would be modified by cell wall enzymes during cell
6
growth (Fry, 1995; Pauly, Qin, Greene, Albersheim, Darvill, & York, 2001; Vierhuis et al.,
2001).
Arabinoxylans, which are major dietary fibre supplements in the diet, are widely distributed
in all major cereal grains, including wheat, barley, oats, maize, psyllium, and flaxseed. They
consist of β-(1,4)-D-xylopyranosyl backbone with α-L-arabinofuranosyl attached to some of the
xylopyranosyl residues at O-2 and/or O-3 positions. The neutral fraction from flaxseed mucilage
is mainly composed of arabinoxylans, which are substituted at O-2 and/or O-3 positions by 1~3
sugar residues (Qian, 2014). Likewise, the structural differences of arabinoxylans from maize,
rice, and wheat brans on in vitro fermentation profiles have been studied by Rose, Patterson, and
Hamaker (2010).
β-Glucans occur in the subaleurone and endosperm cell walls of cereals, including oats,
wheat and barley (Cui, Wu, & Ding, 2013). They are homo-polysaccharides with (1,3), (1,4), or
(1,6)-linked β-D-glucopyranosyl residues. The content of β-Glucans varies from around 1% in
whole wheat to as high as 11% in barley (Cui, 2005). The cholesterol-lowering health claim of
oat products is specifically attributed to (1,3), (1,4)-linked β-D-glucans based on animal studies
and clinical trials (Wood et al., 1994; Health Canada, 2007).
Galactomannans are distributed in guar plant, tara shrub, fenugreek plant, the seeds of
legumes, and the stems of some other herbs (Cui, Wu, & Ding, 2013). They consist of
β-(1,4)-D-mannosyl backbone branched at O-6 with α-D-galactopyranosyl residues, and the
7
mannose to galactose ratio is dependent on plant origin (Xing, Cui, Nie, Phillips, Goff, & Wang,
2013).
1.2.3.2. Pectin
In cell walls of dicotyledons, two different layers can be recognized, and the external one is
mostly composed of pectin, constituting the middle lamella (Zykwinska, Rondeau-Mouro,
Garnier, Thibault, & Ralet, 2006). Coexisting with the cellulose and hemicelluloses, the pectin
appears to share the same space with cellulose/hemicellulose networks (Albersheim et al., 1996).
There are three major pectic polysaccharides, which are homogalacturonan (HG),
rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II). HG consists a linear
α-(1,4)-D-galacturonan backbone without any branch, while RG-I consists of the repeating
(1-4)-α-D-GalpA-(1,2)-α-ʟ-Rhmp disaccharide units with arabinans and/or galactans attaching to
the rhamnose residues at the backbones (Albersheim et al., 1996; Cui, Wu, & Ding, 2013). The
acidic fraction from flaxseed mucilage is mainly composed of RG-I, which has higher content of
mono-galactosyl branches attaching to O-3 positions of rhamnosyl residues with limited HG
residues (Qian, Cui, Nikiforuk, & Goff, 2012). However, the structure of RG-I from soybean
seeds was reported to have no attached HG (Huisman et al. 2001). Neutral sugar side-chains
contribute to polymer entanglement, and both galactans and arabinans have been discovered in
isolated cell wall components (Renard and Jarvis, 1999; Hwang, Pyun, & Kokini, 1993).
8
Arabinans are generally considered to be associated with RG-I in the pectic network with a
backbone of (1,5)-linked α-ʟ-arabinofuranosyl units (Gomez et al., 2009; Westphal et al., 2010).
As shown in Table 1.1, arabinans branch mostly on O-3, or both O-2 and O-3, while O-2
branching can be found in mung bean (Swamy & Salimath, 1991), mustard seed (Tharanathan,
Bhat, Krishna & Paramahans, 1985), dehulled rapeseed (Eriksson, Andersson, Westerlund,
Andersson & Aman, 1996), honey locust seed (Navarro et al., 2002), horsebean root (Joseleau,
Chambat & Lanvers, 1983), and prickly pear (Habibi et al., 2005). Oilseeds (e.g. flaxseed,
rapeseed, soybean, etc.), cabbage, and some fruits (e.g. apple, grape) had higher molar
percentage of branched chains than other plant sources (Siddiqui & Wood, 1974; Norton &
Harris, 1975; Aspinall, & Cottrell, 1971; Aspinall & Fanous, 1984). Less branched arabinans
were found in cowpea, Schizolobium parahybae and Cassia fastuosa seed (Petkowicz,
Sierakowski, Ganter & Reicher, 1998), pea hull (Ralet, Saulnier & Thibault, 1993), and apple
juice (Churms et al., 1983). It is worth noting that pectins with neutral branches could prevent
cell wall components (e.g. homogalacturonans, cellulose fibrils) from tightly associating with
each other (Jones, Milne, Ashford, & McQueen-Mason, 2003), and maintain the flexibility
during cell growth, lipid storage, and water deficit stress (Moore, Farrant, & Driouich, 2008).
Table 1.1. Linkage type of arabinans from different plant sources
Linkage type (molar
ratio)
/ Plant source (reference)
T-Araf
1,5Araf
1,3,5
-Araf
1,2,3,5
-Araf
T-Ara
p
1,2Araf
1,3Araf
1,2,5
-Araf
Soybean
(Aspinall, & Cottrell, 1970)
4
3
1.5
1
-
-
-
0.6
9
Cowpea (Vigna sinensis)
(Muralikrishna &
Tharanathan, 1986)
Pigeon pea (Cajanus
cajan) cotyledon
(Swamy & Salimath, 1991)
2
5
0.6
0.4
-
-
-
0.3
1
3.5
0.2
0.5
0~0.5
-
-
1
Mung bean (Vigna
radiata) cotyledon
(Swamy & Salimath, 1991)
Lupin (Lupinus
angustifolius) cotyledon
(Cheetham, Cheung &
Evans, 1993)
Pea hull
(Ralet et al., 1993)
Olive
(Vierhuis, Korver, Schols &
Voragen, 2003)
Olive pulp
(Cardoso et al., 2007)
Olive pomace
(Cardoso et al., 2002)
2
3.5
0.5
0.4
-
-
-
1.5
3
4
3
0.1
-
-
-
-
1
5
0.4
0.1
-
-
-
-
4
4
1.5
0.3
-
0.1
0.3
0.2
4
4
3
-
-
-
1.5
-
4
5
3
-
-
-
1
-
Quinoa (Chenopodium
quinoa) seed
(Cordeiro et al., 2002)
Mustard seed meal
(Tharanathan et al., 1985)
Rapeseed (Brassica
campestris)
(Siddiqui & Wood, 1974)
Rapeseed (Brassica napus)
(Norton & Harris, 1975)
1
5
1
-
-
-
0.4
-
4
2
0.6
0.8
0.8
-
-
1.6
4
2.6
2.5
1.5
-
-
-
-
3
3
2.5
1.6
-
-
-
-
Dehulled rapeseed
(Eriksson et al., 1996)
Almond (Prunus dulcis)
seed
(Dourado et al., 2004)
4.5
0.4
0.1
0.4
0.2
-
-
3.5
4
2.5
1.5
1.5
-
-
0.1
-
Honey locust (Gleditsia
triacanthos) seed
1
1
0.1
0.2
-
-
-
0.7
10
(Navarro et al., 2002)
3
2
-
1
-
-
-
1
0.1
10
-
-
-
-
-
-
Cassia fastuosa seed
(Petkowicz et al., 1998)
Sugar beet
(Oosterveld, Beldman,
Schols & Voragen, 2000)
0.8
8~10
0.4
0.1
0.8
-
-
0.3
4
4
3
0.5
-
-
0.1
-
Horsebean (Vicia faba)
root
(Joseleau et al., 1983)
Marshmallow root
(Capek et al., 1983)
Carrot
(Capek et al., 1983)
Parsnip
(Siddiqui & Emery, 1990)
Cabbage
(Stevens & Selvendran,
1980)
Apple
(Aspinall & Fanous, 1984)
Grape berries pulp
(Saulnier, Brillouet &
Joseleau, 1988)
1.5
6
1
0.2
-
-
-
2.5
4
3
1
1.5
0.1
-
0.1
1
4
4
2
0.5
-
-
0.6
-
3
5
2
0.5
-
-
-
-
4
3
1.5
2
-
-
-
-
3.5
3
2
1
-
-
0.5
-
1.5
3
1
0.1
-
-
0.2
-
Lemon peel
(Aspinall & Cottrell, 1970)
3
4
1.5
1.2
-
-
-
0.5
Prickly pear (Opuntia
ficus-indica) water extract
(Habibi et al., 2005)
Prickly pear (Opuntia
ficus-indica)
arabinan-rich pectin
(Habibi et al., 2005)
Apple juice
4
0.7
0.1
0.2
-
-
1.6
4
4
4
1.3
1
-
-
-
1
0.1
5
0.2
-
0.2
-
-
-
Nata Karanja
(Caesalpinia bonduc) seed
(Mandal et al., 2011)
Schizolobium parahybae
seed
(Petkowicz et al., 1998)
11
(Churms et al., 1983)
Grape juice
(Villettaz, Amado &
Neukom, 1981)
Red Wine
(Belleville, Williams &
Brillouet, 1993)
White willow (Salix alba)
bark
(Karacsonyi, Toman,
Janecek & Kubackova,
1975)
2
4
3
-
-
-
-
-
0.2
3
0.2
-
-
-
-
-
4
2.5
1
1.5
0.2
-
0.4
0.6
1.2.4. Previous research on the structure of flaxseed dietary fibres
Previous research on flaxseed dietary fibres mainly focused on soluble flaxseed gum from
mucilaginous layer of whole seed or flaxseed hull, which is summarized in Table 1.2.
Table 1.2. Previous research on flaxseed dietary fibres
Source
Flaxseed
mucilage
Flaxseed
mucilage
Flaxseed
mucilage
Flaxseed
mucilage
fractions
Flaxseed
mucilage
Flaxseed gum
Study/Description
Glucose, galactose, arabinose and xylose were identified;
mucilage fraction had 17% pentose, and acidic part was
separated.
Identification of aldobiouronic acid during hydrolysis:
glucosidic linkage between D-galacturonic acid and
L-rhamnose.
Reference
Non-cellulosic mucilage was heterogeneous
polysaccharides composing of acidic and neutral fractions.
Bailey, 1935
Monosaccharide composition and linkage information of
neutral and acidic polysaccharide fractions.
Monosaccharide composition of neutral and acidic
polysaccharide fractions; linkage pattern of separated
arabinoxylan and rhamnogalacturonan.
Molecular weight, monosaccharide composition, linkage
12
Neville, 1913
Anderson and
Crowder, 1930
Erskine and Jones,
1957; Hunt and
Jones, 1962
Muralikrishna et al.,
1987
Cui, Mazza,
from mucilage
pattern, and 13C NMR spectra of acidic fraction and neutral Biliaderis et al.,
fraction.
1994
Flaxseed
mucilage
Composition of monosaccharides and galacturonic acid,
and 13C NMR spectra of mucilage fractions.
Water-soluble
polysaccharides Monosaccharide composition of different flaxseed
from flaxseed
cultivars.
mucilage
Flaxseed gum
13
C NMR spectra and monosaccharide composition of
different flaxseed cultivars.
Water-soluble
polysaccharides Polysaccharides were made of "slightly stiff random coil
from flaxseed
molecules".
meal
Fedeniuk and
Biliaderis, 1994
Oomah et al., 1995
Cui, Kenaschuk, &
Mazza, 1996; Cui,
& Mazza, 1996
Goh et al., 2006
Flaxseed
mucilage
Linkage pattern, molecular weight, monosaccharide
composition of rhamnogalacturonan I and arabinoxylan.
Flaxseed gum
from hull
Detailed structure and conformation study of acidic fraction Qian et al., 2012;
(AFG) and neutral fraction (NFG) of flaxseed mucilage.
2014
Flaxseed meal
Structure and repeating units of xyloglucans from whole
flaxseed meal, and possible existence of arabinogalactan.
Naran et al., 2008
Ray et al., 2012
1.2.5. Application of flaxseed dietary fibres and their potential products
Whole or ground flaxseed has been added to various food materials for preparing
value-added food products as shown in Table 1.3.
Table 1.3. Previous research on the application of flaxseed dietary fibres
Flaxseed component
Effect/Benefit
Product
13
Reference
Flaxseed gum
Flaxseed gum
flaxseed flour
flaxseed flour
flaxseed flour
Ground flaxseed
Ground flaxseed
Ground flaxseed
Ground flaxseed &
flaxseed oil
Flaxseed meal &
flaxseed oil
Whole and crushed
flaxseed & flaxseed
oil
Fat-free flaxseed
powder
Stable at concentration > 0.45%
(w/w), pH >2.8.
Salad
dressing
Flaxseed gum reduced the creaming
of cloudy carrot juice better than
Juice
pectin and guar gum.
After heating at 100°C, 15 min, 60%
antifungal activity was retained.
Noodle
Pasta was darker and redder with the
increase of flaxseed flour; cooked
firmness was reduced.
Pasta by flaxseed flour had lower
cooked firmness and cooking loss,
and lower microbial counts during
storage.
Muffin with 50% ground flaxseed
were rated optimum in shape,
tenderness and texture compared
with 30% ground flaxseed and flour.
Muffin with 20% roasted flaxseed
powder had better overall acceptable
quality; no significant nutrition loss.
Lignan and ALA were stable during
32-week storage of
flaxseed-fortified macaroni.
ALA remained stable during
processing and cooking fortified
with ground flaxseed.
Storage stability unaffected by the
addition of flaxseed meal.
Flaxseed rolls retained moisture and
softness more efficiently; No
off-odors were detected during 6
days at 22°C.
Lignan was stable during normal
baking temperatures and storage.
14
Stewart and Mazza,
2000
Qin et al., 2005
Xu et al., 2008
Pasta
Sinha and Manthey,
2008
Pasta
Manthey et al., 2008
Muffin
Alpers and
SawyerMorse, 1996
Muffin
Sudha et al., 2010
Hall et al., 2005
Macaroni
Spaghetti
Manthey et al., 2002
Whole-wheat Shearer and Davies,
muffin
2005
Bread roll
Pohjanheimo et al.,
2006
Graham bun,
bread &
muffin
Hyvarinen et al.,
2006
The other potential products of flaxseed dietary fibres are listed below:
(1) Flaxseed hull: lignan-enriched bakery product, lignan tablet, laxative tablet with high soluble
and insoluble dietary fibres.
(2) Flaxseed meal or ground flaxseed: high dietary fibre donut, bagel, muffin; whole wheat bread
and other bakery product with nutty flavor and low gluten.
(3) Whole flaxseed kernel: cake, snack bar, salad topping, bakery and roasted products
(value-added flaxseed kernel products with high ALA and dietary fibres).
(4) Flaxseed gum: stabilizer, water-holding and suspending agent, cake, cheese spread, ice cream,
sour cream, whipping cream, frozen dessert, sauce, etc.
(5) Soluble flaxseed kernel dietary fibres: low caloric food or beverage, salad dressing, oil and
flavor emulsion, bakery fillings, icing, confectionaries, processed meat product, pie filling, etc.
(6) Insoluble flaxseed dietary fibres: bulking and laxative agent, drug therapy for constipation.
1.2.6. Structural and conformational characterization of polysaccharides
Monosaccharide compositions, linkage types, sugar rings, anomeric configurations,
sequences of sugar residues, repeating units, and substitutions are the most essential information
in order to characterize the primary structure of a polysaccharide. As the orientations of
monosaccharides are restricted by the torsion angles (e.g. φ, ψ and/or ω) through the glycosidic
bonds in a polysaccharide (Cui, 2005), conformational (i.e. advanced structural) characteristics
are also important in order to bridge the gap between a polysaccharide and its functionality.
15
1.2.6.1. High Performance Anion Exchange Chromatography (HPAEC)
HPAEC is widely used technique for the determination of monosaccharide composition.
The principle is based on that monosaccharides are ionized in the alkaline solution, and thus they
can be separated by an ion exchange column (Hardy, Townsend, & Lee, 1988). Sodium
hydroxide is utilized as the eluent to separate monosaccharides, and pulsed amperometric
detector (PAD) is widely used as the detector for HPAEC system. As sugars cannot be directly
detected by UV or fluorescence detectors, the separation of monosaccharides with low detection
limits can be achieved using PAD, which is also suitable for gradient elution (Lee, 1990; Cui,
2005).
1.2.6.2. Methylation/ GC-MS analysis
All free hydroxyl groups of a polysaccharide can be methylated by methyl iodide, and then
hydrolyzed by trifluoroacetic acid (TFA). The hydrolyzed monomers are reduced and acetylated
by acetic anhydride. The resultant partially methylated alditol acetates (PMAA) could be
dissolved in dichloromethane, and analyzed by GC-MS (Taylor and Conrad, 1972; Ciucanu and
Kerek, 1984). As TFA cleaves the glycosidic bonds but leaves methylether linkages intact, the
position of O-acetyl groups after acetylation in PMAA reflect the linkage types and ring sizes,
which can be identified and quantified by GC-MS (Cui, 2005). Relative retention time (RT),
which is relative to 2,3,4,6-Me4-Glucopyranose, can be used to confirmed the deduced linkage
types and compared with literature data. The GC-MS spectra and detailed deduction of different
16
linkage types have been extensively summarized by Carpita & Shea (1988), and RT based on
specific GC columns were also provided.
1.2.6.3. Partial acid hydrolysis and specific enzyme hydrolysis
Glycosidic linkages from furanosyl and deoxy sugars (e.g. arabinose, xylose, fucose and
rhamnose) are easier hydrolyzed by acid than the ones from other sugars. For example, it is
around 5 times faster when hydrolyzing glycosidic linkages from 6-deoxyhexoses by acids than
those from the other hexoses (Lindberg, Lönngren, & Svensson, 1975). Moreover, glycosidic
linkages in the branches are easier hydrolyzed than the linkages in the backbones during mild
acid hydrolysis, given that they all belong to similar acid labile glycosidic linkages or non-acid
labile ones (Round, Rigby, MacDougall, & Morris, 2010). Polysaccharide cleaving enzymes are
also widely used to release specific sugar residues. The high purity of an enzyme is of the utmost
importance for specific cleavage. Specific enzymes can easily release acid resistant glycosidic
linkages, or any other particular types of sugar linkages. However, the enzyme activity might be
inhibited due to steric-hindrance effects in some polysaccharides. Therefore, enzyme hydrolysis
is usually combined with partial acid hydrolysis for better and more specific characterization
(Cui, 2005). A mixture of monosaccharides and oligosaccharides can be separated from higher
molecular weight polymers using ethanol precipitation. The separated fractions can be identified
by NMR (Section 1.2.6.4) and MALDI-TOF-MS (Section 1.2.6.5), which will provide further
evidences to confirm the primary structure of a polysaccharide.
17
1.2.6.4. Nuclear magnetic resonance (NMR) spectroscopy
The linkage patterns deduced by methylation/GC-MS can be further confirmed by NMR
spectroscopy. Moreover, NMR provides information on anomeric configuration, linkage site &
sequence, substitution, and position of appended groups, etc. of a polysaccharide (Agrawal,
1992). From NMR spectra, the number of signals represents different kinds of protons, and the
location indicates magnetic environment (i.e. shielded or deshielded) of a proton. While signal
intensity (i.e. integrated peak area) refers to the number of a specific proton, and signal splitting
shows the number of nearby protons (Bubb, 2003). The 1H and
13
C chemical shifts of sugar
residues from oligosaccharides have been summarized by Agrawal (1992). Because a narrow
signal region (mostly between 3~5.5 ppm) of carbohydrates in 1H NMR spectrum, two and
multi-dimensional NMR techniques have been utilized to improve the sensitivity and resolution,
among which the most common ones are COSY, TOCSY, HSQC/HMQC, HMBC, and NOESY
(Cui, 2005). In general, COSY spectrum reveals the correlation of individual proton to the
protons which are two or three bonds apart; TOCSY correlates all protons in a spin system, thus
the correlations of anomeric protons to other protons can be observed; HSQC/HMQC spectrum
reveals heteronuclear correlation of protons with their neighbouring carbons; HMBC detects
1
H-13C coupling for relatively longer range, which is normally two to three bonds away; NOESY
provides information on the 1H-1H correlation through space via nuclear overhauser effect
(distance within 5 Å), meanwhile, the intra- and inter-residue connectivities could be observed
(Martin, Hadden, & Russell, 2003; Cui, 2005).
18
1.2.6.5.
Matrix-assisted
laser
desorption/ionization
time-of-flight
mass
spectrometry
(MALDI-TOF)
MALDI-TOF is usually combined with partial acid hydrolysis and specific enzyme
hydrolysis for the determination of monosaccharide sequences, repeating units, branch residues,
and modifications (Zaia, 2004). It has been widely used in the characterization of
oligosaccharides derived from xyloglucans (Vierhuis et al., 2001; Hoffman et al., 2005; Peña,
Darvill, Eberhard, York, & O’Neill, 2008). Ions are often observed as sodium and potassium
adducts [M+Na]+ and [M+K]+ from MS1 to MSn. The molecular mass differences of the sugar
residues from pentose, hexose, and deoxyhexose enable accurate elucidation of oligosaccharide
fragments, hence the structure of polysaccharides (Cui, 2005). It is worth noting that MS
fragments are specific for one structure rather than mixtures, and thus the pre-treatment of sugar
sample is of significant importance in avoid of false-positive results (Bauer, 2012).
1.2.6.6. Conformational characterization of polysaccharide and light scattering techniques
The classification of polysaccharide conformation has been extensively summarized
(Burchard, 2005; Burchard, 2008; Cui, 2005). Briefly, polysaccharides include ordered
conformation and disordered conformation. Ordered conformation can be further classified as
ribbon-like, hollow-helix, crumpled-ribbon, periodic, and interrupted types. Compared with
ordered conformation, disordered one has much less regularities, among which random coil is a
major conformational type of many polysaccharides. Conformation has direct impacts on the
19
rheological properties, and surface or interfacial properties of a polysaccharide. Moreover, some
polysaccharide conformation might provide binding sites, which will be recognized by particular
cell surfaces for specific biological functionalities (Rees & Scott, 1971; Burchard, 2008).
Light scattering techniques are widely utilized in the characterization of polysaccharide
conformation. There are two types of light scattering instruments, which are batch mode and
continuous flow/ chromatography mode. They share the same principle: the size and shape of
macromolecules can be measured and determined based on angular dependence of scattering
intensity, and the intensity is directly proportional to the concentration and molecular weight of
polymer in a dilute solution (Cui, 2005; Podzimek, 2011). The light scattering techniques have
been well-developed for synthetic homopolymers and copolymers, as well as several
polysaccharides (Burchard, 2008). Batch mode light scattering is a preferred analytical technique
to measure the absolute molecular weight, radius of gyration (Rg), second virial coefficient (A2),
and hydrodynamic radius (Rh). It allows the accurate measurement of scattering light intensities
in various solutions at the defined angles, and provides dynamic mode (i.e. dynamic light
scattering, DLS) for measuring the fluctuation of the intensity over time (Cui, 2005). Although
chromatography mode light scattering cannot be used for DLS measurement, it offers a fast and
convenient detection, which can also give important conformational parameters (e.g. molecular
weight, Rg, and Mark-Houwink parameter, etc.) of each eluting fraction in a mixture. Light
scattering also provides possibilities to determine translational diffusion coefficient, segmental
motions, and chain association behavior in thermodynamic equilibrium (Burchard, 2008).
20
1.3. Overall objectives
Flaxseed kernel contains about 20% of dietary fibre, most of which cannot be directly
extracted by hot water. However, after being released by chelating agent and alkaline solution
from the network of the cell wall, the extracted flaxseed kernel dietary fibre (FKDF) fractions are
all water soluble. FKDF is abundant in de-oiled flaxseed meal, which is a by-product of flaxseed
oil industry. To promote further utilization of flaxseed, investigation on structure and
functionality of FKDF is an essential step to explore its commercial potentials. There is no
published research focused on FKDF, and the structure-function relationship is still unknown.
In order to promote the potential application of FKDF, and improve the utilization of
soluble dietary fibres/gums from farm wastes and food processing by-products, the overall
objective of the present study is to elucidate the structural and conformational characteristics of
FKDF fractions, as well as to evaluate their physicochemical properties and short-chain fatty
acid (SCFA) profiles during in vitro fermentation, thus establishing the structure-function
relationship of FKDF.
The milestones of this research are:
(1) To develop sequential extraction and fractionation methods to obtain FKDF fractions, and to
analyze physicochemical properties of those fractions for the potential applications.
(2) To elucidate the fine structures of main FKDF fractions, and characterize the conformation
for establishing the relationship between primary structure and conformation of FKDF.
21
(3) To propose a partial cell wall model of flaxseed kernel, and establish the relationship between
cell wall structure and sequential extraction for further utilization of flaxseed and other oilseeds.
(4) To evaluate SCFA profiles of flaxseed dietary fibres through in vitro fermentation, and to
compare them with widely accepted psyllium fibre.
(5) To build partial structure-function relationship of FKDF based on characterization of
structure and conformation, as well as functionalities. To provide theoretical and empirical bases
when utilizing dietary fibres from oilseeds or oilseed by-products for healthier and more
value-added products in related industries.
22
CHAPTER 2. FLAXSED KERNEL DIETARY FIBRES: EXTRACTION,
FRACTIONATION, AND PARTIAL PHYSICOCHEMICAL PROPERTIES
2.1. Introduction
Flaxseed is rich in soluble and insoluble dietary fibres. Previous research on flaxseed
dietary fibres mainly focused on soluble flaxseed gum from the mucilaginous part of whole seed
or flaxseed hull (Muralikrishna, Salimath, & Tharanathan, 1987; Mazza & Biliaderis, 1989; Cui,
Mazza, & Biliaderis, 1994; Fedeniuk & Biliaderis, 1994; Oomah, Kenaschuk, Cui, & Mazza,
1995; Cui & Mazza, 1996; Cui, Kenaschuk, & Mazza, 1996; Naran, Chen, & Carpita, 2008;
Qian, Cui, Wu, & Goff, 2012), as well as aqueous extracted and/or chelator and alkali extracted
dietary fibres from whole flaxseed meal (Goh, Pinder, Hall, & Hemar, 2006; Ray et al., 2013).
Our study discovered that the kernel of flaxseed contained about 20% (w/w) of dietary fibres
(Ding, et al., 2012), which has not been previously reported. In this chapter, five separate FKDF
fractions were obtained using a modified sequential extraction and fractionation method, and
their chemical composition, molecular weight distribution, monosaccharide ratio, as well as
physicochemical characteristics (rheological properties and surface activities) were investigated.
2.2. Methodology
2.2.1. Materials
Flaxseed kernels (~70% purity) were obtained by a patented de-hulling technique (Cui &
Han, 2006) from Natunola Health Inc. (Ontario, Canada); then, pure flaxseed kernels (>99.9%
This chapter has been published as Soluble Polysaccharides from Flaxseed Kernel as a New Source of Dietary Fibres:
Extraction and Physicochemical Characterization (Ding et23
al., 2014).
purity) were collected through further sieving and cleaning. The cultivar of flaxseed was CDC
Sorrel. All chemicals were of reagent grade.
2.2.2. Extraction and fractionation procedure
The extraction and fractionation procedures for FKDF fractions followed that of Dusterhoft,
Voragen, & Engels (1991), Cui, Mazza, Oomah, & Biliaderis (1994), and Selvendran & Stevens
(1985) with modification as shown in Fig. 2.1.
Figure 2.1. Extraction and fractionation procedure of FKDF fractions
(FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides.)
24
2.2.3. Determinations of sugar, uronic acid, dietary fibre, starch, protein, ash, and moisture
Sugar content was determined by phenol-H2SO4 method (Dubois, Gilles, Hamilton, Rebers,
& Smith, 1956) with glucose solutions as standards. Total uronic acid content was determined by
m-hydroxyphenyl colorimetric method (Blumenkrantz & Asboe-Hansen, 1973) with glucuronic
acid and galacturonic acid (1:1) solution as standards. Dietary fibre content was measured by
using a Megazyme total dietary fibre test kit (Megazyme International Ireland, Wicklow, Ireland)
based on AOAC Methods 2009.01 and 2011.25 (McCleary et al., 2010; McCleary et al., 2012).
Total starch content was measured by using a Megazyme total starch test kit (Megazyme
International Ireland, Wicklow, Ireland) based on the method of McCleary, Gibson, & Mugford
(1997). Nitrogen content was determined by NA2100 Nitrogen and Protein Analyzer (Thermo
Quest, Milan, Italy), and then protein content was obtained by a conversion factor of 5.41
(Oomah, Kenaschuk, Cui, & Mazza, 1995). The ash and moisture content was determined
according to the methods of AOAC 930.05 and 934.01, respectively (AOAC, 2000). All
chemical analysis was completed in triplicate.
2.2.4. Scanning electron microscope (SEM)
The de-oiled flaxseed kernels were sputter-coated with 20~30nm of gold by an Anatech
hummer VII Sputter Coater (Alexandra, VA) under vacuum; then the fixed samples were
observed and photographed via a Hitachi S-570 Scanning Electron Microscope (Hitachi S-570
SEM, Tokyo, Japan) at different scales.
2.2.5. Molecular weight distribution
25
Molecular weight distribution was analyzed by high performance size-exclusion
chromatography (HPSEC) system (Shimadzu Scientific Instruments Inc., Maryland, US) with
multiple detectors: a UV detector (Viscotek 2600), a refractive index (RI) detector, a differential
pressure viscometer, a right angle laser light scattering detector and a low angle laser light
scattering detector (Viscotek TDA 305). The column systems combined two AquaGel PAA-M
columns and a PolyAnalytik PAA-203 column (Polyanalytik Canada, London, Canada) in series.
Detectors were calibrated by pullulan standards (JM Science Inc., New York, US). The eluent
was 0.1 M NaNO3 with 0.05% (w/w) NaN3 at a flow rate of 0.6 mL/min, and the columns and
detectors were maintained at 40 °C. The total volume and void volume of the columns were 34.9
mL and 14.2 mL, respectively. Data were analyzed by OmniSEC 4.6.1 software.
2.2.6. Monosaccharide composition analysis
Monosaccharide composition analysis followed that of Cui, Wood, Blackwell, & Nikiforuk
(2000) with minor modification. Polysaccharide samples (around 2 mg) were hydrolyzed in 1
mL 1 M H2SO4 at 100ºC for 2 h, and then diluted 20 times with Milli-Q water. The diluted
samples were filtered and analyzed on a high performance anion exchange chromatography
(HPAEC) system equipped with a pulse amperometric detector (PAD) (Dionex-5500, Dionex
Corporation, California, US). Each sample was washed through a CarboPac PA1 (250 ×4 mm
I.D.) column with 100-300 mM NaOH as gradient eluents at a flow rate of 1.0 mL/min.
Post-column eluent was 600 mM NaOH solution at a flow rate of 0.5 mL/min. Six target
26
monosaccharides (fucose, rhamnose, arabinose, galactose, glucose, and xylose) at various
concentrations were chosen as standards. Each sample was measured in triplicate.
2.2.7. Fourier transform infrared spectroscopy (FTIR) analysis
FKDF fractions were analyzed by a FTS 7000 FT-IR spectrometer with a deuterated
triglycine sulfate (DTGS) detector and a Golden-gate diamond single reflectance attenuated total
reflectance (ATR) cell (Digilab Inc., Massachusetts, US). Each spectrum was collected at
absorbance mode from 1800 to 800 cm−1 at a resolution of 4 cm−1 with128 co-added scans.
2.2.8. Surface tension analysis
Surface tension was determined by Du Nouy ring method (Du Nouy, 1919) with Fisher
Surface Tensiomat (model 21, Fisher Scientific, Toronto, Canada). FKDF fractions were
dissolved in Milli-Q water with the concentration 0.01% - 1.5% (w/v) by constant stirring for 5 h
in 50 mL beakers (PyrexTM, Ontario, Canada) at 25 °C. Solutions were kept steady for 1 h to
reach equilibrium before tests, which were repeated five times.
2.2.9. Rheological properties determination
Rheological properties were analyzed on a ARES rheometer (TA Instrument Inc., Delaware,
US) with a cone and plate geometry (4° cone angle, 50 mm diameter, and 0.047 mm gap). FKDF
fractions were dissolved in Milli-Q water with various concentrations by constant stirring for 5 h
at 25 °C. FKDF fractions were also dissolved in 0.001, 0.01, 0.1, and 1.0 M CaCl2 solutions with
1% (w/v) concentration by constant stirring for 5 h at 25 °C. The temperature was fixed at the
measuring temperature (15, 25, 35, 45, and 55 °C) and equilibrated for 5 min for each
27
rheological test. The steady test was carried out at shear rate from 0.1 to 500 s-1, and dynamic
frequency sweep test was carried out at frequency from 1 - 20 Hz with 2% (w/v) concentration at
25 °C. The applied strain (5%) for frequency sweep test was confirmed within the linear
viscoelastic region (LVR) by strain sweep test. All tests were measured in duplicate.
2.3. Results and discussion
2.3.1. Extraction, fractionation and chemical composition analysis
Chemical composition of flaxseed kernel (FK) and de-oiled FK (cultivar: CDC Sorrel) is
shown in Table 2.1. There was a high content of flaxseed oil (~44%, w/w) followed by protein
(~26%, w/w), and total dietary fibres (~20%, w/w). The separate anatomic parts of whole
flaxseed were observed by scanning electron microscopy (SEM) in Fig. 2a. It was composed of
hull (36~40%, w/w), endosperm, and kernel (55~59%, w/w) (Thompson, & Cunnane, 2003;
Naran, Chen, & Carpita, 2008). The images revealed that the cells of flaxseed kernel were
arranged in a highly ordered, honeycomb-like structure with high content of oil inside the cell
(Fig. 2.2a, 2.2b). After oil extraction and enzymatic hydrolysis of protein, the remaining cell wall
material contained around 72% (w/w) total dietary fibres and 23% (w/w) protein as shown in Fig.
2.2c and 2.2d. The main supporting materials for the cell wall are cellulose and hemicelloses,
which are dietary fibres. Data in Table 2.1 also indicated that the minerals were associated with
the cell wall materials. Most of FKDF, which were linked through non-covalent and covalent
bonds with other tissue constituents, cannot be directly extracted by hot water. After being
released by chelating agent and alkaline solution from the network of the cell wall, the extracted
28
FKDF fractions were all water soluble. It is worth noting that the entangled dietary fibre
fractions (FK-EP, FK-NP, FK-KPI, & FK-KPII) were considered as insoluble dietary fibres
based on AOAC Methods 2009.01 and 2011.25 as shown in Table 2.1.
Figure 2.2. Scanning Electron Microscopy (SEM) images of whole flaxseed (a), bar
represents 100 μm; flaxseed kernel (FK) before oil extraction (b), bar represents 10 μm;
and FK after oil extraction (c, d), bar represents 100 μm (c) and 10 μm (d), respectively.
29
Table 2.1. Chemical composition of flaxseed kernel (FK) and de-oiled FK
Compounda
Content (%, w/w)
Before oil
After oil extraction and
extraction
enzymatic hydrolysis of protein
Lipid
43.77 ± 1.30
NDb
Total Dietary fibre (DF)
19.52 ± 1.56
71.96 ± 0.27
Soluble DF
4.09 ± 0.21
15.11 ± 0.44
Insoluble DF
15.43 ± 1.44
56.85 ± 0.71
Protein
26.29 ± 0.92
22.94 ± 1.01
Ash
4.20 ± 0.05
8.63 ± 1.48
Moisture
5.61 ± 0.24
NDb
a
not detected; data are given as “mean ± SD”, n=3
The chemical compositions and yields of different FKDF fractions are shown in Table 2.2.
FKDF fractions accounted for more than 60% (w/w) of total dietary fibres from flaxseed kernel,
which was calculated based on the extraction yields. As shown in Fig 2.1, the left residue
(Residue V) was mainly composed of cellulose after 4 M KOH extraction, which accounted for
16.54% (w/w) of dry de-oiled FK meal.
FK-KPI had the highest yield followed by FK-KPII, and both of them had higher
percentages of polysaccharides with no protein detected. Alkaline solution extracted fractions
(FK-NP, FK-KPI, and FK-KPII) had relatively higher content of uronic acid (~15%, w/w)
compared with FK-WP and FK-EP. Only traces of starch can be detected in FK-WP and FK-EP
(<1%, w/w). It is worth noting that glucuronic acid and galacturonic acid had much lower
absorbance values compared to other monosaccharides (xylose, arabinose, galactose, glucose,
30
etc.) from neutral sugars by Phenol-H2SO4 method at the wavelength of 490 nm (Cui, 2005).
Thus, sugar contents in Table 2.2 should be close to the neutral sugar contents of the samples.
Table 2.2. Sugar, uronic acid, starch, protein content and yield of FKDF fractions
Fraction a
Sugar (%, w/w)
Uronic acid
Starch
Protein
Yield
(%, w/w)
(%, w/w)
(%, w/w)
(%, w/w)
FK-WP
78.36 ± 4.57
4.74 ± 0.13
0.29 ± 0.03
7.72 ± 2.10
3.05 ± 0.59b
FK-EP
75.02 ± 3.82
4.25 ± 1.12
0.98 ± 0.09
8.44 ± 1.43
1.71 ± 0.38b
FK-NP
80.66 ± 6.88
14.44 ± 2.39
NDc
3.59 ± 1.85
2.57 ± 0.70b
FK-KPI
84.46 ± 8.04
14.47 ± 0.82
NDc
NDc
6.98 ± 1.07b
FK-KPII
81.48 ± 4.17
15.67 ± 0.68
NDc
NDc
5.96 ± 1.40b
a
FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides; data are given as “mean ± SD”, n=3
b
based on the dry weight of de-oiled FK meal
c
not detected
2.3.2. Molecular weight distribution of FKDF fractions
Molecular weight distribution of FKDF fractions was determined by HPSEC. As shown in
Fig. 2.3, both of the polysaccharide and protein peaks of FKDF fractions can be recognized by
refractive index detector. The peaks of protein residues in FK-WP, FK-EP and FK-NP were
recognized by UV detector at 280 nm.
31
Figure 2.3. HPSEC elution profiles of FKDF fractions (RI and UV detector)
FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides.
Based on multiple detectors, the molecular parameters of these fractions were calculated in
Table 2.3. The weight average of molecular weight (Mw) of FKDF fractions, which ranged from
486 kDa to 1660 kDa, increased with the accumulation of ionic strength and pH of the extraction
solvent. The polydispersity index (PDI) value of FKDF decreased in the order: FK-WP (3.08) >
FK-EP (2.75) > FK-NP (1.81) > FK- KPII (1.56) > FK-KPI (1.34), which showed that alkaline
solution extracted polysaccharides had narrow polydispersity.
32
Table 2.3. The weight average molecular weight (Mw), number average molecular weight
(Mn), intrinsic viscosity ([η]) and polydispersity index (Mw/Mn) of FKDF fractions
Fraction
Mw (kDa)
Mn (kDa)
[η] (dL/g)
Mw/Mn
FK-WP
486.0
157.8
3.30
3.08
FK-EP
593.7
215.9
2.58
2.75
FK-NP
704.8
390.3
2.76
1.81
FK-KPI
770.5
577.3
4.41
1.34
FK-KPII
1660
1065
5.22
1.56
FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides.
2.3.3. Monosaccharide analysis of FKDF fractions
Monosaccharide compositions of FKDF fractions were determined by HPAEC-PAD (Fig.
2.4). FKDF fractions were mainly composed of glucose (33~46%, w/w), xylose (24~32%, w/w),
galactose (17~24%, w/w) and arabinose (7~19%, w/w). Relative monosaccharide composition
analysis (Table 2.4) showed that FK-NP, FK-KPI and FK-KPII had relatively lower content of
glucose (33~34%, w/w) compared with FK-WP and FK-EP. The arabinose contents in FK-WP
and FK-EP were low (7~8%, w/w), while alkaline solution extracted fractions had relatively
higher percentage of arabinose (15~19%, w/w).
rhamnose (0.9~2.1%, w/w).
33
FKDF fractions also had trace amounts of
Figure 2.4. HPAEC of FKDF fractions (PAD detector)
(FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides)
Compared with flaxseed mucilage gum, which mainly composed of xylose (38%, w/w),
galactose (22%, w/w), rhamnose (17%, w/w), arabinose (13%, w/w), and glucose (3%, w/w)
(Qian, Cui, Wu, & Goff, 2012), FKDF fractions had extremely higher content of glucose but low
in rhamnose. The reason might be that the cell wall materials in flaxseed kernel need stronger
structure such as hemicelluloses (e.g. xyloglucans) to maintain its integrity, which might contain
higher levels of glucose. According to previous study, major cell wall polysaccharides in food
are
cellulose,
mixed-linkage
β-glucans,
arabinoxylans,
xyloglucans,
glucomannans,
galactomannans, and pectic polysaccharides (Andersson, Westerlund, & Åman, 2006). No
34
mannose was detected among FKDF fractions (Table 2.4), which excluded the presence of
glucomannan and galactomannan.
Table 2.4. Relative neutral monosaccharide composition of FKDF fractions
Fraction a
FK-WP
FK-EP
FK-NP
FK-KPI
FK-KPII
Fucose
0.83±0.09
0.48±0.16
0.68±0.04
0.80±0.10
0.81±0.02
Rhamnose
0.72±0.08
1.15±0.47
2.08±0.77
0.87±0.27
1.53±0.52
Arabinose
7.94±0.17
6.71±1.04
19.18±0.83 15.01±0.86 18.77±2.03
Galactose
23.88±0.30 20.76±1.08 20.96±0.46 17.51±0.85 17.44±3.18
Glucose
40.28±1.34 46.47±2.48 32.82±2.14 33.98±1.03 32.96±5.14
Xylose
26.34±1.05 24.42±3.00 24.29±1.69 31.84±1.35 28.50±0.46
/Monosaccharide (%, w/w)
a
FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides; data are given as “mean ± SD”, n=3
2.3.4. FTIR analysis of FKDF fractions
The FTIR spectra of FKDF fractions are presented in Fig. 2.5. Peaks in 800–1200 cm−1 are
the fingerprint regions of polysaccharides, among which the variance of five fractions indicated
35
they had different structure information. The peak observed at 1617 cm −1 (COO− asymmetric
stretching) and 1414 cm−1 (COO− symmetric stretching) of
FK-NP, FK-KPI and FK-KPII
confirmed the presence of uronic acid, which was also in agreement with the previous results of
uronic acid contents (Table 2.2) and monosaccharide composition (Table 2.4).
Figure 2.5. FTIR spectra of FKDF fractions
(FK-WP: flaxseed kernel water-extracted polysaccharides; FK-EP: flaxseed kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPI: flaxseed kernel 1M KOH-extracted polysaccharides; FK-KPII: flaxseed kernel 4M
KOH-extracted polysaccharides)
2.3.5. Surface activities of FKDF fractions
Soluble polysaccharides are widely added to oil/water emulsions to stabilize the system
(Dickinson, 2009). In order to evaluate the surface activity of FKDF fractions, the surface
tension at different concentration was investigated and the results are presented in Fig. 2.6.
Compared with the surface tension of pure water at the air/water interface (72.8 mN/m), FKDF
36
fractions showed the ability to reduce the surface tension of water. In the concentration range of
0.01% - 1.50% (w/v), surface tension decreased in the order: FK-KPI >> FK-WP > FK-NP >
FK-EP > FK-KPII. Among them, FK-KPI exhibited the best surface activities even though there
was no protein detected (Fig. 3). The surface tension of 0.5% (w/v) FK-KPI solution (56.2 mN/m)
is comparable with guar gum and flaxseed gum solution at the same concentration (Huang,
Kakuda, & Cui, 2001; Qian et al., 2012). Moreover, the surface tension was reduced to 51.8
mN/m with the increase in concentration. It is also worth noting that the monosaccharide ratio of
FK-KPI and FK-KPII was similar, and the ratio of arabinose, galactose, glucose, and xylose was
around 1 : 1 : 3 : 3; however, the surface activities were totally different with each other. The
higher surface activity of FK-KPI might be caused by its hydrophobic residues from ester
linkages released by alkali hydrolysis (Cui, 2005), which contributed to its good amphiphilic
property.
Figure 2.6. Surface tension of FKDF fractions with various concentrations at 25 °C
37
2.3.6. Rheological property of FKDF fractions
Both steady shear flow and dynamic rheology of FKDF fractions are showed in Fig. 2.7a
and 2.7b, respectively. FK-WP, FK-EP, FK-KPI and FK-KPII water solutions exhibited
pseudoplastic flow behaviour, whereas FK-NP was more Newtonian-like fluid at all
concentrations. At 25 °C and shear rate of 50 s-1, FK-EP had the lowest viscosity among FKDF
fractions; FK-KPI and FK-KPII had relatively higher viscosity, which was around 0.02 Pa∙s at
1% (w/v) concentration. It might be due to their higher Mw and intrinsic viscosity as shown in
Table 2.3. The viscosities of FK-KPI and FK-KPII were close to that of flaxseed mucilage gum
(Qian et al., 2012), while their viscosities were much lower than that of most commercial gums
(e.g. xanthan gum, fenugreek gum, guar gum, and locust bean gum, etc.), which ranged from
0.32~0.67 Pa∙s under similar test conditions (Fabek, 2011).
Dynamic rheological properties by frequency sweep test are presented in Fig. 2.7b. Both of
G’ (storage modulus) and G’’ (loss modulus) were increased and became closer with the increase
of frequency; however, crossover points were still not observed when frequency was as high as
20 Hz, which indicated that 2% (w/v) FKDF water solution showed viscoelastic behaviour.
The effects of temperature (Fig. 2.8) and ionic strength on FKDF fractions were also
evaluated (Fig. 2.9) for the potential application in food system. As shown in Fig. 2.8, the
apparent viscosity of 1% (w/v) FKDF solution decreased, but the pseudoplastic flow behaviour
was still maintained with the increase of temperature. It might be due to enhanced polymer chain
flexibility at higher temperature.
38
Divalent salt (Ca2+) caused a decrease of apparent viscosity in FK-EP and FK-KPI solution,
and 0.001 M CaCl2 solution had the lowest viscosity among all tested solutions. It demonstrated
that FK-EP and FK-KPI had weak binding ability of free Ca2+ ions, and the existed ions
decreased the charge repulsion in aqueous solution. On the contrary, Ca2+ increased the apparent
viscosity of FK-WP and FK-NP solution, which indicated a relatively stronger binding ability of
free Ca2+ ions. The apparent viscosity of FK-KPII decreased in 0.001 M CaCl2 solution, while
gradually increased when increasing the ion concentration to 0.1 M, and then decreased in higher
ion concentration. The fluctuation on the apparent viscosity might be due to the mixture of
neutral hemicellulose as well as a lower percentage of charge polysaccharide in FK-KPII, which
showed totally different rheological behaviors under different ion concentrations.
Figure 2.7. Steady shear flow curves of FKDF fractions under different concentrations at
25 °C (a), and dynamic rheology by frequency sweep test of 2% (w/v) FKDF fractions at
25 °C (b).
39
Figure 2.8. Steady shear flow curves of 1% FKDF fractions at different temperatures
Figure 2.9. Steady shear flow curves of 1% FKDF fractions with different concentrations of
Ca2+ at 25 ⁰C
2.4. Conclusions
Five FKDF fractions were obtained with the Mw ranging from 486 kDa to 1660 kDa. SEM
images revealed that FKDF fractions were mostly in the supporting structure of the cell walls.
Monosaccharide compositions varied among FKDF fractions. All FKDF fractions contained
40
higher levels of glucose but low in rhamnose. Alkaline solution extracted FKDF fractions had
higher Mw and higher percentage of arabinose compared with aqueous and EDTA extracts. All
FKDF fractions showed the ability to reduce the surface tension of water, among which FK-KPI
exhibited the highest surface activity making it a potential emulsifier and stabilizer in oil/water
emulsions. The effects of temperature and ionic strength on rheological properties of FKDF
fractions were also evaluated. The low viscosity of flaxseed kernel dietary fibres favours the
fortification of soluble dietary fibres in relatively larger quantities to related foods (beverage,
bakery and dairy products, etc.) for exerting health benefits as well as maintaining good
organoleptic characteristics. Those findings could be useful for their potential application in
related products.
41
CHAPTER
3.
STRUCTURAL
CHARACTERIZATION
OF
RG-I
BRIDGE-LINKED ARABINANS FROM FLAXSEED KERNEL CELL
WALL
3.1. Introduction
Flaxseed (Linum usitatissimum L.) is rich in polyunsaturated fatty acids, which are mostly
located in flaxseed kernel (two cotyledons and embryo) (Vaughan, 1970). In our previous study,
flaxseed kernel was found to contain ~20% (w/w) of dietary fibres, which were mostly in the
supporting structure of the cell walls providing a natural but tight encasement to protect the oil
inside (Cui, 2012; Chapter 2). Five flaxseed kernel dietary fibre (FKDF) fractions were
sequentially extracted, and the physicochemical properties were characterized. Among all FKDF
fractions, 1 M KOH extracted flaxseed kernel polysaccharide (FK-KPI) fraction had the highest
yield and best surface activity (Chapter 2). In order to better understand the cell wall structure of
flaxseed kernel and its roles on the protection of polyunsaturated fatty acids, as well as to
provide a theoretical basis for the interpretation of the physicochemical and physiological
functionalities of FKDF, FK-KPI fraction was chosen as a representative to elucidate the detailed
structures of flaxseed kernel cell wall polysaccharides. The present study focused on the
molecular structures of FK-KPI ammonium sulphate supernatant fraction (KPI-ASF) using
methylation/GC-MS, NMR spectroscopy, and MALDI-TOF-MS analysis techniques.
This chapter has been published as Arabinan-rich Rhamnogalacturonan-I from Flaxseed Kernel Cell Wall (Ding et al., 2015).
42
3.2. Experimental
3.2.1. Materials
Flaxseed kernel 1 M KOH extracted polysaccharides (FK-KPI) was obtained from
sequential extraction as described in 2.2.2. The cultivar of flaxseed was CDC Sorrel. All
chemicals were of reagent grade unless otherwise specified.
3.2.2. Further fractionation of FK-KPI fractions
Two polysaccharide fractions were isolated from FK-KPI by sequentially using 60% (w/v)
ammonium sulphate precipitation as FK-KPI ammonium sulphate supernatant fraction
(KPI-ASF), and gradual ethanol precipitation as FK-KPI ethanol precipitated fraction (KPI-EPF)
as shown in Fig. 3.1.
Figure 3.1. Fractionation of FK-KPI
a
based on the dry weight of FK-KPI
43
3.2.3. Total uronic acid analysis, molecular weight distribution, monosaccharide
composition, and total phenol content determination of KPI-ASF
Total uronic acid analysis, molecular weight distribution, monosaccharide composition, and
have been described in 2.2.3, 2.2.5, and 2.2.6. Briefly, total uronic acid content was determined
by m-hydroxyphenyl colorimetric method (Blumenkrantz & Asboe-Hansen, 1973). Molecular
weight distribution was analyzed by a HPSEC system, and monosaccharide composition was
analyzed by a HPAEC system. Total phenolic content of KPI-ASF was measured by
Folin-Ciocalteu method (Singleton, Orthofer, and Lamuela-Raventós, 1999), which was
expressed as mg gallic acid equivalent per gram dry weight (mg GAE/g DW).
3.2.4. Reduction of uronic acids and methylation/ GC-MS analysis
KPI-ASF was dissolved in deuterium oxide (D2O) and reduced by sodium borodeuteride
(NaBD4) by labelling each reduced uronic acid residue with two deuterium nuclei as KPI-ASF
after reduction (KPI-ASF-R). Both KPI-ASF and KPI-ASF-R were analyzed by methylation/
GC-MS. The procedure of reduction and methylation followed that of previous work (Taylor and
Conrad, 1972; Ciucanu and Kerek, 1984; Cui, 2005). Briefly, 5~10 mg KPI-ASF was dissolved
in 2 mL D2O, and then gradually reduced by NaBD4. After dialysis and vacuum drying, the
reduced sample and untreated sample (2~3 mg each) were dissolved in 0.5 mL anhydrous
DMSO. Around 20 mg dry NaOH powder was added in each vial, and the test solutions were
kept stirring for 3 h. Methyl iodide (0.3~0.6 mL) was added into the solution, stirring for
additional 2.5 h. After removing the salt and drying, the partially methylated samples were
44
hydrolyzed by 0.5 mL 4 M trifluoroacetic acid (TFA) for 6 h at 100 ºC, and TFA was evaporated
by nitrogen. Then, Milli-Q water (0.3 mL), 1% NH4OH (around 3 drops), and NaBD4 (1~5 mg)
were added in each vial, stirring for 12 h at room temperature. Methanol was utilized to remove
the generated Barium, and the following acetylation procedure involved adding 0.5 mL acetic
anhydride in each vial for 2 h at 100 ºC. Finally, a few drops of ethanol were added to stop the
acetylation, and the samples were dried by nitrogen. The resultant partially methylated alditol
acetates (PMAA) were dissolved in CH2Cl2, and injected into a GC-MS system (ThermoQuest
Finnigan, San Diego, CA) with an SP-2330 (Supelco, US) column (30 m x 0.25 mm x 0.2 μm,
160 ºC -210 ºC at 2 ºC/ min, and then 210 ºC -240 ºC at 5 ºC/ min) equipped with an ion trap MS
detector.
3.2.5. Nuclear magnetic resonance (NMR) spectroscopy
KPI-ASF was partially hydrolysed by 0.1 M trifluoroacetic acid (TFA) for 1.5 h at 100 °C,
and then precipitated by 3 volumes of ethanol and freeze-dried as KPI-ASF TFA Hydrolysis
(KPI-ASF-H). KPI-ASF (80 mg) was dissolved in 4 mL D2O (99.9 atom %D), and KPI-ASF-H
(80 mg) was dissolved in 4 mL dimethyl sulfoxide-d6 (DMSO-d6, 99.9 atom %D). They were
freeze-dried (repeated three freeze-thaw cycles), and then transferred into 5 mm NMR tubes. The
1D and 2D NMR spectra were recorded at 600.10 MHz on a Bruker AVIII 600 NMR
spectrometer (Bruker, Germany). The spectra of correlation spectroscopy (COSY), total
correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC) of
KPI-ASF, as well as 1H and nuclear overhauser effect spectroscopy (NOESY) of both KPI-ASF
45
and KPI-ASF-H were collected at 295 K. Trimethylsilyl propionate (TSP) in D2O, and
tetramethylsilane (TMS) in DMSO-d6 were used as chemical shift standards.
3.2.6. MALDI-TOF-MS analysis
The matrix chosen for MALDI-TOF-MS analysis was 2,5-Dihydroxybenzoic (DHB) acid,
which was dissolved in 1 mL 20% (v/v) ethanol solution. KPI-ASF-H0.5h and KPI-ASF-H1.5h
(0.1 M TFA hydrolysis of 0.5 h and 1.5 h) were re-suspended in 1 mL matrix solution, and dried
on a stainless steel plate MALDI prior to analysis. The sample/ matrix mixtures were allowed to
air-dry, and were analyzed in a Bruker Reflex III equipped with a 337 nm nitrogen laser (Bruker,
Germany). Samples were analyzed in positive ion modes scanning from 0 - 10000 m/z using ion
suppression up to 500 m/z. The ion sources 1 and 2 were held at 20 kV and 16.35 kV,
respectively.
The guiding lens voltage was set up at 9.75 kV, and the reflector detection gain
was 5.3 with pulsed ion extraction at 200 ns. Laser energy was equal to ~7 mJ per laser shot.
Spectra were collected from an average of at least 100 shots.
3.3. Results and discussion
3.3.1. Fractionation and chemical composition analysis of FK-KPI
Although FK-KPI had the lowest polydispersity index among five FKDF fractions, it was
composed of more than two different kinds of polysaccharide. FK-KPI was fractionated by
sequential ammonium sulphate precipitation and ethanol precipitation. The two major fractions
were separated based on their different monosaccharide composition as shown in Table 3.1. The
46
yield of KPI-EPF and KPI-ASF was 51.2% (w/w) and 24.4% (w/w), respectively. Since the
structural information and functionality of KPI-EPF, as well as the analysing methods, were
different from that of KPI-ASF, the structural characterization of KPI-EPF will be discussed in
Chapter 4.
Table 3.1. Monosaccharide composition of KPI-EPF, KPI-ASF, KPI-ASF-R, and
KPI-ASF-H
Fractiona
Monosaccharide (mol%b)
Fucose
Rhamnose
Arabinose
Galactose
Glucose
Xylose
Total uronic acid (%, w/wd)
a
KPI-EPF
KPI-ASF
0.93±0.42
0.56±0.28
4.78±2.45
15.40±0.75
44.57±1.73
33.76±0.70
NDc
2.71±0.67
64.24±4.26
11.86±0.59
3.41±2.33
17.78±2.02
6.38±1.51
14.59±0.17
KPI-ASF-R
NDc
5.44±1.33
54.07±1.03
19.92±1.08
3.10±0.14
17.47±0.64
KPI-ASF-H
0.42±0.02
2.48±0.16
4.12±0.11
32.48±0.63
4.02±0.06
56.48±0.88
KPI-EPF: ethanol precipitated fraction of FK-KPI (flaxseed kernel 1 M KOH-extracted
polysaccharides); KPI-ASF: ammonium sulphate supernatant fraction of FK-KPI; KPI-ASF-R:
KPI-ASF after reduction; KPI-ASF-H: KPI-ASF after TFA hydrolysis; bNeutral monosaccharide
was determined by HPAEC-PAD and expressed as relative molar percentage; cND: not detected.
d
Total uronic acid contents of KPI-EPF and KPI-ASF were determined by m-hydroxyphenyl
colorimetric method.
47
3.3.2. Methylation/ GC-MS analysis of KPI-ASF
As KPI-ASF had 14.59 ± 0.17% (w/w) of uronic acid (Table 3.1), the linkage information
could be lost due to the resistance of uronic acid to TFA hydrolysis during methylation analysis.
Thus, the reduction of carboxyl group in KPI-ASF was necessary before methylation analysis.
Based on the GC-MS profiles of PMAA, linkage types of KPI-ASF and KPI-ASF-R can be
deduced. The relative retention time of different PMAAs, as well as the molar percentage of each
monosaccharide can also be utilised to confirm the deduced linkage types.
Linkage types of KPI-ASF and KPI-ASF-R are listed in Table 3.2. The dominate sugar
residues of KPI-ASF were (1,5)-linked-arabinofuranose (19~20 mol%), (1,3,5)-linkedarabinofuranose (18~21 mol%), terminal arabinofuranose (~20 mol%), and (1,2,3,5)-linkedarabinofuranose (~10 mol%). The linkages of (rhamnogalacturonan-I) RG-I backbone, including
(1,2)-linked-rhamnopyranose, (1,2,4)-linked-rhamnopyranose, (1,4)-linked-galacturonic acid,
(1,3,4)-linked-galacturonic acid, can be distinguished when comparing the differences in molar
percentage with and without reduction. The total molar percentages of different monosaccharides
from the methylation/ GC-MS analysis were also confirmed by monosaccharide composition
results using high performance anion exchange chromatography (Table 3.1).
The molar percentage of terminal arabinofuranose and total branching points of
arabinofuranose were ~20 mol% and ~30 mol%, respectively. The molar ratio of terminal
arabinofuranose from GC-MS could be underestimated. The reason might be that terminal
arabinofuranose was relatively easily hydrolysed and destroyed by acid during methylation.
48
Instead of terminal arabinofuranose, phenolic compounds could also be ester-linked to C-2 of
arabinose (Kroon & Williamson, 1996; Levigne, Ralet, Quemener, & Thibault, 2004), which
might cause the molar ratios of total non-reducing terminal residues lower than total branching
points.
Comparing with KPI-ASF and KPI-ASF-R, it was found that the molar percentage of
(1,2,3,5)-linked-arabinofuranose
was
decreased
due
to
the
destruction
of
terminal
arabinofuranose and/ or phenolic ester linkage during reduction process. The molar percentage of
galactopyranose was increased by 8 mol% after reduction, which was derived from the
galacturonic acid units. Rhmnopyranose (4~6 mol%) could be linked with galacturonic acid to
form RG-I. According to previous research, arabinan, galactan, and arabinogalactan could be
linked with RG-I through O-4 of the rhamnopyranosyl residues (Albersheim et al., 1996), and
arabinoxylans might also be covalent linked with RG-I (Tan et al. 2013). Neutral sugar residues
of
xylopyranose,
such
as
terminal
xylopyranose,
(1,4)-linked-xylopyranose,
(1,2,4)-linked-xylopyranose, could be contributed by arabinoxylans and/or xyloglucans. After
the reduction, the molar ratios of terminal xylopyranose and (1,3,4)-linked-galactopyranose were
both increased, which might be released from the galacturonic acid units of xylogalacturonan
(XG).
49
Table 3.2. Linkage type and molar percentage of KPI-ASF and KPI-ASF-R based on
Methylation/ GC-MS analysis
PMAAb
2,3,5-Me3-Araf
T-Araf-(1→
2,3-Me2-Araf
→5)-Araf-(1→
2-Me-Araf
→3,5)-Araf-(1→
Araf-(OAc)5
→2,3,5)-Araf-(1→
Arabinose
KPI-ASFc,
mol%e
19.78
19.34
18.25
11.78
69.15
KPI-ASF-Rd,
mol%e
12.67
20.06
20.60
1.01
54.34
0.913
1.313
3,4-Me2-Rhmp
→2)-Rhmp-(1→
3-Me-Rhmp
→2,4)-Rhmp-(1→
Rhamnose
2.69
1.47
4.16
3.55
2.46
6.01
1.496
1.716
1.976
2.087
2,3,6-Me3-Galp
→4)-Galp-(1→
2,6-Me2-Galp
→3,4)-Galp-(1→
6-Me-Galp
→2,3,4)-Galp-(1→
2,4-Me2-Galp
→3,6)-Galp-(1→
Galactose
4.84
NDf
2.34
1.44
8.62
7.96
6.20
0.56
1.90
16.62
0.756
1.208
1.593
2,3,4-Me3-Xylp
2,3-Me2-Xylp
3-Me-Xylp
Xylose
T-Xylp-(1→
→4)-Xylp-(1→
→2,4)-Xylp-(1→
4.10
8.10
1.18
13.38
13.06
5.01
0.78
18.85
1.527
2.002
2,3,6-Me3-Glcp
→4)-Glcp-(1→
2,3-Me2-Glcp
→4,6)-Glcp-(1→
Glucose
2.43
2.28
4.71
2.79
1.39
4.18
RTa
0.615
1.124
1.477
1.689
a
Linkage type
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; b PMAA: partially methylated alditol
acetate; c KPI-ASF: ammonium sulphate supernatant fraction of FK-KPI (flaxseed kernel 1 M
KOH-extracted polysaccharides); d KPI-ASF-R: KPI-ASF after reduction; e molar percentage of
each PMAA was based on the percentage of its peak; f ND: not detected.
50
3.3.3. NMR analysis of KPI-ASF
The deduced linkage types of KPI-ASF were confirmed by 1D/ 2D NMR spectroscopy, and
the anomeric configuration can be determined. Compared with literature data (Cordeiro et al.,
2012; Cardoso, Silva & Coimbra, 2002; Cardoso et al., 2007; Dourado, Cardoso, Silva, Gama &
Coimbra, 2006; Gomez et al., 2009; Habibi, Mahrouz & Vignon, 2005; Jones, Milne, Ashford &
McQueen-Mason, 2003), four signals with higher abundance (5.03, 5.08, 5.10, and 5.13 ppm)
were
assigned
to
the
H-1
of
(1,5)-linked-α-ʟ-Arabinofuranose
(A5),
(1,3,5)-linked-α-ʟ-Arabinofuranose (A35), (1,2,3,5)-linked-α-ʟ-Arabinofuranose (A235), and
terminal α-ʟ-Arabinofuranose (AT). Other three signals at low field (4.65, 4.96, and 5.01 ppm)
were assigned to (1,4)-linked-β-D-Galactopyranose (G4), (1,4)-linked-α-D-Galacturonic acid
(GA4), and (1,3,4)-linked-α-D-Galacturonic acid (GA34). Two signals at high field (1.25 and 1.29
ppm)
were
assigned
to
the
H-6
of
(1,2)-linked-α-ʟ-rhamnopyranose
(R2),
and
(1,2,4)-linked-α-ʟ-rhamnopyranose (R24), respectively (Fig. 3.2a).
The 1H and
13
C nuclear magnetic resonance chemical shifts of above mentioned sugar
residues, as well as the coupling interactions were achieved through 2D NMR spectra as shown
in Fig. 3.3 to 3.5. COSY spectra reveal the correlation of individual proton to the protons which
are two or three bonds apart (Martin, Hadden, & Russell, 2003; Cui, 2005). The correlations of
anomeric protons (H-1) to H-2 of A5, A35, A235, and AT can be recognized (Fig. 3a). AT was
chosen as a representative to label the correlations of H-2 to H-3, H-3 to H-4, H-4 to H-5 as
shown in Fig. 3.3a. Likewise, TOCSY experiment correlates all protons in a spin system (Martin
51
et al., 2003; Cui, 2005), thus the correlations of anomeric protons to other protons can be
observed. As shown in Fig. 3.3b, the total correlations of A5, AT, and G4 were recognized and
labelled. However, the correlations of A35 cannot be clearly distinguished due to the overlap in
the region.
(a)
52
(b)
Figure 3.2. 1H NMR spectra of (a) KPI-ASF (in D2O at 295 K), and (b) KPI-ASF-H (in
DMSO-d6 at 295 K)
(a)
53
(b)
Figure 3.3. Parts of (a) COSY and (b) TOCSY spectra of KPI-ASF (in D2O at 295 K)
The assignments of 1H and 13C nuclear magnetic resonance chemical shifts based on HSQC
spectra, which reveal heteronuclear correlation of protons with their neighbouring carbons
(Martin et al., 2003; Cui, 2005), are summarized in Table 3.3 and labelled in Fig. 3.4. The
complete assignments of some sugar residues, such as GA4, R2, and R24, could not be achieved
due to the low abundance. However, the H-6 signals of R2 and R24 in the high field (1.23 and
1.24 ppm) still make them easy to recognize.
54
Table 3.3. 1H/13C NMR Chemical Shifts (ppm) of sugar resides from KPI-ASF (in D2O at
295 K)
Sugar Residues
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5;H-5’/C-5 H-6;H-6’/C-6
A5
→5)-α-ʟ-Araf-(1→
5.08/107.8
4.08/82.2
4.12/79.8
4.20/82.2
3.93; -/66.1
A35
→3,5)-α-ʟ-Araf-(1→
5.10/107.4
4.28/79.6
4.08/82.5
4.30/81.5
3.88; -/66.4
A235
→2,3,5)-α-ʟ-Araf-(1→
5.15/107.1
4.32/82.2
4.03/84.1
4.23/82.2
3.78; -/66.7
AT
T-α-ʟ-Araf-(1→
5.12/107.1
4.08/81.8
3.94/76.5
4.02/83.4
3.82; 3.70/62.3
R2
→2)-α-ʟ-Rhmp-(1→
-/-
4.16/77.5
3.91/68.8
3.51/72.2
-/-
1.23; -/16.6
R24
→2,4)-α-ʟ-Rhmp-(1→
-/-
4.16/77.5
3.91/68.8
3.68/82.0
-/-
1.24; -/16.6
GA4
→4)-α-D-GalpA-(1→
4.96/92.6
3.86/68.8
4.12/70.0
- /-
-/-
-; -/175.6
G4
→4)-β-D-Galp-(1→
4.65/104.5
3.66/71.5
3.76/72.6
4.21/78.5
3.70/74.5
3.54;3.63/62.4
Previous studies were mainly focused on structural elucidation of neutral arabinans from
different plant origins (Capek et al., 1983; Navarro, Cerezo & Stortz, 2002; Vignon, Heux,
Malainine & Mahrouz, 2004; Zykwinska et al., 2006; Simkovic, Uhliarikova, Yadav & Mendichi,
2010; Mandal et al., 2011). The reason might be: (1) chemical shifts of RG-I backbone units
were difficult to be achieved due to low abundance; (2) neutral arabinans had been released as
free polysaccharides. In order to lower the NMR signals of arabinose residues (54~69 mol%),
KPI-ASF was partially hydrolysed by 0.1 M TFA to remove the arabinan side-chains. As shown
in Fig. 3.2b, the signals of 1H spectrum in the anomeric region of arabinose residues (5.0~5.2
55
ppm) in KPI-ASF-H were much lower than the signals in Fig. 3.2a, and the H-1 of R2, R24, GA4,
GA34 and xylose residues can be identified.
Figure 3.4. Parts of HSQC spectra of KPI-ASF (in D2O at 295 K)
NOESY spectra of both KPI-ASF and KPI-ASF TFA Hydrolysis (KPI-ASF-H) are shown
in Fig. 3.5. Observed NOE contacts from anomeric protons (H-1) are summarized in Table 3.4.
NOESY provides information on the 1H-1H correlation through space via nuclear overhauser
effect (distance within 5 Å), meanwhile, the intra- and inter-residue connectivities could be
observed (Cui, 2005). As shown in Fig. 5a, arabinan backbone was confirmed by the cross point
of A5 (H-1) and A35 (H-5), as well as the cross point of A235 (H-1) and A5 (H-5). RG-I
backbone was confirmed by the cross point of GA4 (H-1) and H-2 in R2 & R24. The cross point
of A235 (H-1) and R24 (H-4) revealed that arabinans might directly linked with RG-I backbone
56
through A235. After removing arabinan side-chains by acid hydrolysis, there were stronger
correlation signals of rhamnose and galacturonic acid residues (Fig. 3.5b). The cross points at
low field (5.56 ppm) were assigned to the 1H-1H correlation of O-3 acetylated galacturonic acid
residues (Komalavilas & Mort, 1989).
According to the methylation/GC-MS results, some terminal xylopyranose residues were
released from the galacturonic acid (Table 3.2). We hypothesized that those terminal
xylopyranose
residues
could
be
released
from
xylogalacturonan
(XG)
from
rhamnogalacturonan-II (RG-II). However, there was no direct evidence from 1D/2D NMR
spectra due to the low abundance.
Further investigation is still required to confirm the
existence of XG.
(a)
(b)
Figure 3.5. Parts of NOESY spectra of (a) KPI-ASF (in D2O at 295 K), and (b) KPI-ASF-H
(in DMSO-d6 at 295 K)
57
Table 3.4. Observed Nuclear Overhauser Effect (NOE) contacts from anomeric protons of
KPI-ASF (in D2O at 295 K), and KPI-ASF-H (in DMSO-d6 at 295 K)
Anomeric proton
Glycosyl residue
δH*/δH** (ppm)
→2,3,5)-α-ʟ-Araf-(1→
5.17 / ND
235
A
T-α-ʟ-Araf-(1→
5.15 / ND
T
A
NOE contact protons
Residue, Atom
3.93 / ND
A5, H-5
3.71-3.64 / ND
R24, H-4
4.32-4.28 / ND
A235, H-2;
4.08 / ND
AT, H-2; A35, H-3;
3.94 / ND
AT, H-3
3.94 / ND
AT, H-3
→3,5)-α-ʟ-Araf-(1→
5.12 / ND
4.32-4.28 / ND
A35, H-2
3.95 / ND
A5, H-5
A35
3.95 / ND
A5, H-5
→5)-α-ʟ-Araf-(1→
5.08 / ND
4.12 / ND
A5, H-3
3.95 / ND
A5, H-5
A5
3.88 / ND
A35, H-5
3.78 / ND
A235, H-5
→2,4)-α-ʟ-Rhmp-(1→
5.25 / 5.41
4.40 / ND
GA4, H-4
24
R
4.12/ 4.14
R24, H-2
→2)-α-ʟ-Rhmp-(1→
5.22 / 5.33
4.40 / 4.38
GA4, H-4
R2
4.12/ 4.14
R2, H-2
→4)-α-D-GalpA-(1→
4.96 / 4.91
4.16-4.12 / 4.13
R2, H-2; R24, H-2
GA4
4.01/ 4.06
GA4, H-3
3.89/ 3.93
GA4, H-2
→4)-β-D-Galp-(1→
4.65/ ND
4.20-4.16 / ND
G4, H-4
G4
3.82-3.76 / ND
G4, H-3
3.72-3.64 / ND
R24, H-4
*
Assignment from NOESY of KPI-ASF in D2O at 295 K;
**
Assignment from NOESY of KPI-ASF-H in DMSO-d6 at 295 K;
ND: not determined;
δH*/δH** (ppm)
3.3.4. MALDI-TOF-MS analysis of KPI-ASF
The mass spectra of KPI-ASF showed pentose-rhamnose saccharide series (e.g. 4P+Rhm,
5P+Rhm, 6P+Rhm, etc., P represents pentosyl residues, and number represents units). It can be
further confirmed by saccharide of R, R', U, and U' series in Fig. 3.6. As shown in Table 3.5, R
58
refers to pentoses linked with a RG-I disaccharide unit with mass peak [3P+Rhm+GalA+Na]+ at
742.8 m/z, and R' refers to esterified uronic acid carboxyl groups of R with mass peak
[3P+Rhm+GalA (esterified)+Na]+ at 756.0 m/z. U refers to pentose linked with two units of
rhamnose and galacturonic acid disaccharides with mass peak [3P+2Rhm+2GalA+Na]+ at 785.0
m/z, and U' refers to esterified uronic acid carboxyl groups of U with mass peak
[3P+2Rhm+2GalA (esterified)+Na]+ at 799.7 m/z. The peak-to-peak mass difference of 132 Da
was observed between neighbouring units of R, R', U, and U' series (Table 3.5), which indicated
that the branches of KPI-ASF were mainly pentoses.
Figure 3.6. MALDI-TOF-MS profile of KPI-ASF and KPI-ASF-H
(KPI-ASF-H0.5h refers to 0.1 M TFA hydrolysis of KPI-ASF for 0.5 h as the spectrum in orange;
KPI-ASF-H1.5h refers to 0.1 M TFA hydrolysis of KPI-ASF for 1.5 h as the spectrum in red;
KPI-ASF control refers to no treatment of KPI-ASF as the spectrum in green; R, R', U, and U'
series are summarized in Table 3.5)
59
Table 3.5. Saccharide series and mass peak of KPI-ASF-H in MALDI-TOF-MS spectra
Saccharide series*, mass peak (m/z)
R: [3P+Rhm+ GalA+Na]+,
742.8 m/z
R1: [1P+R] +, 873.6 m/z
R2: [2P+R] +, 1006.1 m/z
R3: [3P+R] +, 1138.4 m/z
R4: [4P+R] +, 1270.9 m/z
R5: [5P+R] +, 1403.3 m/z
R’: [3P+Rhm+GalA (esterified)
+Na]+, 756.0 m/z
U: [2P+2Rhm+2GalA
+Na]+, 785.0 m/z
U’: [2P+2Rhm+2GalA
(esterified)+Na]+, 799.7 m/z
R1’: [1P+R’] +, 887.5 m/z
U1: [1P+U] +, 916.7 m/z
U1’: [1P+U’] +, 930.6 m/z
R2’: [2P+R’] +, 1021.4 m/z
U2: [2P+U] +, 1047.6 m/z
U2’: [2P+U’] +, 1064.5 m/z
R3’: [3P+R’] +, 1152.3 m/z
U3: [3P+U] +, 1180.0 m/z
U3’: [3P+U’] +, 1195.4 m/z
R4’: [4P+R’] +, 1284.7 m/z
U4: [4P+U] +, 1312.4 m/z
U4’: [4P+U’] +, 1327.8 m/z
R5’: [5P+R’] +, 1418.7 m/z
U5: [5P+U] +, 1445.0 m/z
U5’: [5P+U’] +, 1460.9 m/z
* P represents pentosyl residue, and number represents unit; Rhm represents rhamnose, and GalA represents galacturonic acid; R
refers to pentoses linked with a RG-I disaccharide units; R' refers to esterified uronic acid carboxyl groups of R; U refers to pentose
linked with two unis of rhamnose and galacturonic acid disaccharide; and U' refers to esterified uronic acid carboxyl groups of U with
mass peaks.
60
3.3.5. Proposed structure of KPI-ASF
Based on the results of Methylation/ GC-MS, 1D/ 2D NMR, and MALDI-TOF, a possible
structure of KPI-ASF was proposed as RG-I bridge-linked arabinans as shown in Fig. 3.7. The
weight average of molecular weight (Mw) of KPI-ASF was calculated as 850 kDa. The molar
percentages of different sugar residues were quantified by methylation/GC-MS analysis (Table
3.2). The total phenol content of KPI-ASF was calculated to be 0.96 ± 0.01 mg GAE/g DW by
Folin-Ciocalteu method.
As arabinogalactans and arabinoxylans could also be linked with RG-I through covalent
bonds (Tan et al. 2013), R1 (8~12 mol%) in Fig. 3.7 could be arabinoglactans, and/or
arabinoxylans. It is worth pointing out that the molar percentage of total non-reducing terminal
arabinofuranose were less than total branching points of arabinans (Table 3.2), which might be
caused by ester-linked of phenolic compounds to C-2 of arabinose. R2 (1~12 mol%) in Fig. 3.7
could be terminal arabinose and/or ester-linked phenolic compounds. Around 9 mol% of
terminal xylopyranose residues were released from the galacturonic acid when compared with
KPI-ASF and KPI-ASF-R (Table 3.2), and xylogalacturonan (XG) has been reported to be linked
with homogalacturonan (HG) and/or RG-I (Coenen, Bakx, Verhoef, Schols, & Voragen, 2007).
Therefore, R3 (12~18 mol%) in Fig. 3.7 might be xylogalacturonan, which needs to be further
confirmed.
It is worth noting that arabinoxylans and arabinogalactans are also widely distributed in the
cell wall of plants, and those polysaccharides could be linked with pectin through covalent bonds
(Tan et al., 2013), or just be entangled physically with pectin. Some linkage types, such as
(1,2)-linked and (1,2,4)-linked rhamnopyranose in RG-I, and terminal xylopyranose in XG,
which are directly linked with galacturonic acid units, could be underestimated without reduction
61
during methylation/ GC-MS analysis. The quantification of those linkage types should be closer
to the real values after the reduction of uronic acid. However, it was found that some highly
branched linkage types, such as (1,2,3,5)-linked arabinofuranose and (1,2,3,4)-linked
galactopyranose, could be destroyed during the reduction process in our study (Table 2), and
similar results were also found in previous studies (Habibi et al., 2005; Gooneratne, Needs,
Ryden, & Selvendran, 1994). Thus, the linkage types of arabinans and the related RG-I backbone
can be finally determined and quantified when comparing the molar percentages of PMAAs with
and without reduction during methylation/GC-MS analysis.
In contrast with the rhamnogalacturonan-I in flaxseed mucilage, which had around 38%
(w/w) rhanmnose and 39% (w/w) galacturonic acid (Qian et al., 2012; Qian et al., 2013), RG-I in
flaxseed kernel was rich in neutral arabinan side-chains but low in rhanmnose (~5%, w/w) and
galacturonic acid (~15%, w/w). Arabinans could interact with water (Fenwick, Apperley,
Cosgrove, & Jarvis, 1999; Renard and Jarvis, 1999) and maintain flexibility of the cell wall by
preventing homogalacturonan polymers from forming tight associations (Jones et al., 2003).
Arabinans are often substituted with terminal phenolic esters, particularly feruloyl or coumaroyl
esters, which can dimerize oxidatively to form links between polymers (Jones et al., 2003).
Feruloyl esters may have a role in guard cell wall flexibility by providing crosslinks between
arabinans and other wall polymers. It well explains why FKDF fractions are water soluble but
cannot be directly extracted by water. Along with that the polysaccharides (e.g. arabinan-rich
pectin and xyloglucan) in the flaxseed kernel might give its cell wall structure with low water
and oil permeability (Keegstra et al., 1973). Moreover, the higher surface activity of FK-KPI
might be caused by the hydrophobic group from esters and/or attached phenolic compounds in
KPI-ASF, which contributed to the good amphiphilic property.
62
Figure 3.7. Proposed structure of KPI-ASF as RG-I bridge linked arabinans
(RG-I refers to rhamnogalacturonan-I backbone; n: 3~9 (30~90 arabinofuranose units in each
arabinan side-chain), m: 25~50 (50~100 repeating units of RG-I disaccharide), i: unclear; R1
(8~12 mol%) could be arabinoglactan and/or arabinoxylan; R2 (1~12 mol%) could be t-α-L-Araf,
and/or
phenolic
residues;
R3
(12~18
mol%)
could
be
xylogalacturonan
from
rhamnogalacturonan-II.)
3.4 Conclusion
The structure of ammonium sulphate supernatant fraction from 1 M KOH extracted flaxseed
kernel polysaccharides (KPI-ASF) was elucidated by methylation/GC-MS, NMR spectroscopy,
and MALDI-TOF-MS analysis techniques. The dominate sugar residues of KPI-ASF molecule
63
comprised of (1,5)-linked-α-ʟ-Arabinofuranose, (1,3,5)-linked-α-ʟ-Arabinofuranose, (1,2,3,5)linked-α-ʟ-Arabinofuranose, terminal α-ʟ-Arabinofuranose, and rhamnogalacturonan-I (RG-I)
repeating disaccharide units. The total phenol content of KPI-ASF was calculated as 0.96 ± 0.01
mg GAE/g DW by Folin-Ciocalteu method. A possible structure of KPI-ASF was proposed as
RG-I bridge-linked arabinans. As branched arabinans are widely distributed in the cell wall of
oilseeds and legumes, the detailed structures of KPI-ASF might help explain the roles of cell
wall polysaccharides of flaxseed kernel on the protection of polyunsaturated fatty acids.
64
CHAPTER 4. STRUCTURAL CHARACTERIZATION OF
XYLOGLUCANS AND PARTIAL FLAXSEED KERNEL CELL WALL
MODEL
4.1. Introduction
In our previous study, flaxseed kernel dietary fibres (FKDF) were sequentially extracted
from the kernel of flaxseed (Chapter 2), among which 1 M KOH extracted flaxseed kernel
polysaccharide (FK-KPI) fraction was chosen as representative to study the main structure of
FKDF. Two polysaccharide fractions were further isolated from FK-KPI as FK-KPI ammonium
sulphate supernatant fraction (KPI-ASF), and FK-KPI ethanol precipitated fraction (KPI-EPF).
The yields of KPI-ASF and KPI-EPF fractions were 24.4% (w/w) and 51.2% (w/w), respectively
(Chapter 3). The structure of KPI-ASF has been proposed as rhamnogalacturonan-I bridge-linked
arabinans (Chapter 3), which could prevent cell wall components (e.g. homogalacturonans,
cellulose fibrils) from tightly associating with each other (Jones et al., 2003), and maintain the
flexibility during cell growth, lipid storage, and water deficit stress (Moore, Farrant, & Driouich,
2008). As KPI-EPF was the major component of FK-KPI, in order to better understand cell wall
structure of flaxseed kernel and the structure-function relationship of FKDF, this chapter focused
on the detailed molecular structures of KPI-EPF using methylation/GC-MS, NMR spectroscopy,
and MALDI-TOF-MS techniques.
4.2. Experimental
4.2.1. Materials
KPI-EPF was obtained from the sequential extraction of de-oiled flaxseed kernel meal, and
further fractionation of FK-KPI fraction as described in 3.2.2. Further fractionation of other
65
FKDF fractions (FK-WP, FK-EP, FK-NP, & FK-KPII) was performed by gradual ethanol
precipitation, and purified fractions (FK-WPpf, FK-EPpf, FK-NPpf-1, FK-NPpf-2, & FK-KPIIpf)
were collected.
4.2.2. Methylation/GC-MS analysis
The procedure of methylation has been described in 3.2.4. The resultant partially
methylated alditol acetates (PMAA) were analyzed by GC-MS, and the linkage types of
KPI-EPF, FK-WPpf, FK-EPpf, FK-NPpf-1, FK-NPpf-2, and FK-KPIIpf were deduced. Degree of
branching (DB) for KPI-ASF molecule was calculated based on methylation results using the
following equation (Hawker, Lee, & Fréchet, 1991; Qian, Cui, Nikiforuk, & Goff, 2012):
DB 
NT  N B
NT  N B  N L
(4.1)
where NT, NB, and NL are the molar percentages of the terminal, branched, and linear sugar
residues, respectively.
4.2.3. Nuclear magnetic resonance (NMR) spectroscopy
KPI-EPF (80 mg) was dissolved in 4 mL D2O (99.9 atom %D) and freeze-dried (repeated
three freeze-thaw cycles), and then transferred into 5 mm NMR tubes. The 1D and 2D NMR
spectra were recorded at 600.10 MHz on a Bruker AVIII 600 NMR spectrometer (Bruker,
Rheinstetten, Germany). The spectra of 1H,
13
C, correlation spectroscopy (COSY), total
correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and
nuclear overhauser effect spectroscopy (NOESY) were collected at 295 K. Trimethylsilyl
propionate (TSP) in D2O were used as a chemical shift standards.
4.2.4. Hydrolysis of KPI-EPF and MALDI-TOF-MS analysis
KPI-EPF was hydrolysed for 24 h by endo-1,4-β-D-glucanase from Aspergillus niger
(Megazyme International Ireland, Wicklow, Ireland) as KPI-EPF-G24h. KPI-EPF was partially
66
hydrolysed by 0.1 M TFA for 1.5 h at 100 °C as KPI-EPF-H1.5h. KPI-EPF-G24h and
KPI-EPF-H1.5h were analyzed by MALDI-TOF-MS as described in 3.2.6.
4.3. Results and discussion
4.3.1. Methylation and GC-MS analysis of KPI-EPF
Based on the GC-MS profiles of PMAA, linkage types of KPI-EPF can be deduced as listed
in Table 4.1. The dominate sugar residues of KPI-EPF were (1,4,6)-linked-glucopyranose (24.1
mol%), terminal xylopyranose (16.2 mol%), (1,2)-linked-xylopyranose (10.7 mol%),
(1,4)-linked-glucopyranose (10.7 mol%), terminal galactopyranose (8.5 mol%), (1,2) or
(1,4)-linked-galactopyranose (7.4 mol%), and (1,2,3)-linked-xylopyranose (7.2 mol%).
It is worth noting that the PMAAs of 2-linked galactopyranosyl units were symmetrical
equivalents of 4-linked units, thus they cannot be separated chromatographically (Carpita & Shea,
1988), and further confirmation by NMR spectroscopy is necessary. There were also trace
terminal fucopyranose residues (0.36 mol%) detected. Other sugar residues (T-Araf, 3-Xylp,
3-Galp, 6-Galp, 2,3-Galp, and 3,4-Galp), which could be from other mixed or covalent-linked
polysaccharide contaminants, were also listed in Table 4.1.
The molar ratios of total non-reducing terminal residues and total branching points from
dominate sugar residues of KPI-EPF were 28.5 and 31.2%, respectively, which agreed well with
each other. The degree of branching (DB) value for KPI-EPF molecule was calculated to be 0.63
based on Equation (4.1). As “DB = 0” represents linear chain without any branch, and “DB = 1”
represents fully branched structure, the DB value of KPI-EPF indicated that it exhibited a
branched structure.
67
Table 4.1. Linkage type and molar percentage of KPI-EPF based on Methylation/ GC-MS
analysis
RTa
PMAAb
Linkage type
KPI-EPFc (mol%d)
0.761
2,3,4-Me3-Xylp
T-Xylp-(1→
16.20
1.118
2,4-Me2-Xylp
→3)-Xylp-(1→
6.17
1.215
3,4-Me2-Xylp
→2)-Xylp-(1→
10.69
1.612
4-Me-Xylp
→2,3)-Xylp-(1→
7.18
Xylose
40.24
1.537
2,3,6-Me3-Glcp
→4)-Glcp-(1→
10.66
2.041
2,3-Me2-Glcp
→4,6)-Glcp-(1→
24.05
Glucose
34.71
1.077
2,3,4,6-Me4-Galp
T-Galp-(1→
8.49
1.398
2,4,6-Me3-Galp
→3)-Galp-(1→
1.38
1.474
3,4,6-Me3-Galp
→2)-Galp-(1→
7.39
1.657
2,3,4-Me3-Galp
→6)-Galp-(1→
0.75
1.712
4,6-Me3-Galp
→2,3)-Galp-(1→
1.14
1.757
2,6-Me3-Galp
→3,4)-Galp-(1→
2.05
Galactose
0.618
2,3,5-Me3-Araf
21.20
T-Araf-(1→
3.48
Arabinose
0.725
2,3,4-Me3-Fucp
3.48
T-Fucp-(1→
Fucose
a
0.36
0.36
b
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; PMAA: partially methylated alditol
acetate;
c
KPI-EPF: ethanol precipitation fraction of FK-KPI (flaxseed kernel 1 M
KOH-extracted polysaccharides);
d
molar percentage of each PMAA was based on the
percentage of its peak area.
68
4.3.2 NMR analysis of KPI-EPF
The deduced linkage types of KPI-EPF based on GC-MS profiles of the PMAAs was
confirmed by 1D/2D NMR spectroscopy, and their anomeric configurations was deduced.
Compared with literature data (Košťálová, Hromádková, Berit, & Ebringerová, 2014; Wang,
Salazar, Zabotina, & Hong, 2014; Lucyszyn, Lubambo, Ono, Jó, De Souza, & Sierakowski,
2011), five signals at low field (5.25, 5.17, 5.15, 5.10, and 4.95 ppm) in Fig. 4.1 were assigned to
the
1
H
of
terminal
α-ʟ-Fucopyranose
(FT),
(1,2,3)-linked-α-D-Xylopyranose
(X23),
(1,2)-linked-α-D-Xylopyranose (X2), terminal α-L-Arabinofuranose (AT), and terminal
α-D-Xylopyranose (XT), respectively.
Figure 4.1. 1H spectrum of KPI-EPF (in D2O at 295 K)
69
The signal at high field (1.26 ppm) was assigned to the 6H of terminal α-ʟ-Fucopyranose
(FT-6). The signals at the range of 4.7~4.5 ppm were assigned to anomeric proton of galactose
and
glucose
residues,
which were
(1,2)-linked-β-D-Galactopyranose
(Gal2), terminal
β-D-Galactopyranose (GalT), (1,4)-linked-β-D-Glucopyranose (Glc4), and (1,4,6)-linked-β-DGlucopyranose (Glc46), respectively.
The proton chemical shifts as well as the coupling interactions were achieved through
COSY (Fig. 4.2a) and TOCSY (Fig. 4.2b) spectra. The COSY correlations of anomeric protons
(H-1) to other protons of FT, X2/X23, XT, GalT, and Glc4/Glc46 can be observed (Fig. 4.2a).
Likewise, the TOCSY correlations of X2, XT, and Glc4/Glc46 were recognized and labelled in Fig.
4.2b. The assignments of 1H and 13C NMR chemical shifts based on HSQC spectrum (Fig. 4.3)
are summarized in Table 4.2.
It is worth pointing out that some signals from branched arabinose residues were also
detected in HSQC, however, those signals were not confirmed by methylation/GC analysis and
1
H NMR spectrum. They might be contributed by minor arabinan residues from FK-KPI
ammonium sulphate supernatant fraction (KPI-ASF), which were lower than 4.8 mol% in
KPI-EPF according to its monosaccharide composition (Chapter 3). The complete assignments
of some sugar residues, such as 2H of (1,2)-linked-β-D-Galactopyranose (Gal2-2) and 6H of
(1,4,6)-linked-β-D-Glucopyranose (Glc46-6), cannot be clearly distinguished due to the overlap
in the region.
70
(a)
(b)
Figure 4.2. Parts of COSY (a), and TOCSY spectra (b) of KPI-EPF (in D2O at 295 K)
71
Figure 4.3. Parts of HSQC spectrum of KPI-EPF (in D2O at 295 K)
Table 4.2. 1H/13C NMR Chemical Shifts (ppm) of sugar resides from KPI-EPF (in D2O at
295 K)
Sugar Residues
H-1/C-1
H-2/C-2
H-3/C-3
H-4/C-4
H-5;H-5’/C-5
H-6;H-6’/C-6
FT
T-α-ʟ-Fucp-(1→
5.25/102.7
3.77/71.8
3.86/72.2
3.81/74.6
-/-
X23
→2,3)-α-D-Xylp-(1→
5.17/101.8
3.68/82.5
3.92/ -
3.78/76.0
3.73; 3.57/64.0
X2
→2)-α-D-Xylp-(1→
5.15/101.8
3.68/82.5
3.71/75.4
3.78/76.0
3.73; 3.57/64.0
AT
T-α-ʟ-Araf-(1→
5.10/110.1
4.08/83.9
3.94/79.1
4.02/86.2
3.82; 3.70/63.5
XT
T-α-D-Xylp-(1→
4.95/102.0
3.52/72.3
3.70/75.2
3.61/72.3
3.73; 3.57/64.0
Gal2
→2)-β-D-Galp-(1→
4.61/107.3
-/-
3.82/76.2
3.92/70.3
3.67/73.4
3.65; 3.72/61.7
Glc46
→4,6)-β-D-Glcp-(1→
4.57/105.2
3.39/75.6
3.72/75.9
3.67/ 79.6
3.50/74.2
-; - / -
GalT
T-β-D-Galp-(1→
4.55/107.3
3.70/74.4
3.82/76.2
3.92/70.3
3.67/73.4
3.65; 3.72/61.7
Glc4
→4)-β-D-Glcp-(1→
4.54/105.2
3.39/75.6
3.72/75.9
3.67/ 79.6
3.50/74.2
3.72; 3.91/63.6
72
1.26; -/18.7
4.3.3. MALDI-TOF-MS analysis and proposed structure of KPI-EPF
Different substitution patterns of xyloglucans were described as a single letter nomenclature
(Fry et al., 1993). From MALDI-TOF-MS analysis (Fig. 4.4), it was found that xyloglucans from
flaxseed kernel cell wall was mainly composed of XXXG, XXLG, XLLG substitution patterns,
which was in accordance with the proposed structure of xyloglucans from whole flaxseed meal
(Ray et al., 2013). The XXGG/GXXG, GXLG, GLLG, (G)XXFG, (G)XLFG substitution
patterns were also detected but low in abundance. Some possible substitution patterns, such as
GXFG, GLFG, (G)XXLLG, were also listed in Table 4.3. Due to the unspecific cleavage of the
acid to glycosidic bonds, the branches of some sugar units could be destructed. As well, the units
of other polysaccharide mixtures could also be hydrolysed, which might contributed to the mass
spectrum of KPI-EPF-H1.5h fractions, and caused false-positive results. According to
MALDI-TOF-MS analysis, terminal fucopyranose (FT) was mainly (1,2)-linked with galactose in
KPI-EPF. There was around 7.4 mol% of (1,2)-linked-β-D-galactopyranose (Gal2), however,
T-Fucp was only composed of 0.4 mol%. The reasons might be: 1). T-Fucp from the side-chains
of KPI-EPF was relatively easily destroyed by acid during methylation process, and thus its
molar percentage was underestimated according to methylation/GC-MS analysis; 2). other minor
polysaccharide mixtures in KPI-EPF, such as rhamnogalactouoran-I, arabinoxylan and/or
arabinogalactan, could contributed to the relatively higher ratio of Gal2; 3) some other sugar
residues (e.g. xylose, arabinose, etc.) could also be (1,2)-linked with galactose in KPI-EPF,
which needs to be further confirmed.
73
Figure 4.4. MALDI-TOF-MS profile of KPI-EPF
(KPI-EPF-G refers to endo-1,4-β-D-glucanase hydrolysis of KPI-EPF for 24 h as the spectrum in
orange; KPI-EPF-H1.5h refers to 0.1 M TFA hydrolysis of KPI-EPF for 1.5 h as the spectrum in
green; KPI-EPF control refers to no treatment of KPI-EPF as the spectrum in blue)
74
Table 4.3. MALDI-TOF-MS analysis and deduced subunit structures of xyloglucans from
flaxseed kernel cell wall
m/z
791.5
807.7
927.9*
953.7
969.3
1085.9
1102.8
1116.1
1133.6
1232.5
1248.3
1278.2
1395.0
1410.9
KPI-EPF-G24ha
Ion
Structurec
+
M+Na
XXG
+
M+K (weak)
XXG
+
M+Na (weak)
XSG*
+
M+Na
XXGG/GXXG
+
M+K (weak)
XXGG/GXXG
M+Na+
XXXG
+
M+K (weak)
XXXG
+
M+Na
GXLG/ GXXGG
M+K+ (weak) GXLG/ GXXGG
+
M+Na+
XXF
M+Na + XXLG/ GXXXG,
and/or M+K
and/or XXF
+
M+Na (weak)
M+Na++
M+Na
and/or M+K+
+
1441.6 M+Na (weak)
+
1547.2* M+Na (weak)
1558.9 M+Na+ (weak)
1574.1
M+Na+ +
and/or M+K
1586.0 M+Na+ (weak)
1603.2 M+Na+ (weak)
1707.5* M+Na+ (weak)
-
m/z
791.5
804.8
807.7
820.6*
937.1
953.7
969.3
983.0*
1085.9
1099.2
1102.8
1116.1
1133.6
1145.4*
1232.5
1248.3
GLLG
XXFG
XLLG/ GXXLG
and/or XXFG
1261.9
1278.2
1292.0*
1307.7*
1395.0
1410.9
GLLGG
GLXSG*
XLFG
GXLLG
and/or XLFG
GLFGG
GGLLGG
XXLLG*
-
1424.7*
1441.6
1454.4*
1470.3*
1558.9
1574.1
1586.0
1603.2
1617.7*
1707.5*
1719.9*
1736.1*
75
KPI-EPF-H1.5hb
Ion
Structurec
+
M+Na
XXG
M+Na+
FG
+
M+K (weak)
XXG
+
M+Na
GGXG*
M+Na+
XF
M+Na+
XXGG/GXXG
+
M+K
XXGG/GXXG
+
M+Na
GGLG*
M+Na+
XXXG
+
M+Na
XFG
+
M+K
XXXG
+
M+Na
GXLG/GXXGG
M+K+
GXLG/GXXGG
+
M+Na
GGLGG*
+
M+Na+
XXF
M+Na +
XXLG/
and/or M+K
GXXXG,
and/or XXF
M+Na+
GXFG/ LFG
+
M+Na
GLLG
M+Na+
GGFGG*
M+Na+
GGGLGG*
+
M+Na+
XXFG
M+Na + XLLG/ GXXLG
and/or M+K
and/or XXFG
GLFG/
M+Na+
GXFGG*
M+Na+
GLLGG
+
M+Na
GGGFGG*
M+K+
GGGFGG*
+
M+Na+
XLFG
M+Na +
GXLLG
and/or M+K
and/or XLFG
+
M+Na
GLFGG
+
M+Na
GGLLGG
+
M+Na
GGGFGGG*
M+Na+ (weak)
XXLLG*
+
M+Na
GXLFG*
+
M+K (weak)
GXLFG*
* The m/z value might be contributed by other sugar units from contaminants, and could cause the
false positive results.
a
KPI-EPF-G24h: endo-1,4-β-D-glucanase hydrolysis of KPI-EPF for 24 h.
b
KPI-EPF-H1.5h: 0.1 M TFA hydrolysis of KPI-EPF for 1.5 h.
c
G: β-D-Glcp backbone or reducing end; X: T-α-D-Xylp-(1→6)-linked with G; L: T-β-D-Galp-
(1→2)-α-D-Xylp-(1→6)-linked
with
G;
F:
T-α-ʟ-Fucp-(1→2)-β-D-Galp-(1→2)-α-D-
Xylp-(1→6)-linked with G; S: T-α-ʟ-Araf-(1→2)-α-D-Xylp-(1→6)-linked with G.
The structure of KPI-EPF was proposed as xyloglucans with XXXG, XXLG, XLLG
building units, and other possible substitution patterns (e.g. XXGG, GXXG, GXLG, GLLG,
XXFG,
and
XLFG)
as
shown
(1-4)-linked-β-D-Glucopyranosyl
in
backbone
Fig.
was
4.5.
The
calculated
substitution
as
69.3%
rate
of
the
based
on
methylation/GC-MS results. The structure of xyloglucans in various plants, including legumes,
corn, olive, tomato, lettuce, carrot, onion, pepper, liverwort, etc., had been extensively
summarised (Fry, 1989a; Kato, 2001; Vierhuis et al., 2001; Hoffman et al., 2005; Peña et al.,
2008). The aforementioned xyloglucans were extracted from the cell walls of the stems, seeds,
shoots, suspension-cultured cells, and/or other edible parts. Most xyloglucans are composed of
XXXG and XXGG-type structure, as well as XXLG and/or XXFG-type units. The side-chain
distributions of xyloglucans are both species and tissue specific, and the structure would be
modified by cell wall enzymes during cell growth (Fry, 1995; Pauly et al., 2001; Vierhuis et al.,
2001).
76
Figure 4.5. Proposed structure of KPI-EPF as xyloglucans
(The substitution rate of the backbone is 69.3%; R1 could be: T-α-D-Xylp-(1→, or none; R2
could be: T-α-D-Xylp-(1→, T-β-D-Galp-(1→2)-α-D-Xylp-(1→, or T-α-ʟ-Araf-(1→2)-α-DXylp-(1→; R3 could be: T-α-D-Xylp-(1→, T-β-D-Galp-(1→2)-α-D-Xylp-(1→, T-α-ʟ-Fucp(1→2)-β-D-Galp-(1→2)-α-D-Xylp-(1→, or none.)
4.3.4. Distribution of polysaccharides in flaxseed kernel (FK) cell walls, and partial FK cell
wall model
Based on different extracting effects of solvents during sequential extraction, the distribution
of cell wall polysaccharides (i.e. dietary fibre) was studied. To specify: 1). water at various
temperatures extracts unentangled polysaccharides, and breaks H-bonds & van der Waals
interactions to further extract some polysaccharides after physical disruption of the cell walls; 2).
chelating agent (e.g EDTA, & CDTA), which binds divalent cations (e.g. Ca2+, Mg2+, etc.), can
disrupt some ionic bonds, and extract polysaccharides mainly from middle lamella; 3). sodium
carbonate with relatively low concentration (e.g. 50 mM) at 1~20 °C has been widely utilized to
further abstract divalent cations within the cell wall matrix, and cleave some covalent bonds,
such as some ester bonds and polysaccharide-protein glycosic bonds; 4). strong alkalis at various
concentrations, such as 1~4 M NaOH or KOH solution, can further cleave covalent bonds (e.g.
polysaccharide-phenol ester linkages, and phenolic cross-linking bonds, etc.), and swell cellulose
fibrils in the cell wall (Cui, 2005; Selvendran & Stevens,1985; Dusterhoft, Voragen, & Engels,
1991). The working range and involved energy of different interaction forces have been
extensively summarized by Walstra (2002).
77
Figure 4.6. Fractionation of dietary fibres in flaxseed kernel cell walls
a
based on the dry weight of de-oiled FK meal
The distribution of polysaccharides in flaxseed kernel is presented in Fig. 4.6. Based on the
extraction yield, there were around 37% (w/w) of dietary fibres separated from de-oiled flaxseed
kernel meal, among which around 45% (w/w) was cellulose and the left residues. It is worth
pointing out that the yields were calculated based on the dry weights of final collected fractions,
and the loss rates were not considered during the sequential extraction and fractionation
processes. Thus, the content of each fraction presented by its yield (Fig. 4.6) was lower than the
real value in flaxseed kernel cell wall.
Each FKDF fraction was further separated by gradual ethanol precipitation, and purified
sub-fractions were collected and analyzed by methylation/GC-MS. The linkage types and molar
percentages of major FK-WP purified sub-fraction (FK-WPpf), major FK-EP purified
78
sub-fraction (FK-EPpf), first FK-NP purified sub-fraction (FK-NPpf-1), second FK-NP purified
sub-fraction (FK-NPpf-2), and major FK-KPII purified sub-fraction (FK-KPIIpf) were presented.
Based on the same principles when deducing the structure of KPI-ASF and KPI-EPF from
FK-KPI (Chapter 3 & this chapter), the structure of FK-WPpf was deduced as xyloglucans with
50.7 mol% backbone substitution rate (Table. 4.4). As shown in Table 4.5, the structure of
FK-EPpf was deduced as a mixture of xyloglucans (~27 mol%) and highly branched pectins with
arabinans side-chains. The total branching points of FK-EPpf were around 75 mol%. The
structure of FK-NPpf-1 was also deduced as a mixture of xyloglucans (~19 mol%) and highly
branched pectins with arabinans side-chains (Table 4.6). The total branching points of FK-NPpf-1
(~70 mol%) were lower than FK-EPpf, while FK-NPpf-1 had higher ratio of branched arabinans
(~57 mol%) than that of FK-EPpf (~40 mol%). As shown in Table 4.7, the structure of FK-NPpf-2
was deduced as RG-I bridge-linked arabinans, and the total branching points (42 mol%) were
less than FK-EPpf and FK-NPpf-1. Finally, the structure of FK-KPIIpf was deduced as a mixture
of xyloglucan (30~70 mol%), arabinans (10~18 mol%), and/or arabinoxylans (30~60 mol%)
(Table 4.8). It is worth pointing out that all purified sub-fractions accounted for 23% (w/w) to
76% (w/w) of the total weight of original five FKDF fractions (Fig. 4.6), and the mixed
polysaccharides in each fraction were not included causing the loss of some sugar linkage types.
Table 4.4. Linkage type and molar percentage of FK-WPpf based on Methylation/ GC-MS
analysis
RTa
0.798
1.199
1.277
1.686
PMAAb
2,3,4-Me3-Xylp
2,4-Me2-Xylp
3,4-Me2-Xylp
4-Me-Xylp
Xylose
Linkage type
T-Xylp-(1→
→3)-Xylp-(1→
→2)-Xylp-(1→
→2,3)-Xylp-(1→
FK-WPpfc (mol%d)
17.25
3.31
13.97
3.49
38.02
1.614
2,3,6-Me3-Glcp
→4)-Glcp-(1→
12.38
79
2.041
1.130
1.467
1.546
1.776
2,3-Me2-Glcp
→4,6)-Glcp-(1→
Glucose
2,3,4,6-Me4-Galp
T-Galp-(1→
2,4,6-Me3-Galp
→3)-Galp-(1→
3,4,6-Me3-Galp
→2)-Galp-(1→
2,3,4-Me3-Galp
→6)-Galp-(1→
Galactose
24.42
36.80
11.41
1.58
6.42
1.07
20.49
0.648
2,3,5-Me3-Araf
T-Araf-(1→
4.69
Arabinose
4.69
a
b
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; PMAA: partially methylated alditol
acetate; c FK-WPpf: purified fraction of FK-WP (flaxseed kernel water-extracted
polysaccharides); d molar percentage of each PMAA was based on the percentage of its peak
area.
Table 4.5. Linkage type and molar percentage of FK-EPpf based on Methylation/ GC-MS
analysis
RTa
0.792
1.215
PMAAb
2,3,4-Me3-Xylp
3,4-Me2-Xylp
Xylose
1.123
1.539
2,3,4,6-Me4-Galp
3,4,6-Me3-Galp
2.089
1.601
2.109
1.394
Linkage type
T-Xylp-(1→
→2)-Xylp-(1→
T-Galp-(1→
→2)-Galp-(1→
→2,3,4)-Galp-(1→/
6-Me3-Galp
→2,3,4)-GalpA-(1→
Galactose
2,3,6-Me3-Glcp
→4)-Glcp-(1→
2,3-Me2-Glcp
→4,6)-Glcp-(1→
Glucose
Rhmp-(OAc)5 →2,3,4)-Rhmp-(1→
Rhamnose
FK-EPpfc (mol%d)
2.47
3.84
6.32
1.77
0.43
31.59
33.79
6.04
12.99
19.03
0.52
0.52
Araf-(OAc)5
40.34
→2,3,5)-Araf-(1→
Arabinose
40.34
a
b
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; PMAA: partially methylated alditol
acetate; c FK-EPpf: purified fraction of FK-EP (flaxseed kernel EDTA-extracted polysaccharides);
d
molar percentage of each PMAA was based on the percentage of its peak area.
1.782
80
Table 4.6. Linkage type and molar percentage of FK-NPpf-1 based on Methylation/ GC-MS
analysis
RTa
0.788
1.262
PMAAb
2,3,4-Me3-Xylp
2,3-Me2-Xylp
Xylose
Linkage type
T-Xylp-(1→
→4)-Xylp-(1→
FK-NPpf-1c (mol%d)
0.58
2.15
2.73
1.122
2,3,4,6-Me4-Galp
2.64
1.559
2,3,6-Me4-Galp
T-Galp-(1→
→4)-Galp-(1→/
→4)-GalpA-(1→
→2,3,4)-Galp-(1→
2.078
6-Me3-Galp
Galactose
1.208
1.529
1.774
2,3-Me2-Araf
→5)-Araf-(1→
2-Me-Araf
→3,5)-Araf-(1→
Araf-(OAc)5
→2,3,5)-Araf-(1→
Arabinose
6.38
11.57
20.59
1.53
1.74
57.42
60.69
2,3,6-Me3-Glcp
→4)-Glcp-(1→
5.60
2,3-Me2-Glcp
10.39
→4,6)-Glcp-(1→
Glucose
15.99
a
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; b PMAA: partially methylated alditol
acetate; c FK-NPpf-1: purified fraction (#1) of FK-NP (flaxseed kernel Na2CO3-extracted
polysaccharides); d molar percentage of each PMAA was based on the percentage of its peak
area.
1.600
2.103
Table 4.7. Linkage type and molar percentage of FK-NPpf-2 based on Methylation/ GC-MS
analysis
RTa
0.646
1.165
1.527
1.770
PMAAb
Linkage type
2,3,5-Me3-Araf
T-Araf-(1→
2,3-Me2-Araf
→5)-Araf-(1→
2-Me-Araf
→3,5)-Araf-(1→
Araf-(OAc)5
→2,3,5)-Araf-(1→
Arabinose
FK-NPpf-2c, mol%d
7.98
33.79
22.26
13.16
77.18
0.960
3,4-Me2-Rhmp
→2)-Rhmp-(1→
Rhamnose
2.14
2.14
1.121
2,3,4,6-Me4-Galp
T-Galp-(1→
81
1.93
1.559
1.923
2.076
2.190
2,3,6-Me3-Galp
3,6-Me2-Galp
6-Me-Galp
2,4-Me2-Galp
Galactose
→4)-Galp-(1→/
→4)-GalpA-(1→
→2,4)-Galp-(1→
→2,3,4)-Galp-(1→
→3,6)-Galp-(1→
8.27
1.31
4.99
1.98
18.49
2,3-Me2-Xylp
→4)-Xylp-(1→
2.19
Xylose
2.19
a
b
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; PMAA: partially methylated alditol
acetate; c FK-NPpf-2: purified fraction (#2) of FK-NP (flaxseed kernel Na2CO3-extracted
polysaccharides); d molar percentage of each PMAA was based on the percentage of its peak
area.
1.263
Table 4.8. Linkage type and molar percentage of FK-KPIIpf based on Methylation/ GC-MS
analysis
RTa
0.647
1.055
1.537
PMAAb
Linkage type
FK-KPIIpfc (mol%d)
2,3,5-Me3-Araf
T-Araf-(1→
8.14
2,5-Me2-Araf
→3)-Araf-(1→
2.65
2-Me-Araf
→3,5)-Araf-(1→
7.64
18.43
Arabinose
0.797
1.272
1.711
2,3,4-Me3-Xylp
3,4-Me2-Xylp
3-Me-Xylp
Xylose
T-Xylp-(1→
→2)-Xylp-(1→
→2,4)-Xylp-(1→
18.78
13.45
14.30
46.53
1.00
1.537
2.041
2,3,4,6-Me4-Glcp
T-Glcp-(1→
2,3,6-Me3-Glcp
→4)-Glcp-(1→
2,3-Me2-Glcp
→4,6)-Glcp-(1→
Glucose
3.01
5.52
14.93
23.46
1.131
1.732
2,3,4,6-Me4-Galp
2,3,4-Me3-Galp
7.98
1.11
1.824
2,6-Me3-Galp
T-Galp-(1→
→6)-Galp-(1→
→3,4)-Galp-(1→/
→3,4)-GalpA-(1→
2.49
Galactose
11.58
b
RT: relative retention time, relative to 2,3,4,6-Me4-Glcp; PMAA: partially methylated alditol
acetate; c FK-KPIIpf: purified fraction of FK-KPII (flaxseed kernel 4M KOH-extracted
polysaccharides); d molar percentage of each PMAA was based on the percentage of its peak
area.
a
82
The cell wall materials from oilseed and/or legume cotyledons share some similarities
(Chapter 1 & 3), from which the entanglement of cellulose fibrils, xyloglucans, and pectins
(decorated with neutral side-chains of arabinans, galactans, and/or arabinoxylans, etc.), together
with proteins/glycoproteins play an important role to protect the polyunsaturated fatty acids
stored inside the cell, while maintain the flexibility of the cell walls during cell growth. Based on
previous research literatures (Albersheim et al., 1975; Vaughan, 1970; Keegstra et al., 1973; Bair,
1979; Fry, 1989b; Talbott, & Ray, 1992; Albersheim et al., 1996; Cosgrove, 2001; Jones et al.,
2003; O'Neill et al., 2004; Scheller et al., 2007; Tan et al., 2013; Dominguez, Nunez, & Lema,
1994; Rosenthal, Pyle, & Niranjan, 1996; Thompson & Cunnane, 2003; Ulvskov et al., 2005;
Zykwinska, Thibault, & Ralet, 2008), and current study of FK chemical composition (Table 2.1),
SEM images (Fig. 2.2), distribution of FK cell wall polysaccharides (Fig. 4.6), and all deduced
structures of FKDF sub-fractions, the partial FK cell wall model was proposed as shown in Fig.
4.7.
It is worth noting that the carbon skeletons (primary cell walls & middle lamella, as well as
secondary cell walls if present) of the cotyledons in flaxseed kernel cannot be differentiated
based on current study. The existences of arabinoxylan, xylogalacturonan (XG), arabinogalactan
(AG), and arabinogalactan-protein (AGP) in flaxseed kernel cell wall need further investigation.
More evidences are required to confirm some covalent bonds in the pectic network (i.e. HG,
RG-I, & RG-II), such as the covalent cross-linking between RG-I and arabinoxylan, as well as
the possible linkages among other neutral side-chains (arabinans, galactans, & AG) of the pectins
in the plant cell walls. The H-bonds between cellulose fibrils and xyloglucans/arabinans, as well
as phenolic ester bonds along arabinan side-chains, might play vital roles to maintain the
83
flexibility of the cell wall (Keegstra et al., 1973; Jones et al., 2003), and protect its integrity in
response to severe desiccation (Moore et al., 2008).
Figure 4.7. Partial cell wall model from the cotyledons in flaxseed kernel
(*FK-WP:
flaxseed
kernel
water-extracted
polysaccharides;
FK-EP:
flaxseed
kernel
EDTA-extracted polysaccharides; FK-NP: flaxseed kernel Na2CO3-extracted polysaccharides;
FK-KPII: flaxseed kernel 4M KOH-extracted polysaccharides; FK-KPI (flaxseed kernel 1M
KOH-extracted polysaccharides) were in the left parts of the cell walls except the cellulose fibrils;
84
the yields and ratios of all flaxseed kernel dietary fibres are presented in Fig. 4.6; FK-WP was
mainly released from unentangled polysaccharides, and cell wall polysaccharides through
H-bonds with cellulose fibrils after physical disruption of the cell walls;
**AG: arabinogalactan; AGP: arabinogalactan-protein; HG: homogalacturonan; RG-I:
rhamnogalacturonan-I; XG: xylogalacturonan; background picture is the SEM image of flaxseed
kernel after oil extraction and protease hydrolysis.)
4.4. Conclusions
Xyloglucans is widely distributed in flaxseed kernel cell wall, which was around 51.2%
(w/w) in FK-KPI fraction. Flaxseed kernel xyloglucans were mainly composed of XXXG,
XXLG, XLLG building units, as well as other possible substitution patterns (e.g. XXGG, GXXG,
GXLG, GLLG, XXFG, and XLFG). The degree of branching (DB) value for KPI-EPF molecule
was calculated to be 0.63, and the substitution rate of the backbone is 69.3%, which represented
a relatively high branched structure. Xyloglucans can be largely extracted as soluble dietary
fibres (i.e. gums) from farm wastes and food processing by-products of plant origins, and it could
be utilized as thickener, ice-crystal stabilizers, gelling agents, fat replacers, etc. for profitable
uses in food industry (Cui, Wu, & Ding, 2013). Partial cell wall model of flaxseed kernel helps
explain the distribution of FKDF in the cell walls and their roles, as well as provides theoretical
and empirical bases for further utilization of flaxseed and other oilseeds.
85
CHAPTER
5.
CONFORMATION
OF
RG-I
BRIDGE-LINKED
ARABINANS AND XYLOGLUCANS FROM FLAXSEED KERNEL CELL
WALL
5.1. Introduction
Most polysaccharides contain more than one type of glycosidic bond and/or sugar residue,
which is responsible for their diversified conformation (Cui, 2005; Qian, 2014). Non-covalent
bonds (e.g. electrostatic interactions, van der Waals forces, hydrophobic interactions), most of
which are energetically favorable to the structure, might restrict chain flexibility of
polysaccharide (Wang et al., 2005).
In Chapters 3 and 4, the structures of two major fractions of flaxseed kernel dietary fibre
have been proposed as RG-I bridge-linked arabinans (FK-ASF) and xyloglucans (FK-EPF) based
on sequential extraction, methylation/ GC-MS, NMR spectroscopy, and MALDI-TOF-MS
analysis. The conformational properties of KPI-ASF and KPI-EPF was studied and presented in
this chapter using static & dynamic light scattering (SLS & DLS) analysis techniques. The
relationship between primary structure and conformation is also discussed.
5.2. Experimental
5.2.1. Materials
KPI-ASF (i.e. RG-I bridge-linked arabinans) and KPI-EPF (i.e. xyloglucans) were obtained
through further fractionation of FK-KPI fraction as described in 3.2.2.
5.2.2. High performance size exclusion chromatography (HPSEC) and intrinsic viscosity
measurement
86
The elution profile of KPI-ASF and KPI-EPF was obtained by HPSEC system as described
in Chapters 3 and 4. The eluent was 0.1 M NaNO3 with 0.05% (w/w) NaN3 at a flow rate of 0.6
mL/min, and the columns and detectors were maintained at 40 °C. Data were analyzed by
OmniSEC 4.6.1 software, and weight average molecular weight (Mw), radius of gyration (Rg),
and intrinsic viscosity ([η]) were extracted.
The intrinsic viscosities ([η]) of KPI-ASF and KPI-EPF in different solvents were measured
using Ubbelohde capillary viscometer (NO. 75, Cannon Institution Company, USA) at 23 °C in
triplicate, which were calculated by the following equations:

t

0 t0
(5.1)
 sp   rel  1
(5.2)
 rel 
[ ]  lim
c0
 sp
 sp
(5.3)
c
 [ ]  k '[ ]2 c
(5.4)
( l n r e )l
 [ ]  k ' '[ ]2 c
c
(5.5)
c
where ηrel is relative viscosity; η and η0 are zero-shear viscosities of the solution and solvent,
which could be measured by the time it takes for the solution (t) and solvent (t0) to pass through
the Ubbelhode capillary viscometer; ηsp is specific viscosity; [η] is calculated by using the
Huggins (Equation 5.4) and Kraemer (Equation 5.5) relationships (Huggins, 1942; Kraemer,
1938), and k’ and k’’ are Huggins and Kraemer constants, respectively.
5.2.3. Light scattering
Both static light scattering (SLS) and dynamic light scattering (DLS) measurements were
conducted at 637 nm using a Brookhaven BI-200SM laser light scattering instruments including
87
a goniometer, a photomultiplier and a 128-channel BI-9000AT digital autocorrelator
(Brookhaven Instruments, NY, US). SLS measurements were carried out in the angular range of
30-150 º, and toluene was used as a reference with the Rayleigh ratio of 1.398 x 10-5 cm-1. The
solvents and sample solutions were well filtered through 0.45 μm syringe filters before analysis.
Weight average molecular weight (Mw), radius of gyration (Rg), and the second virial coefficient
(A2) were determined by SLS using the following equations:
2
2
K c
1  q Rg

1
R
M w 
3
q

  2A c
2


(5.6)
4n0 sin( 2)
4 2 n0 (dn dc)
N 0 0 4
2
K
(5.7)
0
2
(5.8)
where K is optical contrast factor; c is the polymer concentration; Rθ is the Rayleigh ratio; q is
the scattering vector for vertically polarized light; θ is scattering angle; n0 is the refractive index
of the solvent; dn/dc is the refractive index increment of the solution; N0 is Avogadro’s number,
and λ0 is the wavelength in vacuum (Debye, 1947; Zimm, 1948). The refractive index increment
(dn/dc) values of KPI-EPF in tested solutions were measured by BI-DNDC differential
refractometer (Brookhaven Instruments, NY, US).
The particle size distributions were calculated by the constrained regularization (CONTIN)
method using BIC Dynamic Light Scattering Software. Hydrodynamic radius (Rh) and the size
distribution will be determined by dynamic light scattering (DLS). Rh can be calculated using
Stokes-Einstein equation (Einstein, 1905):
D
kT
6Rh
(5.9)
where D is the diffusion constant; k is the Boltzmann’s constant; T is absolute temperature, and η
88
is the viscosity of solvent. The particle size distributions were calculated by the constrained
regularization (CONTIN) method using BIC Dynamic Light Scattering Software. Finally,
ρ-parameters can be extracted as per Burchard (2005):

Rg
(5.10)
Rh
where ρ is the structure-sensitive parameter; Rg is radius of gyration, and Rh is hydrodynamic
radius as described in Equations (5.6) and (5.9), respectively. After filtrating through 0.45 μm
syringe filters, the recovery rate (R%) of KPI-EPF in each solvent was calculated based on the
equation as below:
R% 
TS a
%
TSb
(5.11)
where TSa is the total sugar content in a given volume of solution after filtration, and TSb is the
total sugar content in a given volume of solution before filtration. Total sugar content was
determined by phenol-H2SO4 method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). All
tests were measured in duplicate at 23 ºC.
5.3. Results
5.3.1. Conformation characterization of KPI-ASF
As polysaccharides could be easily aggregated in water (Fig. 5.1A), various solvents (0.1 M
NaNO3, 6 M NH4OH, and 0.5 M NaOH) were utilized to eliminate aggregation effects in
KPI-ASF solutions. KPI-ASF has aggregates in Milli-Q water and 0.1 M NaNO3 solution (Fig.
5.1A and 5.1B); however, the lower molecular diameter of KPI-ASF in 0.5 M NaOH might be
due to the mild hydrolysis of NaOH to KPI-ASF (i.e. highly branched RG-I with arabinan
side-chains) molecules, which was also confirmed by the relatively higher polydispersity (Fig.
89
5.1D). Thus, the experimental results of KPI-ASF in Milli-Q water, 0.1 M NaNO3, and 6 M
NH4OH were chosen as representatives for further conformational characterization.
Figure 5.1. The molecular size distribution of KPI-ASF (concentration: 0.1 mg/mL,
temperature: 23 ºC) in Milli-Q water (A), in 0.1 M NaNO3 (B), in 6 M NH4OH (C), and in
0.5 M NaOH (D).
90
Figure 5.2. HPSEC elution profile of KPI-ASF
The HPSEC elution profile of KPI-ASF analyzed by multiple-detectors is presented in Fig.
5.2A, from which some conformation parameters, such as weight average molecular weight
(Mw), radius of gyration (Rg), and intrinsic viscosity, were extracted. The stability of KPI-ASF
in 6 M NH4OH within 48 h, which was stored in airtight containers, was evaluated based on
molecular weight distribution (Fig. 5.3A) and hydrodynamic radius (Rh) (Fig. 5.3B). The results
revealed that the molecular weight and Rh of KPI-ASF in 6 M NH4OH is relatively stable when
comparing with 0.5 M NaOH.
According to BI-DNDC differential refractometer, the specific refractive index (dn/dc)
values of KPI-ASF in Milli-Q water, 0.1 M NaNO3, and 6 M NH4OH were determined as
0.150±0.002 mL/g, 0.108±0.001 mL/g, and 0.104±0.004 mL/g, respectively. Based on dn/dc
values, the conformation information based on SLS & DLS, and HPSEC was summarized and
compared in Table 5.1.
91
It should be emphasized that aggregates were prone to be retained by the 0.45 μm
membrane during sample preparation, and final concentration will be changed due to the
different recovery rates of polysaccharides in selected solvents. The recovery rates were
calculated based on Equation (5.11), and final concentrations of KPI-ASF filtrates in different
solutions were determined.
(A)
(B)
Figure 5.3. Stability of KPI-ASF in 6 M NH4OH, (A) HPSEC elution profile of KPI-ASF
dissolving in 6 M NH4OH after 24 h & 48 h, and (B) hydrodynamic radius (Rh) of KPI-ASF
dissolving in 6 M NH4OH after 1 h, 2 h, 4h, 24 h, & 48 h.
92
Table 5.1. Conformational parameters of KPI-ASF using Static and Dynamic Light Scattering (SLS & DLS), and High
Performance Size Exclusion Chromatography (HPSEC)
Solvent
ρ
Intrinsic
Recovery
viscosity (dL/g)
rate (%)
1.00a
-
64.7±5.2c
0.36±0.20
(NA)
0.90a
(NA)
0.59±0.01b
(0.34d)
79.8±0.3c
6.36±0.79
1.21a
0.63±0.01b
84.4±0.7c
MW
Rh
Rg
A2
(kDa)
(nm)
(nm)
(10-4cm3mol/g2)
Water
1168±33
88.70±0.45
88.8±2.4
-5.00±0.96
0.1M NaNO3
861±7
(596.1d)
39.10±0.01
(NA)
35.3±1.0
(49.6d)
6M NH4OH
550.4±5
26.30±0.06
31.8±1.5
a
ρ value was calculated (Equation 5.10) as the ratio of Rg to Rh
b
data obtained from Ubbelohde capillary viscometer at 23 ºC
c
Recover rate was calculated (Equation 5.11) as the ratio of total polysaccharide contents after filtration to that before filtration
d
data obtained from HPSEC at 40 ºC
NA: not available
93
The Mw of KPI-ASF was calculated to be 550 kDa in 6 M NH4OH by SLS (Equations 5.6 –
5.8), and 596 kDa based on multiple-detector calibration by HPSEC. The Mw of KPI-ASF in 0.1
M NaNO3 calculated by HPSEC is comparable with the Mw in 6 M NH4OH calculated by SLS.
As there was still slight degradation of KPI-ASF in 6 M NH4OH, the absolute Mw of KPI-ASF
should ranged from 550 to 596 kDa. Radius of gyration (Rg) of KPI-ASF ranged from 31.8 to 35.3
nm, and hydrodynamic radium (Rh) ranged from 26.3 to 39.1 nm.
The intrinsic viscosity of KPI-ASF in various solvents was determined by Ubbelohde
capillary viscometer (Equations 5.1 – 5.5) at 23 ºC, and the results are also included in Table 5.1.
The intrinsic viscosity, which is inversely proportional to the density of the polymer coil
(Kulicke & Clasen, 2004), reflects how much the polymer is extended at a given chain length
(Cui, 2005). The intrinsic viscosity value ([η]) of KPI-ASF in 0.1 M NaNO3 (0.59 dL/g) was
comparable with that in 6 M NH4OH (0.63 dL/g). Based on HPSEC system, the [η] of KPI-ASF
was calculated to be 0.34 dL/g in 0.1 M NaNO3 at 40 ºC. Compared with [η] in 0.1 M NaNO3
determined by Ubbelohde capillary viscometer, the lower [η] calculated by HPSEC might be
caused by higher temperature and high shear rates from HPSEC columns.
The [η] of KPI-ASF was slightly lower than the [η] of pectins extracted from apple and
citrus at 30 ºC, which were 0.64 and 0.78 dL/g, respectively (Muhidinov et al., 2010). It is worth
pointing out that the Ubbelohde capillary viscometer method did not provide a good linear fit to
the data points of KPI-ASF in water at various concentrations. As KPI-ASF molecules carried
charges, the force of electrostatic expansion became even more dominant in diluted water
solutions leading to sudden increase of intrinsic viscosity (Podzimek, 2011; Koetz, & Kosmella,
2007), which might give a false-positive result.
94
5.3.2. Conformation characterization of KPI-EPF
Various solvents were also utilized to eliminate aggregation effects in KPI-EPF solutions.
As can be seen from Fig. 5.4, there were still aggregates in Milli-Q water (Fig. 5.4A), and 0.1 M
NaNO3 solution (Fig. 5.4B). Only mono-modal size distribution was observed in 0.5 M NaOH
solution with the apparent diameter of 75.7 nm (Fig. 5.4C). It is worth noting that KPI-EPF still
has aggregates in mild alkaline solution, such as 6 M NH4OH (data not shown). The stability of
KPI-EPF in 0.5 M NaOH within 48 h was evaluated based on HPSEC (Fig. 5.5A) and DLS (Fig.
5.5B). The results suggest that the molecular weight and Rh of KPI-EPF are stable in 0.5 M
NaOH within 48 h.
Figure 5.4. The molecular size distribution of KPI-EPF (concentration: 0.1 mg/mL,
temperature: 23 ºC) in Milli-Q water (A), in 0.1 M NaNO3 (B), and in 0.5 M NaOH (C).
95
(A)
(B)
Figure 5.5. Stability of KPI-EPF in 0.5 M NaOH, (A) HPSEC elution profile of KPI-EPF
dissolving in 0.5 M NaOH after 24 h & 48 h, and (B) hydrodynamic radius (Rh) of
KPI-EPF dissolving in 0.5 M NaOH after 1 h, 2 h, 4h, 24 h, & 48 h.
The HPSEC elution profile of KPI-EPF is presented in Fig. 5.6. According to BI-DNDC
differential refractometer, dn/dc values of KPI-EPF in Milli-Q water, 0.1 M NaNO3, and 0.5 M
NaOH were determined as 0.132±0.002 mL/g, 0.0831±0.007 mL/g, and 0.080±0.004 mL/g,
96
respectively. Based on dn/dc values, the conformation parameters of KPI-EPF were summarized
and compared in Table 5.2.
The recovery rates of EPF after filtration in milli-Q water, 0.1 M NaNO3, 0.5 M NaOH were
49.5%, 63.2%, and 86.1%, respectively. The final concentrations of KPI-EPF solutions after
filtration were determined, and the Mw of KPI-EPF was calculated to be 1506 kDa by SLS, and
1462 kDa based on multiple-detector calibration by HPSEC. The higher temperature (40 ºC) and
high shear rates from HPSEC columns might lead to the disaggregation of KPI-EPF in the
HPSEC eluent solution (0.1 M NaNO3), thus the Mw in 0.5 M NaOH calculated by SLS matched
well with that in 0.1 M NaNO3 calculated by HPSEC. Likewise, the apparent molecular weight
(MW) of the mixtures of KPI-EPF and its aggregates were also calculated by SLS as shown in
Table 5.2. As the second virial coefficient (A2) is an indicator for the thermodynamic interaction,
the positive A2 values of 0.5 M NaOH solutions indicated that the interaction between
polysaccharide molecule and solvent was larger than that between two polysaccharide molecules.
The intrinsic viscosities of KPI-EPF determined by Ubbelohde capillary viscometer are
listed in Table 5.2. As NaOH solvent (0.5 M) showed much stronger effects on breaking
hydrogen bonds than Milli-Q water and NaNO3 (0.1 M), the main chains of KPI-EPF molecules
were more flexible in 0.5 M NaOH. Thus, the intrinsic viscosity value ([η]) of KPI-EPF in 0.5 M
NaOH (2.25 dL/g) was lower than that in Milli-Q water (3.88 dL/g) and in 0.1 M NaNO3 (2.82
dL/g). Based on HPSEC system, the [η] of KPI-EPF was calculated to be 2.46 dL/g in 0.1 M
NaNO3 at 40 ºC. The relatively lower [η] calculated by HPSEC might be caused by both of
higher temperature and high shear rates from HPSEC columns, which increased the flexibility of
KPI-EPF main chains.
97
It is worth noting that [η] of KPI-EPF, which was comparable with polysaccharide fractions
from flaxseed mucilage (Qian et al., 2012), was much higher than that of KPI-ASF. The reason
might be: 1). KPI-EPF had higher Mw; 2). KPI-EPF molecule was more extended, and became
less dense than KPI-ASF molecule; 2). the coil expansion of KPI-ASF molecule decreased when
repulsive electrostatic force was shielded by ions due to the ionic groups.
Figure 5.6. HPSEC elution profile of KPI-EPF
98
Table 5.2. Conformational parameters of KPI-EPF using Static and Dynamic Light Scattering (SLS & DLS), and High
Performance Size Exclusion Chromatography (HPSEC)
Solvent
ρ
Intrinsic
Recovery
viscosity (dL/g)
rate (%)
0.66
3.88±0.002b
49.5±15.9c
-0.54±0.33
(NA)
0.82
(NA)
2.82±0.2b
(2.46d)
63.2±4.2c
3.59±0.32
1.44a
2.25±0.04b
86.1±1.9c
MW
Rh
Rg
A2
(kDa)
(nm)
(nm)
(10-4cm3mol/g2)
Water
9660±200
154.80±0.08
102.0±1.8
0.92±0.07
0.1M NaNO3
7290±180
(1462d)
113.10±0.07
(NA)
92.2±2.0
(54.4d)
0.5M NaOH
1506±53
34.20±0.11
49.4±2.2
a
ρ value was calculated (Equation 5.10) as the ratio of Rg to Rh
b
data obtained from Ubbelohde capillary viscometer at 23 ºC
c
Recover rate was calculated (Equation 5.11) as the ratio of total polysaccharide contents after filtration to that before filtration
d
data obtained from HPSEC at 40 ºC
NA: not available
99
5.4. Discussion
Radius of gyration (Rg) and hydrodynamic radius (Rh) vary characteristically with the size
and shape of the macromolecule (Burchard, 2005). The structure sensitive parameter ρ, which is
defined as the ratio of Rg to Rh (Equation 5.10), can be finally extracted. The relationship
between ρ value and molecular architecture has been extensively summarized by Burchard (2005,
2008). Generally, Rh increases with branching due to a higher segment density, and thus
branched chain will have a smaller ρ value than a linear chain.
Based on Equations (5.6) and (5.9) through SLS and DLS measurement, the ρ value range
of KPI-ASF (i.e. RG-I bridge-linked arabinans) were calculated as 0.90~1.21, and that of
KPI-EPF (i.e. xyloglucans) in 0.5 M NaOH was obtained as 1.44. The proposed schematic
conformational models of KPI-ASF and KPI-EPF are shown in Fig. 5.7, and the relationships
between Rg and Rh are also presented.
Figure 5.7. Schematic conformation of KPI-ASF and KPI-EPF
(Rg: radius of gyration; Rh: hydrodynamic radius; ρ: structure-sensitive parameter; ρ of KPI-ASF
ranged from 0.90~1.21, ρ of KPI-EPF was 1.44; the radii of shaded circles are Rh, and the radii
of the circles with solid line are Rg)
100
The ρ value of KPI-ASF is comparable with water soluble soybean polysaccharide (SSPS),
which was calculated as 1.1 (Wang et al., 2005), and both of them represents a highly branched
and more sphere-like molecule. Both of KPI-ASF and SSPS are important polysaccharides from
oilseed cell walls, and they are both pectin-like polysaccharides sharing some common
characteristics in linkage types (Table 1.1).
The conformation of KPI-EPF (i.e. xyloglucans) in 0.5 M NaOH solution, which is more
extended than pectin-like polysaccharides, was proposed as random coil with flexible linear
chain. As the aggregates of KPI-EPF molecules dominated in Milli-Q water and 0.1 M NaNO3
(Fig. 5.4), the low ρ values of KPI-EPF in these two solvents revealed a compact and globular
KPI-EPF conformation. Further investigation is also necessary to further eliminate the
degradation of KPI-ASF in the alkaline solution, while maintain as little aggregation as possible
in the solution.
5.5. Conclusions
The conformational parameters, such as weight average molecular weight (Mw), radius of
gyration (Rg), intrinsic viscosity (η), the second virial coefficient (A2), and structure-sensitive
parameter (ρ) of KPI-ASF and KPI-EPF were determined. The absolute Mw of KPI-ASF ranged
from 550 to 596 kDa, with Rg ranging from 31.8 to 35.3 nm, and Rh ranging from 26.3 to 39.1
nm. The ρ value of KPI-ASF (0.90~1.21) represents a highly branched and more sphere-like
molecular shape. The absolute Mw of KPI-EPF was calculated to be 1506 kDa by SLS, and 1462
kDa based on multiple-detector calibration by HPSEC. The ρ value of KPI-EPF (1.44) represents
a branched and flexible linear-chain conformation.
101
CHAPTER 6. SHORT-CHAIN FATTY ACID PROFILES OF FLAXSEED
DIETARY FIBRES BY IN VITRO FERMENTATION OF PIG COLONIC
DIGESTA
6.1. Introduction
Flaxseed is rich in dietary fibres (22~28%), among which rhamnogalacturonan-I (RG-I) and
arabinoxylans are the major components in flaxseed mucilage (Qian et al., 2012), and RG-I
bridge-linked arabinans and xyloglucans are the major dietary fibres in flaxseed kernel (Chapter
2, 3, & 4). The adequate intakes of dietary fibres are 25 and 38 g for adult women and men,
respectively (IOM, 2005). The healthy benefits of dietary fibres as prebiotics and bioactive
components, as well as their legally permissible claims and overlapping definitions have been
extensively discussed by Phillips (2013).
In typical western diets, around 80% of dietary fibres are non-starch polysaccharide and
resistant starch, and the others are non-digestible oligosaccharides and sugar alcohols, yielding
150~600 mmol short-chain fatty acids (SCFAs) (Wachtershauser, & Stein, 2000). SCFAs could
decrease colon pH and prevent pathogenic bacteria growing; moreover, they increase mucosal
blood flow and the colon mobility, and reduce secondary bile acid formation and osmotic
pressure (Macdonald, Singh, Mahony, & Meier, 1978; Daly, Stewart, Flint, & Shirazi-Beechey,
2001; Cummings, Rombeau, & Sakata, 2004). It was also reported that fermentative production
of butyrate from dietary fibres might decrease the inflammatory response in the colon (Rose et
al., 2007).
SCFAs produced by large intestine microbes are mainly composed of acetic acid, propionic
acid, butyric acid, valeric acid, caproic acid, and other branched fatty acids (e.g. isobutyric acid,
102
& isovaleric acid). It is worth noting that lactic acid and formic acid serve as intermediates in the
metabolism of SCFA and normally do not accumulate in the colon (Macfarlane & Gibson, 2004).
The fermentation profiles of arabinoxylans from agricultural crops (e.g. maize, rice, wheat) and
psyllium fibre have been widely studied (Rose, Patterson, & Hamaker, 2010; Elli, Cattivelli,
Soldi, Bonatti, & Morelli, 2008; Pollet, Van Craeyveld, Van de Wiele, Verstraete, Delcour, &
Courtin, 2012), and they are commonly accepted by the general public as prebiotics (Slavin,
2013b; Kolida, Tuohy, & Gibson, 2002).
There are four major soluble dietary fibre fractions from flaxseed as aforementioned, which
are acidic fraction (FG-AFG) and neutral fraction (FG-NFG) from flaxseed mucilage gum (Qian
et al., 2012), as well as RG-I bridge-linked arabinans and xyloglucans from flaxseed kernel
(Chapter 2, 3, & 4). Soluble flaxseed gum, which is the mixture of AFG and NFG, has been
evaluated using in vitro fermentation model in our research group (Ying, Gong, Goff, Yu, Wang,
& Cui, 2013); however, the detailed structure-function relationship is still unknown. As pigs are
similar to humans regarding the intestinal microbiota and metabolism of digestion system, SCFA
profiles produced by in vitro fermentation of pig colonic digesta on four major soluble flaxseed
dietary fibre fractions (e.g. AFG, NFG, KPI-ASF, & KPI-EPF) were studied with psyllium
arabinoxylans as the reference fibre. Furthermore, partial structure-function relationship of
flaxseed dietary fibres was established in this chapter.
6.2. Experimental
6.2.1. Materials
KPI-ASF (i.e. RG-I bridge-linked arabinans) and KPI-EPF (i.e. xyloglucans) were prepared
as described in 3.2.2. FG-AFG (i.e. RG-I) and FG-NFG (i.e. arabinoxylans) were obtained
through further fractionation of flaxseed mucilage gum through ion exchange column as
103
described by Qian et al. (2012). Psyllium husk was purchased from local market, and psyllium
arabinoxylans were obtained after further water extraction at 70 °C, followed by protease
hydrolysis and dialysis.
6.2.2. Molecular weight and chemical composition analysis
Total sugar, total uronic acid, protein content, molecular weight distribution, and
monosaccharide composition were determined as described in 2.2.3, 2.2.5, and 2.2.6. Briefly,
total sugar content was determined by phenol-H2SO4 method (Dubois et al., 1956). Nitrogen
content was determined by NA2100 Nitrogen and Protein Analyzer (Thermo Quest, Milan, Italy),
and then protein content was obtained by a conversion factor of 6.25. Total uronic acid content
was determined by m-hydroxyphenyl colorimetric method (Blumenkrantz & Asboe-Hansen,
1973). Molecular weight distribution was analyzed by a HPSEC system, and monosaccharide
composition was analyzed by a HPAEC system.
6.2.3. Digesta preparation
Digesta was collected from the central part of colons from three Yorkshire pigs with the
marketing body weight of approximately 60 kg. The pigs were raised at the Arkell Swine
Research Station of University of Guelph. The diet consisted of 59.84% corn, 26% soybean meal,
10% wheat, 1.18% limestone, 1.25% mono/di-calcium phosphate, 0.7% vitamin & mineral
mixture, 0.5% fat, 0.43% salt, and 0.1% lysine-HCl, and the room temperature was controlled at
20 °C. The pigs were cared based on the guidelines from Canadian Council of Animal Care
(Olfert, Cross, & Mcwilliam, 1993), and the use of pigs for this study was approved by the
Animal Care Committee of the University of Guelph. Collected digesta from the same intestinal
region of three pigs was mixed with an equal amount of digesta from each pig, and was stored at
-80°C until use.
104
6.2.4. Medium preparation
Anaerobic incubation medium (AIM) medium was prepared as per Lin et al. (2011). The
ingredients and recipe are listed in Table 6.1, and the added dietary fibres were the sole carbon
and energy source in the medium. Briefly, all ingredients except vitamins and cysteine-HCl were
mixed and dissolved with pH adjusted to 6.8. The solution was heated to boil and flushed with
mixed gas (10% H2, 10% CO2 and 80% N2) in the anaerobic chamber (Coy Laboratory Products
Inc., MI, US) prior to autoclaving for 20 min at 121 °C. Vitamin and cysteine-HCl solutions
were sterilized using 0.22 μm filters, and then flushed with mixed gas before mixing with
autoclaved medium in the anaerobic chamber.
6.2.5. Preparation of in vitro batch culture system
Dietary fibres are the sole carbon source with a concentration of 1% (w/v) in the system,
and 10% (w/v) of digesta was selected for inoculation as per Ying et al. (2013). To specify, the
digesta (0.6 g) were aliquoted into 15 mL Eppendorf tubes, and then dietary fibre samples (0.06
g) were added and mixed with 6 mL AIM, dissolving for 1 h in the anaerobic chamber. The
culture tubes were placed in incubator at 37 °C for 72 h, and culture fluid (1 mL) were collected
at 24 h, 48 h, and 72 h for further analysis. Psyllium fibre was chosen as the positive control, and
a negative control (NC) is only digesta in AIM without carbohydrate added, which was used to
quantify the original SCFAs in the pig colonic digesta at different time points. The above
operations were all conducted in the anaerobic chamber (10% H2, 10% CO2 and 80% N2). Each
treatment was performed in triplicate.
6.2.6. Measurement of short-chain fatty acids (SCFAs)
Collected samples were centrifuged at 14000 rpm, 4 °C for 10 min to precipitate bacteria
cells, and the supernatant and precipitate were separated and stored at -20 °C until further use.
105
The supernatant was filtered through a 0.22 um syringe filter for the measurement of dietary
fibre degradation and SCFAs. The filtered samples were diluted and injected into a GC-MS
system (HP6890, Agilent Technologies, US) with a SupelcoS-24108 GC column (60 m x 250
μm x 0.25 μm, Supelco, Sigma-Aldrich, US) and flame ionization detector (FID). Injector and
detector temperatures were maintained at 200 °C and 250 °C, respectively. The oven temperature
was increased from 100 °C to 200 °C at a rate of 10 °C/min, and held at 200 °C for 10 min. The
peaks were identified and quantified by comparing their retention times with volatile free acid
standard mix (Sigma-Aldrich, US) including acetic acid, propionic acid, isobutyric acid, butyric
acid, isovaleric acid, valeric acid, isocaproic acid, caproic acid, heptanoic acid with the
concentrations ranging from 1 and 10 mM. 2-Ethylbutyric acid (Sigma-Aldrich, US) was added
as an internal standard with the concentration of 5 mM. The net production of each SCFA was
calculated after subtracting the original SCFA concentration in each tested tube at 0 h. Data was
analyzed using the HPCHEM software (Agilent Technologies, US). All tests were conducted in
triplicate, and data are expressed as the mean ± standard deviation (STD). One-way analysis of
variance (ANOVA) was performed using SigmaPlot 12.5 (Systat Software Inc., US), and
differences were considered significant at p < 0.05.
106
Table 6.1. Composition of anaerobic incubation medium (AIM) in 1 L Milli-Q water
Mineral
Vitamin
Other
CaCl2
50 mg
Biotin
0.05 mg
Cysteine-HCl
1000 mg
CoCl·6H2O
2 mg
Calcium D-pantothenate
2 mg
Resazurin
1 mg
FeSO4·7H2O
20 mg
Cobalamine
0.005 mg
K2HPO4
900 mg
Folic acid
0.05 mg
MgSO4
50 mg
Nicotinamide
2 mg
MnSO4·H2O
20 mg
Para-aminobenzoic acid
0.1 mg
NaCl
900 mg
Pyridoxine-HCl
2 mg
Na2CO3
4000 mg
Riboflavin
2 mg
(NH4)2SO4
900 mg
Thiamine-HCl
2 mg
ZnSO4·7H2O
20 mg
107
6.3. Results and Discussion
6.3.1 Chemical composition and monosaccharide ratio of dietary fibres
Chemical compositions of psyllium fibre, KPI-ASF & KPI-EPF from flaxseed kernel, and
FG-AFG & FG-NFG from flaxseed mucilage are shown in Table 6.2. The total sugar contents
were all higher than 90% (w/w). FG-AFG had the highest content of uronic acid (38.7%, w/w)
followed by KPI-ASF (14.6%, w/w). Although both of FG-AFG and KPI-ASF are RG-I from
flaxseed, KPI-ASF from flaxseed kernel had much higher molar ratio of neutral arabinan
side-chains (Chapter 3). From the monosaccharide composition in Table 6.2, it can be deduced
that extracted psyllium fibre were mainly arabinoxylans with a 1 : 4 ratio of arabinose to xylose.
FG-NFG has also been demonstrated as arabinoxylans with a 1: 3.4 ratio of arabinose to xylose.
The molecular weight of psyllium arabinoxylans were comparable with flaxseed mucilage
arabinoxylans; however, they were distinctively different in rheological properties (Guo, Cui,
Wang, Goff, & Smith, 2009; Qian et al., 2012), and psyllium arabinoxylans had higher apparent
viscosities than flaxseed mucilage arabinoxylans under similar tested conditions.
6.3.2 Effect of different dietary fibres on the Production of SCFAs
The production of SCFAs by in vitro fermentation of pig colonic digesta was identified and
quantified by GC. The GC profiles of SCFA standards are presented in Fig. 6.1, and the fitting
equation based on peak areas of SCFAs at various concentrations are provided in Table 6.3.
Lactic acid and formic acid are considered as intermediate acids in the metabolism of SCFAs
(Macfarlane & Gibson, 2004), thus they were not included in the current research.
108
Table 6.2. Chemical composition and molecular weight of dietary fibre samples
Sample
Psyllium
Fibre
/ Composition (%, w/w)
KPI-ASF
KPI-EPF FG-AFGa
FG-NFGa
Protein
-
-
-
8.1%
-
Total Sugar
>90%
>90%
>90%
>90%
>90%
Uronic Acid
<1.0%
14.6%
6.4%
38.7%
1.8%
Molecular Weight
1550 kDa
1510 &
341 kDa
1470 kDa
596 kDa 1462 kDa
Monosaccharideb
Fucose
-
-
0.9%
14.7%
-
Rhamnose
0.8%
2.9%
0.6%
38.3%
-
Arabinose
18.7%
61.9%
4.2%
2.9%
20.2%
Galactose
3.6%
14.1%
16.6%
35.2%
7.9%
Glucose
-
4.0%
48.1%
-
3.7%
Xylose
76.1%
17.2%
29.7%
7.9%
68.2%
a
Qian et al., 2012
b
Relative neutral monosaccharide composition
The production of SCFAs of the cultures grown with psyllium fibre and flaxseed dietary
fibres during in vitro fermentation is presented in Table 6.4. Comparing with negative control
(NC) without carbon sources, all the cultures with added dietary fibres (1%, w/w) showed
increased levels of total SCFAs. There is no significant difference of SCFA production during 24
h fermentation. After 48 h fermentation, the acetic acid production from KPI-EPF, as well as
butyric acid production from FG-NFG, were significantly higher than NC (p<0.05). After 72 h
fermentation, the acetic acid and total SCFA production from psyllium fibre and all flaxseed
dietary fibres showed significant difference with NC. The butyric acid production from FG-NFG
109
was still significantly higher than NC after 72 h fermentation, and the propionic acid production
from FG-AFG exhibited significant difference with NC (Table 6.4).
Table 6.3. Fitting equation and R2 of SCFA standards in GC analysis
SCFA standard
Fitting Equation
R2
Acetic Acid
y = 7.373x – 2.8868
0.9630
Propionic Acid
y = 18.98x – 7.9694
0.9766
Isobutyric Acid
y = 27.303x – 9.976
0.9925
Butyric Acid
y = 26.978x – 10.565
0.9832
Isovaleric Acid
y = 33.308x – 11.188
0.9935
Valeric Acid
y = 32.853x – 12.04
0.9900
Isocaproic Acid
y = 35.349x – 12.04
0.9946
Caproic Acid
y = 35.352x – 12.545
0.9942
Heptanoic Acid
y = 37.056x – 11.736
0.9967
x: concentration of SCFA in the solution (mM)
y: peak area of SCFA in GC analysis
110
Figure 6.1. Profiles of SCFA standards in GC analysis
111
Table 6.4. SCFA production of different dietary fibres by in vitro fermentation of pig colonic digesta
SCFA
Time
Acetic Acid
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
Propionic Acid
Isobutyric Acid
Butyric Acid
Isovaleric Acid
Valeric Acid
Isocaproic Acid
Caproic Acid
NC
(Negative
Control)
24.99±3.49a
35.75±5.47b
14.08±4.72b
3.21±0.88a
8.82±1.73ab
6.25±1.23b
0.15±0.26a
0.53±0.02a
0.50±0.9a
1.31±0.35a
2.01±0.28b
1.68±0.52b
0.38±0.01ab
0.33±0.29a
0.56±0.33a
0.82±0.09a
-
Psyllium Fibre
(Arabinoxylans)
61.55±28.17a
78.73±21.81ab
56.16±12.36a
6.06±3.88a
9.75±3.60ab
7.08±4.28b
0.55±0.02a
0.52±0.001a
0.14±0.24a
3.52±1.95a
3.40±1.22ab
1.80±1.02b
0.53±0.03
0.46±0.01ab
0.11±0.2a
1.41±0.72a
1.14±0.36a
0.44±0.45
0.40±0.02
0.46±0.03
0.53±0.03
0.57±0.003
112
Concentration (mM)*
KPI-ASF
KPI-EPF
(RG-I bridge
(Xyloglucans)
linked arabinans)
44.18±5.74a
43.38±5.29a
63.01±4.37ab
80.39±13.94a
72.13±7.93a
72.85±14.87a
1.73±0.82a
1.11±0.97a
ab
6.90±3.44
5.74±1.77b
13.04±1.61ab
11.65±5.86ab
0.13±0.22a
0.14±0.23a
0.28±0.24a
0.43±0.03a
0.39±0.01a
0.16±0.27a
1.33±0.46a
1.26±0.49a
2.17±0.69ab
3.58±1.01ab
2.86±0.39b
2.55±1.94b
0.39±0.04
0.28±0.25
ab
0.43±0.05
0.48±0.04a
a
0.42±0.01
0.30±0.27a
0.59±0.19a
0.42±0.37a
0.93±0.35a
1.57±0.47a
1.17±0.25
0.95±1.06
0.39±0.02
0.39±0.03
0.41±0.02
0.26±0.23
0.46±0.01
0.45±0.02
0.49±0.03
0.48±0.01
FG-AFG
(RG-I)
55.91±18.92a
54.61±10.72ab
74.27±1.73a
6.79±3.59a
15.64±2.82a
25.95±0.36a
0.28±0.25a
0.40±0.02a
0.49±0.02a
2.13±1.50a
1.97±0.87b
3.97±0.45ab
0.25±0.22
0.24±0.21b
0.48±0.05a
0.82±0.76a
0.72±0.26a
1.97±0.42
0.25±0.21
0.47±0.03
0.46±0.01
FG-NFG
(Arabinoxylans)
39.72±0.35a
50.77±1.84ab
66.26±2.47a
4.57±0.04a
10.86±4.78ab
19.92±11.26ab
0.40±0.01a
0.43±0.01a
0.50±0.01a
2.84±0.23a
4.67±1.15a
6.81±1.82a
0.37±0.02
0.40±0.01ab
0.47±0.004a
0.83±0.01a
1.09±0.04a
1.62±0.24
0.45±0.001
0.45±0.01
Heptanoic Acid
Total SCFA
72 h
24 h
48 h
72 h
24 h
48 h
72 h
30.23±4.70a
48.30±4.53a
22.83±6.35c
0.42±0.02
0.26±0.23
0.43±0.02
74.81±34.46a
95.46±26.95a
66.15±11.55b
0.44±0.01
0.37±0.01
0.38±0.01
49.57±7.38a
74.98±9.05a
90.45±10.11ab
0.44±0.05
0.13±0.22
0.24±0.21
47.55±7.37a
93.18±16.9a
88.88±24.14ab
0.57±0.07
66.64±25.23a
74.29±14.7a
107.71±3.07a
0.47±0.01
49.18±0.21a
68.68±4.08a
96.05±15.76ab
* NC: negative control; data are expressed as the mean ± standard deviation (STD); different letters indicate significant differences
(one-way ANOVA test, p<0.05) in SCFA production at different time points.
Table 6.5. Percentage of SCFA production of different dietary fibres by in vitro fermentation of pig colonic digesta
SCFA
Acetic Acid
Propionic Acid
Isobutyric Acid
Butyric Acid
Time
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
NC
(Negative
Control)
82.90±3.88%bc
73.76±5.20%ab
60.92±4.50%c
10.51±1.58%c
18.43±4.29%ab
28.15±5.25%b
0.47±0.81%a
1.11±0.08%b
2.25±0.43%b
4.30±0.66%ab
Psyllium Fibre
(Arabinoxylans)
82.46±1.93%bc
82.57±0.48%ab
84.51±6.87%a
7.60±2.16%bc
10.04±0.98%ab
11.33±8.35%a
0.83±0.32%a
0.57±0.17%a
0.18±0.31%ab
4.55±0.74%ab
% of total SCFA*
KPI-ASF
KPI-EPF
(RG-I bridge
(Xyloglucans)
linked arabinans)
89.30±1.83%ab
91.61±4.14%a
84.41±4.68%b
86.37±0.75%b
79.76±0.71%ab
82.86±5.03%ab
3.38±1.22%ab
2.15±1.86%a
ab
8.90±3.71%
6.06±0.83%b
14.41±0.52%ab
12.64±2.88%a
0.23±0.40%a
0.27±0.47%a
0.35±0.30%a
0.47±0.06%a
0.43±0.04%ab
0.13±0.23%a
2.62±0.59%b
2.58±0.72%b
113
FG-AFG
(RG-I)
FG-NFG
(Arabinoxylans)
84.93±4.27%abc
73.54±0.16%a
68.97±0.40%b
9.72±1.86%c
21.10±0.46%a
24.10±0.35%ab
0.36±0.31%a
0.54±0.08%a
0.45±0.01%ab
2.88±1.26%b
80.77±0.37%c
74.21±6.98%a
69.93±8.64%b
9.29±0.06%c
15.58±6.05%ab
19.86±8.17%ab
0.82±0.02%a
0.63±0.05%a
0.53±0.09%ab
5.77±0.49%a
Isovaleric Acid
Valeric Acid
Isocaproic Acid
Caproic Acid
Heptanoic Acid
Total SCFA
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
4.21±0.98%b
7.43±1.26%b
0.79±0.06%b
1.25±1.08%a
1.82±0.94%a
1.71±0.23%b
100%
100%
100%
3.50±0.30%b
2.60±1.08%a
0.80±0.30%
0.51±0.16%ab
0.14±0.25%a
1.85±0.26%a
1.18±0.05%ab
0.60±0.57%
0.62±0.28%
0.52±0.18%
0.80±0.30%
0.63±0.18%
0.64±0.08%
100%
100%
100%
2.85±0.65%b
3.16±0.12%a
0.80±0.11%
0.57±0.02%ab
0.47±0.03%a
1.17±0.22%a
1.21±0.37%ab
1.28±0.14%
0.81±0.13%
0.54±0.05%
0.93±0.15%
0.66±0.04%
0.49±0.05%
100%
100%
100%
3.79±0.41%b
2.63±1.32%a
0.55±0.48%
0.52±0.06%ab
0.31±0.27%a
0.82±0.72%a
1.66±0.22%b
0.92±0.90%
0.82±0.13%
0.31±0.28%
0.95±0.13%
0.53±0.09%
0.51±0.07%
100%
100%
100%
2.56±0.68%b
3.68±0.31%a
0.31±0.27%
0.30±0.26%a
0.44±0.03%a
1.02±0.89%a
0.95±0.16%a
1.83±0.34%
0.37±0.33%
0.77±0.27%
0.64±0.12%
0.53±0.05%
100%
100%
100%
6.75±1.25%a
7.01±0.74%b
0.75±0.03%
0.59±0.05%ab
0.49±0.08%a
1.69±0.02%a
1.59±0.04%b
1.69±0.03%
0.91±0.003%
0.66±0.05%
0.49±0.07%
100%
100%
100%
* NC: negative control; data are expressed as the mean ± STD; different letters indicate significant differences (one-way ANOVA test,
p<0.05) in SCFA production at different time points.
114
Figure 6.2. Acetic acid production by in vitro fermentation of pig colonic digesta
(* indicates significant difference with NC regarding SCFA production at different time points
through one-way ANOVA test, p<0.05; NC: negative control; KPI-ASF: RG-I bridge-linked
arabinans from flaxseed kernel; KPI-EPF: xyloglucans from flaxseed kernel; FG-AFG: RG-I
from flaxseed mucilage gum; FG-NFG: arabinoxylans from flaxseed mucilage gum.)
115
Figure 6.3. Total SCFA production by in vitro fermentation of pig colonic digesta
(* indicates significant difference with NC regarding SCFA production at different time points
through one-way ANOVA test, p<0.05; NC: negative control; KPI-ASF: RG-I bridge-linked
arabinans from flaxseed kernel; KPI-EPF: xyloglucans from flaxseed kernel; FG-AFG: RG-I
from flaxseed mucilage gum; FG-NFG: arabinoxylans from flaxseed mucilage gum.)
116
The acetic acid and total SCFA production were chosen as representatives, and the
production of SCFAs was compared as shown in Fig. 6.2 and Fig. 6.3. After 24 h fermentation,
the cultures grown with psyllium fibre had the highest content of acetic acid and total SCFAs
among all samples, followed by the ones with FG-AFG and KPI-ASF. Compared with flaxseed
dietary fibres, psyllium arabinoxylans were relatively faster fermentable dietary fibres (peaked at
48 h fermentation), and showed different trends with FG-NFG even though the latter was also
composed of arabinoxylans. Both of KPI-ASF and FG-AFG showed similar trends on total
SCFA production during 72 h fermentation. The fermentability rates of KPI-EPF (i.e.
xyloglucans) were between psyllium arabinoxylans and flaxseed RG-I, while the culture grown
with KPI-EPF exhibited the highest level of acetic acid after 48 h fermentation.
The percentages of SCFAs produced during in vitro fermentation are shown in Table 6.5,
thus the influence of different dietary fibres on SCFAs can be evaluated. Acetic acid was the
major acid produced during in vitro fermentation, which accounted for 60.92~91.61% of total
SCFA production in all the cultures. KPI-EPF had the highest percentage of acetic acid
production after 24 h fermentation, which was significantly higher than NC and FG-NFG. After
48 h and 72 h fermentation, the percentages of acetic acid production were gradually decreased
(except psyllium fibre) due to the increases of other SCFAs, especially propionic acid.
The relatively higher standard deviation of SCFA production might be caused by the
uneven distribution of microbes in the digesta. Moreover, the higher viscosity of psyllium fibre
might have negative effects on the quantification of SCFAs during sample filtration. Further
117
investigation is still required with enlarged-scale in vitro batch culture systems and more
frequent sampling, as well as more digesta samples collected from the pigs at different ages and
body weights.
6.3.3. Partial structure-function relationship between dietary fibre and fermentation
profiles
Better understanding the relationship between dietary fibre structure and fermentation
profiles will further promote the development of dietary fibres or fibre mix for optimum colonic
health (Rose et al., 2007). The molecular structures of psyllium arabinoxylans and flaxseed
dietary fibres have been proposed (Chapter 3 & 4; Yin, Lin, Nie, Cui, & Xie, 2012; Qian, 2014),
which are summarized in Fig. 6.4. The molecular weight and chemical composition of those
dietary fibres have been summarized in Table 6.2.
Psyllium fibre and FK-NFG were mainly composed of arabinoxylans with comparable
molecular weight ranges and similar arabinose-to-xylose ratios, but they were distinctively
different in rheological properties. Compared with arabinoxylans from flaxseed mucilage,
psyllium arabinoxylans were relatively faster fermentable dietary fibres. Both arabinoxylans
promoted butyric acid production, and they were more effective than other tested dietary fibres
during 48 h fermentation. Moreover, butyric acid production from flaxseed mucilage
arabinoxylans still ranked the highest after 72 h fermentation due to its slower fermentability
than psyllium arabinoxylans.
118
Figure 6.4. Molecular structures of psyllium arabinoxylans (Yin et al., 2012), KPI-ASF & KPI-EPF from flaxseed kernel
(Chapter 3 & 4), and FG-AFG & FG-NFG from flaxseed mucilage (Qian, 2014)
(KPI-ASF: RG-I bridge-linked arabinans; KPI-EPF: xyloglucans; FG-AFG: RG-I; FG-NFG: arabinoxylans)
119
Both of KPI-ASF and FG-AFG contained RG-I backbones; however, the flaxseed mucilage
RG-I had much higher uronic acid content (38.7%, w/w). Extracted RG-I fraction from flaxseed
kernel were rich in neutral arabinan side-chains (> 50 mol%), but relatively lower in uronic acid
content (14.6%, w/w). They showed similar trends on acetic acid, propionic acid, and total SCFA
production, but the cultures grown with flaxseed mucilage RG-I had higher level of SCFA
production (66.64~107.71 mM) than the ones with flaxseed kernel RG-I (49.57~90.45 mM)
during 72 h fermentation. Moreover, flaxseed mucilage RG-I promoted propionic acid more
effectively than other tested dietary fibres during 72 h fermentation. As arabinans from flaxseed
kernel RG-I are often substituted with terminal phenolic esters, these substituting groups might
play some roles on the fermentation profiles, which still needs further investigation.
The fermentability rates of flaxseed kernel xyloglucans were between psyllium
arabinoxylans and flaxseed RG-I. The cultures grown with flaxseed kernel xyloglucans had the
highest percentage of acetic acid production after 24 h fermentation, and they had the highest
level of acetic acid after 48 h fermentation. Together with flaxseed RG-I (i.e. FK-ASF &
FG-AFG), all of them promoted acetic acid production more effectively than psyllium and
flaxseed mucilage arabinoxylans during 72 h fermentation.
It was reported that the changes of molecular weight and viscosity of dietary fibres affected
SCFA production (Nyman, 2003; Olano-Martin, Mountzouris, Gibson and Rastall, 2000).
Decreasing molecular weight of sugar beet arabinans decreased the production of acetic acid,
while it increased the production of propionic acid (Al-Tamimi, Palframan, Cooper, Gibson, &
120
Rastall, 2006). In addition, the branches of dietary fibres played important roles on fermentation
profiles. The cultures grown with more unsubstituted xyloses had faster fermentability rates than
the ones with more densely branched regions of arabinoxylans from maize, rice, and wheat bran
during 24 h fermentation (Rose et al., 2010).
The molecular weight and relative monosaccharide composition of dietary fibres from
different origins seem to have no significant impact on fermentation profiles based on current
research. The relationship between the colonic microbial communities and flaxseed dietary fibres
is still yet to be answered. Moreover, the effects of physicochemical properties (e.g. molecular
weight, apparent viscosity, uronic acid content, substitution, etc.) of dietary fibres with similar
molecular structure, such as pectins from different origins, on colonic microbial fermentation
require further study in order to fully understand the structure-function relationship.
6.4. Conclusions
All fibre grown cultures showed increased levels of total SCFA content. Acetic acid was the
major SCFA produced, followed by propionic acid and butyric acid during 72 h fermentation.
The acetic acid production from KPI-EPF, and butyric acid production from FG-NFG were
significantly higher than NC after 48 h fermentation. The acetic acid and total SCFA production
from all tested dietary fibres were significantly higher than NC after 72 h fermentation.
Psyllium fibre had the highest level of total SCFA after 24 h and 48 h fermentation. Flaxseed
dietary fibres were relatively slower fermentable dietary fibres as compared with psyllium fibre,
121
and all flaxseed dietary fibres had higher level of total SCFA production than psyllium fibre after
72 h incubation. Both psyllium and flaxseed mucilage arabinoxylans promoted butyric acid
production, and they were more effective than other tested dietary fibres during 48 h
fermentation. Flaxseed mucilage RG-I promoted propionic acid more effectively than other
tested dietary fibres during 72 h fermentation.
Flaxseed proved to be a promising source of dietary fibre, containing relatively slow
fermentable dietary fibres. They sustained the production of SCFAs with prolonged fermentation,
and provided more carbon sources for the microbiota in the distal regions of the colon with
physiological benefits. Current research are hoped to further promote the utilization of flaxseed
dietary fibres, as well as to optimize colonic health based on more balanced diets with both grain
and oilseed.
122
CHAPTER 7. GENERAL DISCUSSION AND CONCLUSION
Canada leads the world in producing flaxseed, and Canadian exportation covers more than
half of the global flaxseed trade. In the 2014/2015 crop year, the total flaxseed production and
exportation of Canada are estimated to be 952 and 700 kilotonnes, increasing by 17% to the
highest level since 2010 (Agriculture and Agri-Food Canada, 2014). The world production of
flaxseed is going to increase by 16% in the 2014/2015 crop year, and China has the largest
growth in imports. In the 2015/2016 crop year, the flaxseed production and exportation of
Canada are forecast to increase by 15% and 29%, respectively (Agriculture and Agri-Food
Canada, 2015).
The GRAS status of flaxseed has been confirmed by FDA in 2009, and a
cholesterol-lowering health claim of ground flaxseed has been approved (Health Canada, 2014).
As a rich source of omega-3 fatty acids (>50% of total fatty acids), flaxseed is also high in
dietary fibres but low in starch and sugars, making it an ideal plant-based ingredient in daily
vegetarian diets and nutrition therapies. When flaxseed is eaten whole, the seed coat hardly
allows the utilization of omega-3 fatty acids, dietary fibres, and protein in the kernel; however,
ground flaxseed can be easily oxidized due to the high percentage of PUFA. Thus, flaxseed
de-hulling technique is a promising way to provide healthier and more value-added flaxseed
products for the consumers.
123
Our study discovered that the kernel of flaxseed contained about 20% of dietary fibre, and
SEM images revealed that flaxseed kernel dietary fibres are mostly located in the supporting
structure of the cell walls. Cotyledons and embryo from oilseed kernel are the major oil storage
tissue in flaxseed, canola seed, and sunflower seed, etc., and their carbon skeletons were mainly
composed of dietary fibres with the storage starch largely decreasing during oilseed maturation
(Murphy, Rawsthorne, & Hills, 1993; Bewley, & Black, 1994). Besides the largest flaxseed
producing country, Canada is also the largest single producer of canola, and a large producer of
some other oilseeds (e.g. soybean, safflower, and sunflower, etc.). Adding value to the
agriculture/oilseed by-products becomes even more important nowadays due to the increasingly
global demand for environmentally-friendly bioproducts.
The relationships between structure of extracted dietary fibres (Chapters 3~5) and their
functionality (i.e. techno-functionality and biofunctionality) (Chapters 2 & 6), as well as the
relationships between functionality and application, are the key features when bridging the gap
between theoretical research of dietary fibres and market-ready products. The current research on
flaxseed kernel dietary fibres (FKDF) provides theoretical and empirical bases to explore their
commercial potential. Enhanced understanding of molecular structure, cell wall model,
physicochemical properties, and in vitro fermentation profiles are expected to promote the
value-added utilization of dietary fibres from flaxseed and other oilseeds.
The following conclusions can be derived from the present study:
124
(1) Five FKDF fractions were obtained by sequential extraction of de-oiled flaxseed kernel
meal. SEM images revealed that FKDF fractions were mostly in the supporting structure of the
cell walls. All FKDF fractions showed the ability to reduce the surface tension of water, among
which FK-KPI and FK-NP exhibited higher surface activity, making them potential emulsifiers
and stabilizers in O/W emulsions. The effects of temperature and ionic strength on rheological
properties of FKDF fractions were evaluated. The low viscosity and bland taste of flaxseed
kernel dietary fibres favours the fortification of soluble dietary fibres in relatively larger
quantities to related foods for exerting health benefits as well as maintaining good organoleptic
characteristics.
(2) The structures of two sub-fractions (KPI-ASF & KPI-EPF) from 1 M KOH extracted
flaxseed kernel polysaccharides were elucidated by methylation/GC-MS, NMR spectroscopy,
and MALDI-TOF-MS analysis techniques. A possible structure of KPI-ASF was proposed as
RG-I bridge-linked arabinans, and its distributions and linkage types in other plants were
125
summarized. As branched arabinans are widely distributed in the cell wall of oilseeds and
legumes, the detailed structures of KPI-ASF might help explain the roles of cell wall
polysaccharides of flaxseed kernel on the protection of polyunsaturated fatty acids. A possible
structure of KPI-EPF was proposed as xyloglucans with XXXG, XXLG, XLLG building units,
as well as other possible substitution patterns (e.g. XXGG, GXXG, GXLG, GLLG, XXFG, and
XLFG), where “G” = an unsubstituted β-D-glucopyranosyl unit, “X” = terminal
α-D-xylopyranosyl residue (1-6)-linked with “G”, “L” = a terminal β-D-galactopyranosyl residue
(1-2)-linked with “X”, and “F” = a terminal α-ʟ-Fucopyranosyl residue (1-2)-linked with “L”
(Fry et al., 1993).
(3) The conformation of KPI-ASF and KPI-EPF were studied for better understanding the
relationships between primary structure and conformation. The absolute Mw of KPI-ASF ranged
from 550 to 596 kDa, with Rg ranging from 31.8 to 35.3 nm, and Rh ranging from 26.3 to 39.1
126
nm. The ρ value of KPI-ASF (0.90~1.21) represents a highly branched and more sphere-like
molecular structure than KPI-EPF. The absolute Mw of KPI-EPF was calculated to be 1506 kDa
by SLS, with Rg of 49.4 nm and Rh of 34.2 nm. The ρ value of KPI-EPF (1.44) represents a
branched and flexible linear-chain conformation. The schematic conformational models of
KPI-ASF and KPI-EPF were also proposed.
(4) The cell wall materials from oilseed and/or legume cotyledons share some similarities,
from which the entanglement of cellulose fibrils, xyloglucans, and pectins (decorated with
neutral side-chains of arabinans, galactans, and/or arabinoxylans, etc.), together with
proteins/glycoproteins, play an important role to protect the polyunsaturated fatty acids stored
inside the cell, while maintaining the flexibility of the cell walls during cell growth. Partial cell
wall model of flaxseed kernel helps explain the distribution of FKDF in the cell walls and their
roles. Furthermore, it provides theoretical and empirical bases for further utilization of flaxseed
and other oilseeds.
127
(5) Results from in vitro fermentation profiles revealed that all flaxseed fibre grown cultures
showed increased levels of total short-chain fatty acid (SCFA) content, and acetic acid was the
major SCFA produced, followed by propionic acid and butyric acid during 72 h fermentation.
The acetic acid production from flaxseed xyloglucans was significantly higher than negative
control (NC) after 48 h fermentation. The acetic acid and total SCFA production from all tested
flaxseed dietary fibres were significantly higher than NC after 72 h fermentation. Flaxseed
proved to be promising dietary fibre sources, which contained relatively slowly fermentable
dietary fibres as compared with psyllium fibre. They sustained the production of SCFAs with
prolonged fermentation, and thus might provided more carbon sources for the microbiota in the
distal regions of the colon in vivo, with physiological benefits. Future studies are hoped to
128
optimize colonic health based on more balanced diets with both grain and oilseed. The
relationships between the colonic microbial communities and flaxseed dietary fibres, as well as
the effects of physicochemical properties of dietary fibres on colonic microbial fermentation, still
require further investigation.
129
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