structure-function relationship of flaxseed gum from
TRANSCRIPT
Structure-Function Relationship of Flaxseed Gum
from Flaxseed Hulls
By
Ke-Ying Qian
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
© Ke-Ying Qian, January, 2014
ABSTRACT
STRUCTRUE-FUNCTION RELATIONSHIP OF FLAXSEED GUM
FROM FLAXSEED HULLS
Ke-Ying Qian Co-advisor: Prof. H. Douglas Goff
University of Guleph, 2014 Co-advisor: Prof. Steve W. Cui
Soluble dietary fibre with low viscosity can be included in the diet in a significant
amount to show health benefits without over-texturization. Soluble flaxseed gum has
recently been the subject of increasing research attention due to its low viscosity, which is
favored as a potential fibre fortifier. In this work, soluble flaxseed gum (SFG) extracted
from flaxseed hulls was fractionated into a neutral (NFG) and an acidic (AFG) fraction
gum using ion exchange chromatography. The physicochemical properties, structure and
conformation of both fractions were investigated. The protein and uronic acid content
were 11.8 and 38.7 % in SFG, and 8.1 and 23.0 % in AFG, respectively. NFG contained
no protein or uronic acid. NFG and AFG exhibited pseudoplastic and Newtonian flow
behavior, respectively. The ranking of intrinsic viscosities (mL g-1) in a decreasing order
was: SFG (446.0) >NFG (377.5) >AFG (332.5). AFG was expected to have higher chain
flexibility due to its lower value of Huggins constant (0.16) compared to that of NFG (0.54)
and SFG (0.48). AFG had a highly branched rhamnogalacturonan-I backbone substituted
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at O-3 of →2)-α-L-Rhap-(1→ which was mostly terminated by a monomeric sugar unit
(19.6 % T-α/β-D-Galp, 4.5 % T-α-L-Fucp, and 3.1 % T-β-D-Xylp) or occasionally by a
longer side chain with a similar structure as its backbone. NFG contained β-1,4-linked
xylose backbone being mono-, di- or unsubstituted at O-2 and/or O-3 positions by one to
three linearly- linked sugar residues (24.8 % arabinose, 15.5 % xylose and 9.4 %
galactose/glucose). Static and dynamic light scattering results showed that NFG exhibited
random coil conformation with a molecular weight of 616±19 kDa. The star-like
conformation of AFG (with a molecular weight of 285±25 kDa) also coincided with the
presence of longer side chains along its rather flexible RG-I backbone. The random coil
and star-like conformation of NFG and AFG, as well as their relatively small molecular
sizes, explained the overall low viscosity of flaxseed gum extracted from flaxseed hulls.
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Acknowledgements
Firstly, I would like to thank my advisor Prof. H. Douglas Goff and my co-advisor
Prof. Steve. W. Cui for providing me the opportunity to work on this project. They both
inspired me with their constant enthusiasm and dedication to academia and their
willingness to deliver knowledge. I would like to thank them for their intellectual advices
throughout the project, and their sincerity, great tolerance and patience in helping me
overcome obstacles in experiments and writing. Special thanks are due to Prof. H. Doug
Goff for his effort on final editing of all my manuscripts, and to Prof. Steve Cui for his
insightful suggestions to my personal growth.
Sincere appreciations also extend to my committee members Prof. Milena Corredig
and Prof. Rickey Yada, who generously provided insightful suggestions and comments on
my project and this thesis, and supported me to pass my oral qualification.
I have a number of people to thank in Agriculture and Agri-Food Canada. I want
acknowledge Dr. Qi Wang, Dr. Ying Wu and Prof. Shao-Ping Nie for their knowledge and
sincere advice based on their knowledge and first-hand experience. I owe my special
thanks to technician John Nikiforuk, who put the most effort in obtaining the optimum
condition and all NMR data of my samples in this thesis, technician Ben Huang, Cathy
Wang for their technical support and willingness to share their experience, and technician
Edita Verespej (in Food Science Department, University of Guelph) for her technical
assistance. I extend my heartfelt gratitude to my friends and lab-mates, Dr. Qing-Bin Guo
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and Hui-Huang Ding. With heavy burden on their own shoulders, they took me as a
“brother” and constantly encourage and supported me in all ways throughout all adversity
regardless of my response. I wish I could have returned more. I am grateful to my
lab-mates Dr. Ji Kang, Dr. Xiao-Hui Xing and Dr. Jun-Yi Yin, and my close friend Dr.
Gui-Ying Mei for her trust and support and the memorable time we spent together.
Sincere acknowledgements also go to Prof. Fei Song, Prof. C. Bram Cadsby and their
adorable kids Mia and Benjamin, who treated me as a family member with caring respect
and trust. I thank my dear friend Jennifer Teng for her comfort and deep understanding of
my life, and my ex-roommates Xingyao Ling, Liang Li, Chen Ma, Yaning Shi for their
care and company. My heartfelt gratitude also turns to Yu Liu for sharing her genius sense
of humor and charming experiences of graduate study in her book Send You a Bullet, and
Steve Flowers and Dr. Bob Stahl for their effective treatments in regaining my confidence
and content provided in their book: Living with Your Heart Wide Open.
Endless acknowledgements extend to my kind and caring parents and elder brother
for providing me with the most freedom to push my boundaries with their unconditional
love and support.
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Table of Content ABSTRACT ....................................................................................................................... ii
Acknowledgements .......................................................................................................... iii
Table of Content ............................................................................................................... vi
List of Tables ..................................................................................................................... ix
List of Figures .................................................................................................................... x
Chapter 1. Introduction ................................................................................................... 1
1.1 Significance of soluble dietary fibre and research focus ........................................... 1
1.2 Overall objectives ...................................................................................................... 3
Chapter 2. Literature Review .......................................................................................... 5
2.1 Flaxseed, soluble flaxseed gum and their health benefit ........................................... 5
2.2 Natural occurrence and extraction of flaxseed gum .................................................. 6
2.3 Fractionation of flaxseed gum and the structure of two fractions ............................. 8
2.4 Rheological properties of flaxseed gum .................................................................... 9
2.5 Structure-function relationship ................................................................................ 10
Chapter 3. Flaxseed Gum from Flaxseed Hulls: Extraction, Fractionation and
Characterization ............................................................................................................. 14
3.1. Introduction ............................................................................................................ 14
3.2. Methodology .......................................................................................................... 16
3.2.1 Materials ............................................................................................................ 16
3.2.2 Extraction of gum .............................................................................................. 16
3.2.3 Fractionation and purification of gum ............................................................... 17
3.2.4 Physico-chemical analysis ................................................................................. 19
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3.2.5 Functional properties ......................................................................................... 22
3.3. Results and discussions .......................................................................................... 23
3.3.1 Extraction, fractionation and chemical composition ......................................... 23
3.3.2 Physical characterization ................................................................................... 26
3.3.3 Rheological properties ....................................................................................... 28
3.3.4 Functional properties ......................................................................................... 34
3.4. Conclusions ............................................................................................................ 39
Chapter 4. Structural Elucidation of the Acidic Fraction Gum ................................. 40
4.1 Introduction ............................................................................................................. 40
4.2 Experimental ........................................................................................................... 41
4.2.1 Materials ............................................................................................................ 41
4.2.3 Methylation analysis .......................................................................................... 42
4.2.3 NMR analysis ..................................................................................................... 42
4.3 Results and discussion ............................................................................................. 43
4.3.1 Methylation and GC-MS of partially methylated alditol acetate (PMAA) of the
acidic fraction gum ..................................................................................................... 43
4.3.2 1D and 2D NMR analysis of the acidic fraction gum ........................................ 45
4.4 Conclusions ............................................................................................................. 59
Chapter 5. Structure Elucidation of the Neutral Fraction Gum ................................ 60
5.1 Introduction ............................................................................................................. 60
5.2 Experimental ........................................................................................................... 61
5.2.1 Materials ............................................................................................................ 61
5.2.2 Methylation analysis .......................................................................................... 61
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5.2.3 NMR analysis ..................................................................................................... 61
5.3 Results and discussion ............................................................................................. 62
5.3.1 Methylation and GC-MS of partially methylated alditol acetate (PMAA) of the
neutral fraction gum .................................................................................................... 62
5.3.2 1D and 2D NMR analysis of the neutral fraction gum ...................................... 64
5.4 Conclusions ............................................................................................................. 75
Chapter 6. Conformation of Neutral and Acidic Fraction Gums ............................... 76
6.1. Introduction ............................................................................................................ 76
6.2. Experimental .......................................................................................................... 78
6.2.1. Materials ........................................................................................................... 78
6.2.2. Solution preparation .......................................................................................... 78
6.2.3. Light scattering measurements ......................................................................... 79
6.3. Results and discussion ............................................................................................ 79
6.3.1 The presence and elimination of aggregates ...................................................... 79
6.3.2 Static light scattering .......................................................................................... 81
6.3.3 Dynamic light scattering .................................................................................... 83
6.4. Conclusions ............................................................................................................ 86
Chapter 7. Concluding Discussion ................................................................................. 88
Reference.......................................................................................................................... 91
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List of Tables
Table 3.1. Yield and chemical components of flaxseed gum and its fractions (%) ........... 24
Table 3.2. Neutral monosaccharide in flaxseed gum and its fractions (%) ........................ 25
Table 3.3. The intrinsic viscosity ([η]), Huggins constant ( HK ), critical concentrations
(ccr) and coil overlap parameter values ((c[η])cr) at critical concentrations of SFG, NFG
and AFG ............................................................................................................................. 31
Table 4.1. Partially Methylated Aditol Acetate (PMAA) sugar residue derivatives of the
acidic fraction gum ............................................................................................................ 44
Table 4.2. 1H/13C NMR chemical shifts for the acidic fraction gum in D2O at 70oC (ppm)53
Table 4.3 Heteronuclear (HMBC) and homonuclear (TOCSY & NOESY) connectivities
in the acidic fraction gum (ppm) ........................................................................................ 54
Table 5.1. Partially Methylated Aditol Acetate (PMAA) sugar residue derivatives of the
neutral fraction gum ........................................................................................................... 63
Table 5.2. 1H/13C NMR Chemical Shifts for the neutral fraction gum (ppm) ................... 72
Table 5.3. Heteronuclear (HMBC) and homonuclear (NOESY) connectivities in the
neutral fraction gum (ppm) ................................................................................................ 73
Table 6.1. Molecular characteristics of the neutral (NFG) and acidic (AFG) fractions
obtained from static and dynamic light scattering ............................................................. 83
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List of Figures
Fig. 1.1. Structure of flaxseed .............................................................................................. 7
Fig. 1.2. The occurrence of mucilage in flaxseed ................................................................ 7
Fig. 3.1. Flow chart for extraction, fractionation and characterization of soluble flaxseed
gum .................................................................................................................................... 17
Fig. 3.2. Molecular weight distributions of polysaccharides (a) and their protein fractions
(b) ....................................................................................................................................... 27
Fig. 3.3. The steady shear flow curves (a) and dynamic rheological properties (b) of
soluble flaxseed gum and its two fractions ........................................................................ 28
Fig. 3.4. The intrinsic viscosity (a) and the dependence of viscosity on concentration (b-d)
of SFG, NFG and AFG ....................................................................................................... 32
Fig. 3.5. Reduction of surface tension of dispersions by soluble flaxseed gum and its
fractions .............................................................................................................................. 36
Fig. 3.6. The shifts of mean diameter (D[4,3]) and particle size distribution profiles of
emulsions stabilized by soluble flaxseed gum and its fractions within 24h after
emulsification with 2 % wt of canola oil ........................................................................... 38
Fig. 4.1. 1H (a) and 13C (b) NMR spectra of the acidic fraction gum (ppm) ..................... 45
Fig. 4.2. Key fragments of COSY spectrum of the acidic fraction gum ............................ 46
Fig. 4.3. Key fragments of TOCSY spectrum of the acidic fraction gum ......................... 47
Fig. 4.4. Key fragments of HMQC spectrum of the acidic fraction gum .......................... 48
Fig. 4.5. Key fragment of HMBC spectrum of the acidic fraction gum ............................ 49
xi
Fig. 4.6. Key fragment of NOESY spectrum of the acidic fraction gum ........................... 50
Fig. 4.7. Schematic representations of the conventional (a) and recently proposed
alternative (b) structures of pectin ..................................................................................... 58
Fig. 4.8. Proposed repeating unit of the acidic fraction gum ............................................. 58
Fig. 5.1. 1H (a) and 13C (b) spectra of the neutral fraction gum ......................................... 65
Fig. 5.2. COSY spectrum of the neutral fraction gum ....................................................... 66
Fig. 5.3. TOCSY spectrum of the neutral fraction gum ..................................................... 67
Fig. 5.4. HMQC spectrum of the neutral fraction gum ...................................................... 68
Fig. 5.5. HMBC spectrum of the neutral fraction gum ...................................................... 69
Fig. 5.6. NOESY spectrum of the neutral fraction gum .................................................... 70
Fig. 5.7. Proposed repeating unit of the neutral fraction gum ........................................... 71
Fig. 6.1. Molecular size distribution of acidic fraction gums in different solvents ........... 80
Fig. 6.2. Static light scattering data presented in Zimm plot obtained from the neutral
(NFG, a) and acidic (AFG, b) fraction gums in 0.5 M NaOH at 25 °C ............................. 82
Fig. 6.3. The angular dependence (a: NFG, 0.2 g/L; c: AFG, 0.2 g/L) and concentration
dependence (at 90o, b: NFG; d: AFG) of the hydrodynamic radius (Rh) of neutral (NFG)
and acidic (AFG) fractions determined in 0.5 M NaOH. .................................................. 84
Fig. 6.4. Schematic representation of molecular structure (a & b) and conformation (c & d)
of neutral (NFG) and acidic (AFG) fraction gums ............................................................ 85
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Chapter 1. Introduction
1.1 Significance of soluble dietary fibre and research focus
The latest renewal of dietary fibre (DF) definition in the 2009 meeting of Codex
Alimentarius included natural and synthetic carbohydrate polymers that are resistant to
human gastrointestinal enzymes (Ciucanu et al., 1984; Eastwood et al., 1992; Kendall et
al., 2010). DF can be sub-divided into soluble (SDF) and insoluble (IDF) fractions based
on its hot water extractability. SDFs (except low molecular weight (MW) SDFs) exhibit
viscous and/or gelling properties, and consequently can attenuate postprandial blood
glucose and insulin levels and lower serum cholesterol (Dikeman et al., 2006; Wood,
2007) which are the leading preventive parameters against diabetes (Brennan, 2005) and
cardiovascular diseases (CVD) (Theuwissen et al., 2008).
Despite large variation in molecular structures of SDF, the common mechanism of
controlling postprandial serum glucose has been underlined: the hydration of
viscosity-altering SDFs (at reasonable intake levels) influences the digesta viscosity and
thus the ease of mixing, diffusion and adsorption, hence mediating glycemic and
insulinemic responses linked to the risks of type 2 diabetes (Dikeman et al., 2006). In
contrast, the cholesterol-lowering effects are primarily based on the capacity of SDF to
form a thick unstirred water layer that reduces the (re)absorption of intestinal fats
(cholesterol and bile acids) and/or binding bile acids during formation of micelles. The
latter will lead to an increased fecal excretion of bile acid which, in turn, may drive
2
hepatic conversion of cholesterol into bile acids and absorption of serum low density lipid
(LDL)-cholesterol to compensate the hepatic free cholesterol pools (Denis et al., 2007;
Theuwissen et al., 2008). As a result, depletion of the body’s serum LDL-cholesterol
occurs. Another common hypothesis is that the increased luminal viscosity by the
hydration of SDF may impede the movement of cholesterol, bile acid and other lipid,
resulting in a lower absorption and higher excretion of these components (Eastwood et al.,
1992; Viuda-Martos et al., 2010).
Along with the above two beneficial physiological effects, short-chain fatty acids
(SCFA) produced by colonic fermentation of SDF (including low MW SDF) have drawn
increasing attention for their diverse roles in promoting health. SCFA (acetate, propionate,
and butyrate) acidify the colon environment, which may increase the development of
beneficial bacteria such as bifidobacteria and lactobacilli, reduce the pathogenic bacteria
(Swennen et al., 2006), as well as increase binding of bile acid and absorption of mineral.
Individually, propionate has been suggested to exhibit cholesterol-lowering properties by
offsetting the hyperlipidemic effect of acetate (Wong et al., 2006). Butyrate has
preventive effects on colon cancer and inflammation via regulating cell proliferation and
differentiation, although the mechanisms have not been clearly defined (Hamer et al.,
2008). The SCFA profile varies depending on both structure (Glitsø et al., 1999; Glitsø et
al., 2000; Rose et al., 2009) and average degree of polymerization (avDP) (Hughes et al.,
2007; Van Craeyveld et al., 2008), and also the essential enzymes existing in
corresponding bacterial varieties. Moreover, slowly fermented dietary fibres with
3
sustaining SCFA production are desirable as most fermentation happens in the proximal
colon, yet most colonic disease occurs in the distal colon (Ferguson et al., 2000).
Increasing the intake of SDF has become recognized to prevent CVD, which remains
the leading cause of mortality in the world, especially in the lower socio-economic status
groups. Approximate one third (17.5 million) of people died from CVD in 2005
(Viuda-Martos et al., 2010). However, the challenge of increasing fibre consumption in
the western diet has been the balance between nutritional functionality and sensory
satisfaction. As SDF has to be incorporated at a significant concentration to be
physiologically effective (Giacco et al., 1998), meanwhile such high level of SDF, as a
thickener or gelling agent, will inevitably influence the sensory properties and induce an
over-texturization, high viscosity or phase separation. Various mechanical and
(bio)chemical approaches have been developed to improve the physiological and sensory
properties of SDFs via modifying their granule diameter and/or MW (Yu et al., 2008).
Due to the close relation between the possible mechanisms of each beneficial
physiological effect of soluble dietary fibre and its physicochemical characteristics, the
relationship between the structure of soluble fibres and their rheological behaviour or
physiological benefits has drawn increasing attention in both academic and industrial
fields for better explorations of novel fibres and their potential in applications.
1.2 Overall objectives
The renewed interest in flaxseed as a food source is due to its health benefits
4
attributed to its components including lignans, α-linolenic acid, and flaxseed gum (SFG)
(Hall Iii et al., 2006). Our interests in flaxseed gum are primarily due to its low viscosity,
which is favoured for incorporation into food without an over-texturization, as well as the
high availability of sources and the ease of extraction. Differing from previous gum
extraction out of the whole seed, we extracted SFG from the mucilage enriched
by-product, flaxseed hulls, for lower costs and higher yield. To date, several previous
reports have been published on the structure of flaxseed gum (Naran et al., 2008) or its
two fractions(Cui et al., 1994a; Warrand et al., 2005c), yet their fine structures and
conformation are still not clear.
The overall objective of the current study is to investigate the structure-function
relationship of flaxseed gum and its fractions. The milestones of this work are:
1) To extract the gum from flaxseed hulls and to separate the soluble gum (SFG)
into neutral and acidic fractions using ion exchange chromatography
2) To examine the chemical, physicochemical, rheological, and functional
characteristics of SFG and its two fractions
3) To elucidate the linkage patterns and fine structure of neutral and acidic fractions
using methylation analysis and 1D/2D NMR spectroscopy
4) To investigate the conformation of neutral and acidic fractions using light
scattering
5) To reveal the relationship between the molecular structure, conformation and the
rheological behavior of neutral and acidic fractions
5
Chapter 2. Literature Review
2.1 Flaxseed, soluble flaxseed gum and their health benefit
Flax (Linum usitatissiumum L.) was first introduced to North America as a fibre crop,
but its value and importance as an oil source quickly became apparent (Cunnane et al.,
1995). Canada is the leading country in producing and exporting oil-type flaxseed.
Western Canadian flaxseed is composed of 45% oil and 23% protein (1990-2008 means)
(www.grainscanada.gc.ca). The renewed interest in flaxseed as a food source is due to its
health benefits attributed to its components including lignans (secoisolariciresinol
diglucoside (SDG) being the predominant form), α-linolenic acid (ALA), and soluble
flaxseed gum (SFG) (Hall Iii et al., 2006). Flaxseed gum exhibited much lower viscosity
over a range of shear rate from 10 to 1,000 s-1, in comparison to locust bean gum, guar
gum and xanthan gum at the same concentration (0.3%) (Mazza et al., 1989). Its low
viscosity is favoured for fortification of fibre in food without leading to
over-texturization.
Although health benefits of flaxseed and its food application have been extensively
investigated (Bassett et al., 2009; Bloedon et al., 2004; Mentes et al., 2008; Prasad, 2009;
Rendon-Villalobas et al., 2009; Warrand et al., 2005b), very few investigations have
specifically focused on the role of soluble gum in promoting human health (Fodje et al.,
2009; Thakur et al., 2009). Based on in vitro test results, soluble flaxseed gum showed
higher bile acid binding capacity and generated higher amount of acetate and propionate
6
compared with that of flax meal, wheat and rye bran, due to its significantly higher
soluble fibre content and solubility (Fodje et al., 2009). Inclusion of flaxseed in broiler
chicken diet was also found to be able to significantly increase the viscosity of ileal
digesta and the number of lactobacilli (Alzueta et al., 2003).
Despite the strong gel-forming ability of psyllium (Yu et al., 2003), the similarity in
molecular structure of the neutral fraction of flaxseed gum (NFG) and psyllium (Edwards
et al., 2003) implies that SFG may exhibit slow fermentability as psyllium does..
2.2 Natural occurrence and extraction of flaxseed gum
Flax mucilage (soluble flaxseed gum, SFG) occurs mainly at the outermost layer of
hull as shown in Fig. 1.1 (Thompson et al., 2003) & 1.2 (Cunnane et al., 1995).
This fibre-rich hull is able to release mucilaginous material (gum) easily when soaked
in water. A patented dehulling process (Cui & Han, 2006) could be performed to separate
flaxseed into a kernel (63%) fraction and a hull (37%) fraction (Cui, 2000). Therefore, to
reduce the costs and increase the gum yield, it is better to extract gum from the
by-product, hulls, but not from the whole flaxseed.
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Fig. 1.1. Structure of flaxseed
(A-C, three views of a flaxseed (approx.7x): A, seed; B, transverse section; C, longitudinal section. D,
transverse section (x400); E, longitudinal section (x400), showing testa, endosperm and cotyledon
cells. Abbreviations: cots, cotyledons; es, endosperm; g, germ; t, testa. Figure is from Thompson &
Cunnane, 2003)
Fig. 1.2. The occurrence of mucilage in flaxseed
(The line drawing of flaxseed is from Cunnane & Thompson, 1995)
Kernel
Mucilage Round cells Fiber cells Cross cells Pigment cells Endosperm
Seed Hull
8
The composition and yield of flaxseed gum varies with agronomic practices,
climate, genotype, and extraction condition. A broad variation in extraction yield (3.6~8%)
from flaxseed was found due to different geographical regions and cultivars (Cui et al.,
1996b; Oomah et al., 1995). Soaking flaxseed with a water: seed ratio of 13 at 85 to 95°C
and pH from 6.5 to 7.0 for 3 h was found to be the optimum gum extraction condition to
achieve higher yield and quality, using response surface methodology (Cui et al., 1994b).
However, higher extraction temperature induced higher protein content and a brown color
(Mazza et al., 1989).
2.3 Fractionation of flaxseed gum and the structure of two fractions
Flaxseed gum is reported to be composed of a neutral (NFG) and an acidic (AFG)
fraction, with some proteinaceous contaminants. Fractionation of NFG and AFG from
SFG was performed using ion exchange chromatography (Cui et al., 1994a; Warrand et
al., 2003). NFG is composed of a high MW arabinoxylan (AX), whereas AFG is mainly
composed of a low MW pectin-like rhamnogalacturonan. The structure of SFG (Naran et
al., 2008) and its two fractions (Cui et al., 1994a) were revealed via methylation analysis.
In AFG, nonreducing terminal L-galactose is attached at the O-3 position of the
rhamnosyl residues instead of at the typical O-4 position, whereas the NFG has a highly
branched xylan backbone with nonreducing terminal L-arabinosyl units attached at the
O-2 and/or O-3 positions.
An alternative method has been published recently to separate neutral fraction from
9
SFG via depectination using pectinase (Guilloux et al., 2009). The removal of acidic
fraction was not complete as indicated from the 1D NMR spectrum of the resultant
neutral fraction. However, compared with this method, separation with ion exchange
chromatography is tedious and time-consuming. Each method has its own advantages. To
evaluate the efficiency in separation of neutral from acidic fraction, the discrepancy in
composition, MW distributions and rheological properties of each fraction from different
methods needs to be compared. A fast and efficient fractionation will be favored if a
solution is available to improve either of the two methods.
2.4 Rheological properties of flaxseed gum
Flaxseed gums exhibit various rheological properties due to different cultivars,
extraction methods and measuring conditions. The viscosity of gum solutions exhibits
Newtonian-like behaviour at concentrations below 0.2% and pseudoplasticity at
concentrations above 0.2% (Mazza et al., 1989). Similar behaviour was also found in
crude (CFG), dialysed (DFG) and neutral (NFG) fractions. However, acidic (AFG)
fraction shows much lower viscosity and Newtonian-like behaviour at concentrations
from 0.3 to 2.0%. Small deformation oscillation rheological measurements of 2 % gum
solutions showed that CFG and DFG dispersions exhibited “weak gel” properties,
whereas NFG showed a viscoelastic fluid behaviour comparable to that of guar gum (Cui
et al., 1994a).
Intrinsic viscosity is a parameter reflecting the hydrodynamic volume occupied by the
10
polymer, the value of which depends primarily on the molecular size and chain
conformation of the polymer, as well as the solution quality. Previous work (Cui et al.,
1996c) showed that the intrinsic viscosity (in 1 M NaCl) of flaxseed gums from five
genotypes varied from 434.0 to 657.8 mL/g. Among these cultivars, crude gum (CFG)
from the cultivar, Norman, was further fractionated into NFG and AFG. The values of
intrinsic viscosity (in 1 M NaCl) of CFG, NFG and AFG were 483.0, 530.4 and 248.4
mL/g, respectively. In contrast, the values of gum Arabic, guar gum and xanthan gum
were 14.4, 1135, and 1355 mL/g, respectively (Cui et al., 1996c). The intrinsic viscosity
of flaxseed gum decreases with the increase of ionic strength, and higher value (1030±20
mL/g) of intrinsic viscosity of flaxseed gum from flaxseed meals have been reported
when it was dissolved in MilliQ water (Goh et al., 2006).
2.5 Structure-function relationship
Polysaccharides have great diversity in physical properties including solubility,
water-holding capacity, flow behaviour, gelling potential, and surface or interfacial
properties. They provide various functions in food applications as stabilizers, thickening
and gelling agents, crystallization inhibitors, and encapsulating agents, etc. The
functional properties exhibited by food polysaccharides depend on their molecular
structures and shapes, molecular weight and molecular weight distributions, as well as
their concentrations (Wang et al., 2005a). The discussions below focus mainly on the
overall relationships between rheological properties of the polysaccharides and their four
11
levels of structure.
Physical properties of a polysaccharide can be attributed to the various levels of
molecular organizations. The primary structure of a polysaccharide consists of its
monosaccharide composition, the ring size and anomeric configuration of each monomer,
the covalent linkage pattern and sequence, and substitution (Cui, 2005). The primary
structure is essentially rigid and is the basis for polysaccharide classification and their
three-dimensional shapes, in solid state and in solution. (Lapasin et al., 1995a; Morris et
al., 1979) In a homopolysaccharide chain, which contains only one type of monomeric
unit, four characteristic conformations (or secondary structures) can be recognized: type
A-extended ribbon-like chains, type B-hollow helices, type C-rigid and crumpled ribbons,
and type D-flexible coils or loosely joined chains. However, for most polysaccharides
composed of more than one sugar and/or more than one type of glycosidic bond, the
sequence can be periodic, interrupted or aperiodic and is likely to have an irregular
corresponding conformation. Energetically favorable non-covalent interactions, such as
van der Waals attractive forces, hydrogen bonding, charge and hydrophobic interactions
(Wang et al., 2005a), between shaped chains restrict chain flexibility and result in ordered
tertiary structures such as nested ribbons, multiple helices or aggregates of mixtures.
These tertiary structures exist in solution as the primary structural elements to form
aggregates and cause high viscosity or network formaiton. Tertiary structures may further
associate to establish higher molecular organization, often referred to as quaternary
12
structures.
Molecular weight and molecular weight distribution influence the shape and size of
polysaccharide chains and their behaviour in solution (Launay et al., 1985). In dilute
solution, the intrinsic viscosity, a measure of the hydrodynamic volume occupied by an
isolated polysaccharide, depends primarily on molecular weight, chain rigidity and
solvent quality. On the other hand, the viscosity of a concentrated solution mainly derives
from the interactions or fluctuating entanglement between chains. Hence, the low
shear-rate Newtonian viscosity η0 of undiluted solution is higher and increases more
drastically with concentration for high-molecular-weight polysaccharides than
low-molecular polysaccharides.
The aforementioned four main regular chain-shape types were also found somehow to
correlate with particular biological functions (Rees et al., 1971). Skeletal polysaccharides
capable of forming fibrous aggregates by tight packing are usually of type A-extended
ribbon-like shaped chains. They are mainly derived from 1,4-linkages, which are
prevalent in natural materials such as cellulose, chitin and xylans. Storage and network
polysaccharides are often of type B-hollow helices mainly composed of α-1,4-glucans
such as amylase, amylopectin, and β-1,3-glucans. The β-glucans from cereal grains
contain both periodic chain portions of type A (β-1,4-linkages) and type B (β-1,3-linkages)
and can serve structural and reserve functions during dormancy and germination,
respectively. Chains of type C shape are unnatural and rare in natural polysaccharides.
13
Loosely joined polysaccharides with type D shapes are mainly composed of
1,6/5-linkages and widely found in branched polysaccharides such as amylopectin and
various plant gums. These 1,5/6-linked residues introduce “flexible joints” into bush-like
molecules and result in a sponge-like instead of stiff textures.
14
Chapter 3. Flaxseed Gum from Flaxseed Hulls: Extraction,
Fractionation and Characterization
3.1. Introduction
Flax (Linum usitatissiumum L.) was first introduced to North America as a crop for
structural fibres, but its value and importance as an oil source quickly became apparent
(Cunnane et al., 1995). Canada is the leading country in producing and exporting oil-type
flaxseed. Western Canadian flaxseed is composed of 45% oil and 23% protein (1990-2008
means) (www.grainscanada.gc.ca). The renewed interest in flaxseed as a food source is
due to its health benefits attributed to its components including lignans
(secoisolariciresinol diglucoside (SDG) being the predominant form), α-linolenic acid,
and soluble flaxseed gum (SFG) (Hall Iii et al., 2006). In comparison to locust bean gum,
guar gum and xanthan gum at a concentration of 0.3 % (w/v), SFG exhibited much lower
viscosity over a range of shear rate from 10 to 1,000 s-1 (Mazza et al., 1989). Its low
viscosity might be favoured in dietary fibre fortification in food without leading to an
over-texturization, when a significant concentration of fibre is required to show health
benefits.
In vitro fermentation results showed that SFG exhibited higher bile acid binding
capacity and generated higher amount of acetate and propionate compared with that of
equivalent amount of flax meal, wheat and rye bran, due to its significantly higher soluble
fibre content and solubility (Fodje et al., 2009). Inclusion of flaxseed in the diet of broiler
15
chickens significantly increased the viscosity of ileal digesta and the number of lactobacilli
(Alzueta et al., 2003). The high bile acid binding capacity will lead to an increased fecal
excretion of bile acid, which may lower serum cholesterol (Denis et al., 2007;
Theuwissen et al., 2008). The high short-chain fatty acids (acetate, propionate and
butyrate) productivity and selective stimulation of growth and/or activity of probiotics
(Gibson et al., 2004) of SFG also showed its potential as a good source of prebiotics. The
abundance of two predominant bacteria divisions in the human gut, the Bacteroidetes and
the Firmicutes, were found to have positive and negative correlation with percentage loss
of body weight (Ley et al., 2006).
Flax mucilage (soluble flaxseed gum, SFG) occurs mainly at the outermost layer of
hull. This fibre-rich hull is able to release mucilaginous material (soluble gum) easily
when soaked in water. Earlier research analyses were based on the gum extracted from
the whole seed (Cui et al., 1994a; Diederichsen et al., 2006; Mazza et al., 1989;
Muralikrishna et al., 1987; Naran et al., 2008; Oomah et al., 1995; Warrand et al., 2003,
2005a, 2005c) or flax meal (Fedeniuk et al., 1994; Mueller et al., 2010). With the success
of a patented dehulling process (Cui et al., 2006), the whole flaxseed could be efficiently
separated into a kernel (~63%) fraction and a hull (~37%) fraction (Cui, 2000) in large
scale. Investigating the composition, structure, physicochemical and rheological
properties of SFG from hulls will assist in exploring its potential for the food industry.
In this current study, the gum (SFG) extracted from flaxseed hulls was further
separated into neutral (NFG) and acidic (AFG) fraction gums using ion exchange
16
chromatography (IEC). The chemical (neutral monosaccharide composition, uronic acid
and protein content), physical (molecular weight distribution and heat stability),
rheological (viscosity, viscoelasticity, intrinsic viscosity and critical concentration,) and
functional (surface tension and emulsification) characteristics of SFG and its two
fractions are reported.
3.2. Methodology
3.2.1 Materials
Flaxseed hull (variety Bethune) was supplied by Natunola Health Biosciences
(Winchester, Ontario, Canada).
3.2.2 Extraction of gum
The extraction of the gum was conducted at room temperature as shown on the flow
chart in Fig. 3.1.
A batch of 500 g of flaxseed hull was soaked in 6 L of distilled water overnight
under gentle stirring. A coarse filtration followed using cheesecloth to separate the hull.
The filtered mucilage was collected and centrifuged (Beckman Coulter, Mississauga,
Ontario) at 27,000 g and 25°C for 25 min, to eliminate insoluble particles. The
supernatant was then thoroughly mixed with one volume of 100% ethanol to precipitate
the polysaccharide. The precipitate was recovered by centrifugation of the mixture at
3,000 g for 10 min and redissolved in distilled water at approximately 4-5 (w/v) % before
lyophilization.
17
Fig. 3.1. Flow chart for extraction, fractionation and characterization of soluble
flaxseed gum
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum; IEC: ion-exchange
chromatography; Buffer 1: 20 mM Tris/HCl, pH 8).
3.2.3 Fractionation and purification of gum
3.2.3.1 Fractionation
The procedure of fractionation followed that of Warrand et al. (2003) with minor
modification: the washing solution of 0.1 M NaOH was replaced by 2 M NaCl and 1M
NaOH. A flow chart of the fractionation procedure is also shown in Fig. 3.1. For each
fractionation, 0.35 g of SFG was dissolved in 1 L of buffer 1 (20 mM Tris/HCl, pH 8) at
80°C for 1 h. After cooling down to room temperature, the SFG solution was loaded onto
the pre-equilibrated column (XK 50 Column, GE Healthcare, packed with 900 mL of
Q-Sepharose fast flow as matrix) at a flow rate of 10 mL min-1 for 100 min and flushed
Concentration (vacuum, 50 oC)
Functional Properties (Surface property; Emulsification)
18
continuously with 2 L of buffer 1 for 200 min. The eluate (denoted as NFG) was collected
between 25 and 150 min after the sample loading, when ~90 % of NFG was eluted out.
The column was successively washed with 1 L of buffer 2 (20 mM Tris/HCl + 1 M NaCl,
pH 8) at the same flow rate (10mL min-1). The eluate (denoted as AFG) was collected
between 55 and 110 min after buffer 2 was loaded, during which period most of the acidic
fraction (AFG) was eluted out. The column was sequentially flushed with 1 L of each of
the following three washing solutions: 2 M NaCl, 1 M NaOH and 2 M NaCl, and
re-equilibrated with 2-3 L of buffer 1 for next use. The acidic and neutral fractions were
then concentrated by rotary vacuum evaporator at 50°C, dialyzed (MW cut-off 3.5 kDa,
Fisher Scientific) against distilled water at 25°C for 72 h, and freeze-dried.
3.2.3.2 Purification
The protein fractions in both SFG and AFG were removed via protease hydrolysis,
and the two resultant fractions were denoted as SFGnP and AFGnP. Aqueous solutions of
SFG or AFG (1%, wt) were prepared by dissolving the polysaccharide in phosphate
buffer (80 mM, pH 7.5) at 80°C for 1 h with constant stirring before cooling down to
60°C. Stock solution of purified protease ( Megazyme cat. no. E-BSPRT, 350 tyrosine U
mL-1) was mixed thoroughly (0.2 mL g-1 gum or 70 U g-1 gum) with the gum solution and
incubated at 60°C for 30 min. The mixture was heated at 80°C for 10 min to inactivate
the enzyme. The resultant solution was dialyzed (MW cut-off 3.5 kDa, Fisher Scientific)
against distilled water at 25°C for 72 h, and freeze-dried.
19
3.2.4 Physico-chemical analysis
Monosaccharide analysis of SFG and its fractions was conducted on a high
performance anion exchange chromatography system (Dionex, DX500 Sunnyvale, CA,
USA) coupled with a pulsed amperometric detector (HPAEC-PAD) (Cui et al., 2000).
Hydrolysis was conducted as previously published (Wu et al., 2009). Uronic acid analysis
was conducted using the m-hydroxyphenyl colorimetric method (Blumenkrantz et al.,
1973). Protein analysis was conducted using NA2100 Nitrogen and Protein Analyzer
(Thermo Quest, Milan, Italy). The protein content (%) was obtained by multiplying the
nitrogen content (%) by 6.25. All chemical analyses were completed in triplicate.
The molecular weight (MW) distribution and heat stability of SFG, NFG and AFG
were measured using an HPSEC system consisting of an HPLC system with degasser,
autosampler and refractive index detectors (Optilab Rex, Santa Barbara, CA, USA). To
eliminate baseline variations, the mobile phase (100 mM sodium nitrate + 5 mM Sodium
azide) was degassed and filtered through 0.2 µm filters (Millipore, Fisher Scientific).
Before analysis, the gums were dissolved in the mobile phase at 1 g L-1, and heated at
80°C for 1 h under gentle stirring. To measure the heat stability of gums, heating duration
was extended to 3 h at 80°C. Samples were filtered through disposable 0.45 µm filters
(Millipore, Fisher Scientific) before injection, and aliquots (100 µL) of samples were
injected into the column (Shodex OHpak SB-806M HQ, 7.8 mm ID×300 mm, column
temperature at 40 oC) and eluted at a flow rate of 0.6 mL min-1 for 60 min. Pullulan 800
(MW 788,000), 400 (MW 404,000), 100 (MW 112,000), 50 (MW 47,300), 20 (MW
20
22,800) were used as MW markers to calibrate the column.
To investigate the association of protein and polysaccharides, aliquots (20 µL) of 1g
L-1 gum solutions prepared at the same condition as for MW distribution were injected into
a HPLC system (Waters 600E, Waters Ltd., Toronto, ON, Canada) with refractive index
detector (Waters 410) coupled with UV detector (Waters 486) and with the same column
for MW distribution but at column temperature of 25 oC. Separation was carried out at a
flow rate of 1.0 mL/min in the mobile phase (100 mM sodium nitrate + 5 mM sodium
azide).
Both steady shear and oscillation measurement of flaxseed gum and its fractions
were carried out on a strain-controlled rheometer (ARES, TA Instruments, New Castle,
DE, USA) using parallel-plate geometry (50 mm diameter, 1 mm gap size). The viscosity
of sample solutions of six concentrations (1, 5, 10, 15, 20, 25 g L-1) was measured at
25°C in shear rates ranging from 0.01 to100 s-1. Viscoelastic properties of samples were
measured at 20 g L-1 and 25 °C.
Intrinsic viscosity and critical concentration measurement followed that of (Doyle et
al., 2009). Gum dispersions were heated at 80°C for 1h and cooled down. Salt solutions
(0.2 M) were prepared individually before mixing with gum solutions under constant
stirring at ambient temperature, and salt concentration in each final sample was adjusted
to 0.1 M. Viscosity of solutions was measured using double wall couette geometry (inner
and outer cup radii were 27.94 and 34 mm; inner and outer bob radii were 29.5 and 32
mm, and 1 mm gap) on a strain-controlled rheometer (ARES, TA Instruments, New
21
Castle, DE, USA). The sample (7.5 mL) was left unperturbed for 15 min before each
measurement. All measurements were made in duplicate at 25 °C.
Intrinsic viscosity ( ][η ) was calculated using the following relationships (Harding,
1997).
srel ηηη /= (3.1)
1−= relsp ηη (3.2)
cKc Hspred ⋅+== 2][][ ηηηη (3.3)
cKc Krelinh ⋅+== 2][][)ln( ηηηη (3.4)
where η and sη are the zero shear viscosities (the constant apparent viscosities in a
limited Newtonian region at very low shear rates) of the solution and solvent (MilliQ
H2O); relη and spη are two dimensionless parameters of relative and specific viscosity;
and redη and inhη represent reduced viscosity and inherent viscosity, respectively.
HK (generally positive) and KK (generally negative) are Huggins and Kraemer constants
in Huggins and Kraemer equations (Eqs 3.3 and 3.4). Both equations are valid only for
solution viscosity up to twice of the solvent viscosity (i.e. 2≤relη ). Beyond this region,
with concentration increase polymer-polymer interactions will progressively become
significant, thus higher-order terms (c2, c3, etc.) will no longer be negligible. A lower
limit of 2.1≥relη is also required to eliminate the increasing errors in spη as spη
approaches 0. Concentrations of each gum fraction adopted were confined to this region
( 22.1 ≤≤ relη ). The value of intrinsic viscosity ( ][η ) was reported as the mean of both
22
intercepts in linear functions (3) and (4) by plotting cspη and
crelηln against c and
extrapolating each linear trendline to zero concentration.
3.2.5 Functional properties
3.2.5.1 Surface activity
The surface tension of the air-water interface was measured by the Du Nouy ring
method using the Fisher Surface Tensiomat (model 21, Fisher Scientific, Nepean, ON)
(Izydorczyk et al., 1991). SFG and its fractions were dissolved in 20 mL of distilled water
at various concentrations, heated at 80ºC for 1 h under gentle stirring and then left
unperturbed for 2 h before measurements. The surface tension (dynes cm-1) of distilled
water and gum solutions were measured every 5 min for 6 times at 25ºC.
3.2.5.2 Emulsifying properties
Emulsions were prepared by mixing canola oil (2 % wt) with SFG or its fraction
dispersions (preheated at 80°C for 1 h, final gum concentrations were adjusted to 0.44 %
wt) using Polytron (Kinematica GmbH, Brinkmann homogenizer, Switzerland) at medium
speed (level 5) for 5 min, followed by homogenization (Nano DeBEE Electric Bench-top
Lab Homogenizer, BEE International, South Easton, MA, USA) with 2 passes at 5,000
psi.
The particle size distribution of the emulsions was measured using integrated light
scattering (Mastersizer X, Malvern Southborough, MA) (Khalloufi et al., 2008). The
measurements were performed within one day after emulsion preparation as phase
23
separation occurred in all the emulsions within 24 h. The emulsifying stability was
determined by comparing the shifts of the oil droplet size distributions.
Duncan’s multiple-range tests were conducted using SAS software to examine the
changes in monosaccharide composition of soluble flaxseed gum or the acidic fraction
before and after protein removal. Thus the influence of protein removal processing on
monosaccharide composition of each gum fraction could be evaluated.
3.3. Results and discussions
3.3.1 Extraction, fractionation and chemical composition
The composition and yield of flaxseed gum has been reported to vary with extraction
conditions, as well as culture environment and genotype. Gum yield from flaxseed
increased from 4 to 9.4 % when extraction temperature increased from 25 to 80 oC
(Fedeniuk & Biliaderis, 1994). Broad variations of 3.6~8.0 % among 109 cultivars (80 oC,
2h) and 5.4~7.9 % among 12 genotypes (85 oC, 3h) were also found by Oomah et al.
(1995) and Cui et al. (1996), respectively. Soaking flaxseed with a water: seed ratio of 13
at 85 to 95°C and pH from 6.5 to 7.0 for 3 h was found to be the optimum gum extraction
condition to achieve higher yield and quality, using response surface methodology (Cui et
al., 1994b). However, higher extraction temperature induced higher protein content and a
brown color (Fedeniuk and Billiaderis, 1994). Considering the browning may affect the
properties of the polysaccharides, in the present work, the extraction of the gum was
conducted at room temperature. The yield and chemical composition of SFG, NFG and
24
AFG are shown in Table 3.1.
Table 3.1. Yield and chemical components of flaxseed gum and its fractions (%)
SFG NFG AFG
Yield 9.7 ± 0.3 a 23.2 ± 0.9 b 27.3 ± 3.0 b
Protein 11.8 ± 1.0 nd 8.1 ± 0.4
Uronic Acid 23.0 ± 0.1 1.8 ± 1.0 38.7 ± 1.0
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum; a: yield was based on
flaxseed hull mass; b: yield was based on SFG mass; nd: not detectable; data were on a dry basis)
The yield of SFG is 9.7 % of the hull mass. The recovery of the chromatographic
fractionation was approximately 50 %; the loss of half of the SFG may partially be due to
physical blockage of large particles in the column and/or insufficient unbinding process
while washing AFG out. The uronic acid content in AFG was about 20 fold of that in
NFG, suggesting an efficient separation of NFG from AFG. NFG was free of protein,
while AFG (8.1%) contained less protein than SFG (11.8%). After protein removal from
SFG and AFG, SFGnP and AFGnP were obtained with their protein content decreased to
an undetectable level.
Relative neutral monosaccharide composition of SFG and its fractions is shown in
Table 3.2.
25
Table 3.2. Neutral monosaccharide in flaxseed gum and its fractions (%)
SFG SFGnP NFG AFG AFGnP
Fucose 7.0 ± 0.2c 7.0 ± 0.4c nd 14.7 ± 0.3b 16.5 ± 0.1a
Rhamnose 16.5 ± 0.6c 14.5 ± 0.3c nd 38.3 ± 2.3a 32.8 ± 0.7b
Arabinose 12.7 ± 0.1b 12.6 ± 0.5b 20.2 ± 0.1a 2.9 ± 0.4c 3.0 ± 0.1c
Galactose 22.4 ± 1.0c 22.5 ± 0.7c 7.9 ± 0.1d 35.2 ± 0.4b 39.7 ± 0.4a
Glucose 2.7 ± 0.1b 3.1 ± 0.3ab 3.7 ± 0.4a nd nd
Xylose 38.6 ± 1.2b 40.9 ± 2.1b 68.2 ± 0.6a 8.9 ± 1.3c 7.9 ± 0.3c
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum; data are given as
“mean±SD”, n=3; data in the same row followed with the same superscript letter are not significantly
different by Duncan’s multiple-range test (p≤0.05); nd: not detectable.)
No significant differences were found between SFG and SFGnP, implying the
removal of protein via enzymatic hydrolysis from these two fractions did not modify their
neutral sugar composition. However, the same enzymatic hydrolysis procedure changed
the three main components in AFG: small portion of rhamnose was possibly removed
causing the relative increase in the other two (galactose and fucose). These changes in
SFG and SFGnP were not significant, since AFG occupied only ~27 % of SFG. The
neutral monosaccharide composition of SFG, NFG and AFG was comparable with
previous results (Cui et al., 1994a; Fedeniuk et al., 1994). NFG was mainly composed of
26
xylose (68.2 %) and arabinose (20.2 %) therefore being identified as arabinoxylans
(Warrand et al., 2003). Its minor components included galactose (7.9 %) and glucose
(3.7 %), but no rhamnose or fucose. In contrast, AFG mainly consisted of rhamnose
(38.3 %), galactose (35.2 %) and fucose (14.7 %). With most galactose and all fucose
existing as terminals in side chains (Naran et al., 2008), AFG was referred to as
rhamonogalacturonans for its high content of rhamnose and galacturonic acid (38.7 %,
Table 1.). The presence of small amounts of xylose (8.9 %) and arabinose (2.9 %) in AFG
might be due to the residual fraction of NFG (Cui et al., 1994), or might be derived from
side chain components covalently linked to its backbone (Naran et al., 2008).
3.3.2 Physical characterization
The molecular weight (MW) distribution of SFG, NFG and AFG are shown in Fig.
3.2(a). NFG consisted of one fraction with high MW of approximately 1,470 kDa. By
contrast, AFG consisted mainly of 3 portions of MW: 1,510, 341 and 6.6 kDa, respectively.
When subjected to heat at 80°C from 1 to 3 h, the MW of NFG decreased from 1,470 to
850 kDa. Similarly, a significant reduction of intermediate MW (340 kDa) polymers and
the appearance of additional small MW molecules (2.6 kDa) also implied the degradation
of AFG.
27
Fig. 3.2. Molecular weight distributions of polysaccharides (a) and their protein
fractions (b)
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum; the DRI and UV curves
exhibited the molecular weight distribution of polysaccharide and protein, respectively; the unit of
numbers in (a) is kDa; the column temperatures were 25 and 40 oC in (a) and (b), respectively.)
The UV and DRI response in Fig. 3.2(b) depicts the association of protein and
polysaccharides. No protein was detected in NFG. The major peak of high-MW protein
eluted at around 32 min, substantially later than two major high-MW polysaccharide
peaks (at ~26 and ~30 min, respectively) in both SFG and AFG. The differences in elution
time between these fractions of protein and polysaccharides suggested that most of the
protein fractions were not covalently linked to the polysaccharides in both SFG and AFG,
although trace amount of low-MW protein and polysaccharides were eluted out
simultaneously at around ~37min, making it difficult to reveal their association. That the
28
protein could be completely removed by protease hydrolysis (in Section 3.3.1) also
supported this conclusion, as it was reported that inherent protein could be very difficult
to remove (Garti et al., 1994).
3.3.3 Rheological properties
3.3.3.1 Viscosity and viscoelasticity
Flaxseed gum exhibited relatively low viscosity. At a concentration of 0.3 % (w/v),
the viscosity of flaxseed gum was only around half of that of guar gum and locust bean
gum (Mazza et al., 1989). The steady shear flow curves and dynamic rheological
properties of SFG and its fraction solutions at 25°C from this study are shown in Fig. 3.3.
Fig. 3.3. The steady shear flow curves (a) and dynamic rheological properties (b) of
soluble flaxseed gum and its two fractions
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum.)
SFG and NFG exhibited pseudoplastic flow behaviour at concentrations above 0.5
and 1.0 %, respectively, over a wide range of shear rate; whereas the steady shear flow
29
curves of AFG were more Newtonian-like at all concentrations examined. This was in
good agreement with those reported earlier by Cui et al. (1994). Mazza et al. (1989) also
found that flaxseed gum solutions exhibited pseudoplastic flow behaviour and
Newtonian-like behaviour at concentrations below and above 0.2 %, respectively.
All three dispersions showed liquid-like behaviour, as the loss modulus (G″)
exceeded the storage modulus (G') over the entire frequency range investigated (Fig. 3.3b).
In previous work by Cui et al. (1994), dispersions of crude, dialyzed flaxseed gums and
neutral fractions all exhibited “weak gel” properties. The discrepancy may be due to the
different sources of raw materials and extraction procedures.
3.3.3.2 Intrinsic viscosity
Intrinsic viscosity, denoted as [η], is a parameter reflecting the hydrodynamic volume
occupied by the polymer. The value of [η] primarily depends on the molecular size and
chain rigidity of the polymer, as well as the solution quality (Lapasin et al., 1995b).
Intrinsic viscosity increases with molecular weight (MW) according to the
Mark-Houwink relationship,
αη KM=][ (3.5)
where K is a constant, M is the average MW and, α is an exponent constant related to
the chain flexibility. For polyelectrolytes, the presence of counterions in the solution will
affect their intrinsic viscosity primarily through the reduction of intramolecular repulsion
among groups with the same charges. With increasing concentration of salts (i.e.
increasing ionic strength), the [η] of both SFG and AFG will decrease significantly as
30
they contain acidic groups (see uronic acid content in Table 1). The relation between [η]
and ionic strength has been proposed by Pals and Hermans as follows (Harding, 1997):
5.0][][ −∞ += SIηη (3.6)
where ∞][η is the intrinsic viscosity at infinite ionic strength, I is the ionic strength, and
S is a criterion only for the comparison of stiffness among polymers with the same MW,
as well as in the same solvent counterion environment. The “Smidsrod” stiffness
parameter, B, is defined by
υη )]([ 1.0=⋅= IBS (3.7)
where 1.0][ =Iη is the intrinsic viscosity at 0.1 M ionic strength, and 1.03.1 ±=υ . This
modified parameter B could be used to compare the relative stiffness of polymers even
without knowing their MW. It has been reported that flaxseed polysaccharide was
between a flexible and semi-flexible polymer with a B value of ~0.018, which lies
between ~0.005 (for xanthan gum with stiff conformation) and ~0.045-0.065 (for
carboxymethylcellulose with a flexible chain) (Goh et al., 2006). As a rough guide,
Huggins constant, HK , also reveals the general conformation of a polymer. The value of
HK can be as high as ~2 for uncharged spheres. Lower value is expected for more
extended biopolymer, such as ~0.35 for flexible biomolecules (Harding, 1997). Goh et al.
(2006) reported the HK value for flax meal polysaccharide equal to 0.34±0.05, which
also implied its flexible conformation. The values of HK for SFG, NFG and AFG are
shown in Table 3.3. NFGHK , (0.54) was slightly higher than SFGHK , (0.48) and both were
31
much higher than AFGHK , (0.16), implying the chain flexibility of SFG was between that
of NFG and AFG, as SFG is composed of both fractions.
Table 3.3. The intrinsic viscosity ([ŋ]), Huggins constant ( HK ), critical
concentrations (ccr) and coil overlap parameter values ((c[ŋ])cr
) at critical
concentrations of SFG, NFG and AFG
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum)
The Huggins and Kraemer plots of SFG, NFG and AFG are shown in Fig. 3.4(a) and
the intrinsic viscosities ([η], in 0.1 M NaCl) of SFG, NFG and AFG are 446.0, 377.5 and
332.5 mL g-1, respectively (Table 3.3).
NFG had a larger intrinsic viscosity than AFG, probably due to its higher molecular
rigidity and average MW (Fig. 3.2). Although SFG had similar molecular rigidity to NFG
and a similar MW distribution to AFG (both contained low-MW fraction), it had larger
intrinsic viscosity than both. This might be due to the presence of the synergistic
SFG NFG AFG
[ŋ] (mL g-1) 446.0 ± 3.4 377.5 ± 5.7 332.5 ± 6.8
HK / 0.48 0.54 0.16
ccr (g L-1) 6.61 6.68 6.43
(c[ŋ])cr / 2.95 2.52 2.14
32
interaction between NFG and AFG. Higher intrinsic viscosities of deacetylated
xanthan-guar mixtures have been reported in comparison with those expected on the
assumption of no synergistic interactions (Khouryieh et al., 2007). Goh et al. (2006)
reported a linear relation between [η] of flaxseed gum and 5.0−I as follows:
5.05.0 )9.05.52()4289(][][ −−∞ ⋅±+±=+= ISIηη , according to which the value of [η] in
0.1 and 1 M NaCl would be approximately 455 and 342 mL g-1, respectively. The former
value was comparable to the [η] of SFG (446.0 mL g-1 in 0.1 M NaCl), but higher values
of [η] (in 1 M NaCl) ranged from 434.0 to 657.8 mL g-1 for five genotypes of flax were
also reported (Cui et al., 1996c).
Fig. 3.4. The intrinsic viscosity (a) and the dependence of viscosity on concentration
(b-d) of SFG, NFG and AFG
(SFG: soluble flaxseed gum; NFG and AFG: neutral and acidic fraction gum; viscosity was measured in 0.1
M NaCl (a-d). Reduced viscosity (ηred = ηsp/c) and inherent viscosity ( ηrel = (ln ηrel)/c) versus concentration
are plotted with empty and solid symbols (a), respectively; the common intercept gives [η], and the positive
and negative slopes are KH[η]2 and KK[η]2, where KH and KK are Huggins and Kraemer constant,
respectively (a). n1 and n2 represent the slope of each region; the gum concentration for the circles within
the squares in figure (b-d) equaled 5 g L-1) .
33
3.3.3.3 Critical concentrations
The dependence of viscosity on concentration and intrinsic viscosity could be
expressed by a power law relationship (Morris et al., 1981a):
nsp ck ])[( ηη ⋅⋅= (3.8)
where spη is the “zero-shear” specific viscosity and can be obtained by Eq (3.1) and
(3.2); the degree of space occupancy (c[η]) is also called the coil overlap parameter, in
which c is the concentration and [η] is the intrinsic viscosity; the value of (lg k) and n
equal the extrapolated interception and the slope of the double logarithmic plots (Fig. 3.4
(b-d)) for SFG, NFG and AFG. A drastic change of slope was found in each of the three
fractions (Fig. 3.4 (b-d)) at a specific degree of space occupancy ((c[η])cr). The
corresponding concentration at this point is called the critical concentration (ccr).
Solutions with concentrations below and above ccr are referred to as dilute and
semi-dilute solutions, respectively. In dilute solution, the increase in viscosity is caused
by interfering the flow of solvent by the separated polymer chains and the increment is
proportional to the increment in volume fraction occupied by these polymers; rheological
behaviour in semi-dilute solutions, however, primarily depends on the polymer-polymer
interaction, as the overlap and interpenetration of neighbouring polymer chains are no
longer negligible (Morris et al., 1981b). In practice, there is a transition zone around the
cross of the two linear trendlines for dilution and semi-dilution regions, where the data
points of viscosity obtained from measurements slightly depart from the expected values
34
on each trendline. Therefore critical concentration is an approximate measure for the
onset of overlap and entanglement between polymer chains. The critical concentrations
(ccr, mL g-1) for SFG (~6.61), NFG (~6.68) and AFG (~6.43) were similar to each other,
irrespective of their significant differences in intrinsic viscosities (Table 3.3), and the
corresponding values of (c[η])cr fell within a range from 2.14 to 2.95, not far away from
the values reported between 2.5 and 4 (Morris et al., 1981a). The values of n1 were
confined to 1.08-1.21 (Fig. 3.4) and were comparable with the value (1.1~1.4) reported
(Doyle et al., 2009; Morris et al., 1981; (Wang et al., 2005b)). The n2 for SFG (3.28) and
NFG (3.51) were close to the typical value (~3.3) for various disordered polysaccharides
(Morris et al, 1981), though higher value (n2 = ~5) was also found in the chain of several
galactomannans (Doyle et al., 2009) due to the “hyperentanglement” of unsubstituted
mannan regions. Compared with SFG and AFG, a much lower value of n2 (2.17) detected
for AFG may probably also be due to its relatively higher flexibility and thus be less
likely to form an entanglement network, just as implied from its lower value of
AFGHK , (0.16).
3.3.4 Functional properties
Most water soluble polysaccharides are known to act as stabilizers in oil-in-water
emulsions, only a few can act as emulsifiers mainly due to the presence of hydrophobic
moieties, such as proteinaceous materials. Gum arabic is the most used polysaccharide as
an emulsifier because of the presence of proteinaceous moieties in its structure. The
35
polysaccharide chains can then absorb to the oil phase via the protein structure (Garti et
al., 2001). By contrast, a protein-rich fraction, which was separated from crude guar gum,
contained higher protein (10.6 %) but showed much lower surface activity and
emulsifying stability than both the protein-depleted fraction (0.8 %) and crude gum
(3.0 %), indicating the protein did not play any significant role in their stabilizing action
(Garti et al., 1994). The adsorption mechanism, however, for either protein or the gums
with trace protein left are still not clear.
3.3.4.1 Surface activity
The protein fraction in both SFG and AFG were completely removed via protease
hydrolysis. Two resultant fractions were denoted as SFGnP and AFGnP, respectively. The
surface tension of dispersions decreased slightly with the addition of SFGnP, AFGnP and
NFG (0.01~ 0.5 %, w/v) (Fig. 3.5). SFG and AFG reduced the surface tension to
0.055±0.001and 0.056±0.00 N/m 1, respectively at a concentration of 0.5 %, which is
comparable with the value of the solution containing 0.5 % of guar gum (Huang et al.,
2001). After protein removal, SFGnP (0.062±0.001 N/m) and AFGnP (0.065±0.002 N/m)
reduced the surface tension to the same level as NFG (0.063±0.003 N/m) did at an
equivalent concentration of 0.5 %, implying that the protein fractions in both SFG and
AFG were responsible for their higher surface activity.
36
Fig. 3.5. Reduction of surface tension of dispersions by soluble flaxseed gum and its
fractions
(SFG and SFGnP: soluble flaxseed gum with and without protein; NFG: neutral fraction gum; AFG and
AFGnP: acidic fraction gum with and without protein; surface tension of distilled water: 0.072±0.001 N/m.
Surface tension of each fraction at each concentration was measured for six times and presented as the
mean value with error bars showing the standard deviations.)
3.3.4.2 Emulsifying stability
The volume–length diameter, D[4,3], is sensitive to the presence of large particles
(i.e., sensitive to flocculation and coalescence), thus is commonly used in expressing the
mean particle size of a polydisperse emulsion to indicate the stability of an emulsion. It is
the sum of the volume ratio of droplets in each size-class multiplied by the mid-point
diameter of the size-class (McClements, 2005):
∑∑∑===
==11
3
1
4]3,4[i
iii
iii
ii ddndnD φ (3.9)
where D[4,3] is the volume fraction-length mean diamerter, ni, di and φ represent the
number, mid-point diameter, and the volume fraction of each size-class, respectively.
The top five factors affecting the rate of creaming, thus the stability of an emulsion,
0.075
0.070
0.065
0.060
0.055
0.050
Surfa
ce T
ensi
on
(N/m
)
37
are rheology of the continuous phase, droplet volume fractions, the density difference
between phases, droplet size and its distribution (Hill, 1998). In a preliminary experiment,
an emulsion containing 10 % wt oil and 1.0 % wt SFG showed larger D[4,3] (2.4 µm) but
was stable for more than 30 days, whereas in another emulsion containing 5 % wt oil and
0.5 % wt of SFG, though smaller D[4,3] (2.0 µm) was found, phase separation occurred
within 4 days after emulsification, indicating the higher viscosity by addition of 1% SFG
retarded the rate of flocculation and coalescence. To avoid the viscosity effect, 0.50, 0.48
and 0.44 % wt of SFG, AFG and protein-free gum fractions (SFGnP, AFGnP and NFG)
(containing identical amount (0.44 % wt) of pure gum for each fraction, which is below
their critical concentrations (6.4-6.7 g L-1)) were included to prepare dilute gum solutions.
Before mixing with 2 % wt of canola oil, the zero-shear viscosity (mPa.s) of the
continuous phase of emulsions with SFG (7.4±0.1) and SFGnP (7.5±0.1) were two-fold
of that of NFG (3.7±0.1), AFG (3.6±0.1) and AFGnP (3.5±0.1). The shifts of droplet size
distributions and D[4,3] values of emulsions with the above five fractions are shown in
Fig. 3.6. Monodispersed droplet size distribution and stable D[4,3] values were found in
emulsions containing SFG and AFG from 3 to 24 h after emulsification, whereas the
volume fraction (%) of the second peak (larger size particles), as well as the D[4,3]
values, increased obviously within 24 h for all three protein-free fractions (SFGnP, NFG
and AFGnP), which again confirmed that the protein fractions in SFG and AFG
contributed to their better emulsifying stabilities than the other three protein-free fractions,
in spite of their distinctions in viscosity.
38
Fig. 3.6. The shifts of mean diameter (D[4,3]) and particle size distribution profiles of
emulsions stabilized by soluble flaxseed gum and its fractions within 24h after
emulsification with 2 % wt of canola oil
(SFG and SFGnP: soluble flaxseed gum with and without protein; NFG: neutral fraction gum; AFG and
AFGnP: acidic fraction gum with and without protein. Each mean diameter was measured in triplicate and
presented as the mean value with error bars showing the standard deviations.)
39
3.4. Conclusions
Flaxseed gum may become a significant source of soluble fibre for both its
availability and low viscosity. Two fractions, neutral (NFG) and acidic (AFG), of soluble
flaxseed gum (SFG) from hulls were effectively separated using ion exchange
chromatography. NFG, consisting of only one polymer with high MW, was free of protein
and contained <2 % of uronic acid. AFG mainly consisted of polymers with different
MW associated with 8 % of protein which was not covalently linked. Both fractions were
stable at 80°C for 1 h, however, extended heating resulted in degradations of the
polymers. The reduced surface activity and emulsifying stability after complete removal
of protein content from SFG (11.8 %) and AFG (8.1 %) using protease revealed the
contribution of the protein fractions to their emulsification, regardless of their distinctions
in molecular conformation (molecular mass and chain flexibility) and rheological
properties. However, the differences between these two fractions in MW distribution and
chain stiffness affected their intrinsic viscosity and viscosity dependence on the
concentration in both dilute and semi-dilute solutions.
40
Chapter 4. Structural Elucidation of the Acidic Fraction Gum
4.1 Introduction
In our previous study, soluble flaxseed gum extracted from flaxseed hulls was
separated into a neutral and an acidic fraction using anion exchange chromatography
(Qian et al., 2012b). The acidic fraction gum (AFG) might be favored for its low viscous
properties as a potential dietary fibre fortifier to be included into food systems in a large
amount without over-texturization. Methylation analysis in previous studies showed the
main linkages of the AFG to include →4) -α-D-GalpA-(1→, and linear or branched
→2)-α-L-Rhap-(1→ at O-3 position (Cui et al., 1994a; Naran et al., 2008).
Rhamnogalacturonan-I (RG-I) backbone features its repeating units of alternatively
distributed →2)-α-L-Rhap-(1→ and →4)-α-D-GalpA-(1→. RG-I, RG II and
homogalacturonan (HG) comprise the three main structural elements of pectin. In general,
pectins encompass hetero-polysaccharides containing at least 65% of galacturonic
acid-based units according to the FAO and EU stipulation(Willats et al., 2006). Arabinose
or galactose is the second most abundant neutral sugar component in pectins with no
more than 4% of rhamnose and fucose typically (Stephen, 1995). Although AFG was
referred to as pectic polysaccharides, RG-I (Naran et al., 2008), this fraction differed
from pectins in its higher rhamnose content and lower galacturonic acid content.
Interest in polysaccharides as potential sources for anti-tumor agents increased in
recent decades due to their fewer side effects. Modified pectin was reported to show (in
41
vitro) anti-tumor bioactivities probably due to the existence of galactans from side chains
in the hairy region (Glinsky et al., 2009; Morris et al., 2011). Revealing the fine structure
of polysaccharides assists in further tracing their potential functionalities and/or
bioactivities. The detailed structure of AFG has not been reported. This study focused on
the structure of AFG using methylation-GC-MS analysis and 1D/2D NMR spectroscopy
including homonuclear 1H/1H correlation spectroscopy (COSY, TOCSY) and nuclear
overhauser effect spectroscopy (NOESY), heteronuclear 1H/13C multiple-quantum
coherence spectroscopy (HMQC) and heteronuclear 1H/13C multiple bond correlation
spectroscopy (HMBC).
4.2 Experimental
4.2.1 Materials
Soluble flaxseed gum was isolated by aqueous extraction from flaxseed hulls
(variety Bethune) supplied by Natunola Health Biosciences (Winchester, Ontario,
Canada). The acidic fraction gum (AFG) was separated from soluble flaxseed gum using
anion-exchange chromatography and purified by protease hydrolysis to remove
proteinaceous contaminant (Qian et al., 2012b). All chemicals were of reagent grade
unless otherwise specified.
42
4.2.3 Methylation analysis
The acidic fraction gum (AFG) was reduced into neutral polysaccharides before
methylation. The procedure of reduction and methylation followed that of previous work
(Kang et al., 2011). The resulting partially methylated alditol acetates (PMAAs) were
injected into a GC-MS system (ThermoQuest Finnigan, San Diego, CA) with an SP-2330
(Supelco, Bellefonte, Pa) column (30 m×0.25 mm, 0.2 mm film thickness, 160-210 oC at
2 oC /min, and then 210-240 oC at 5 oC /min) equipped with an ion trap MS detector.
4.2.3 NMR analysis
Sample was dissolved in deuterium oxide (D2O, 80 oC, 1 h) and lyophilized for three
times to replace the exchangeable protons with deuterons before being finally redissolved
in D2O (3 %) for NMR analysis. High-resolution 1H and 13C NMR spectra were recorded
in D2O at 500.13 and 125.78 MHz, respectively, on a Bruker ARX500 NMR
spectrometer operating at 25 oC. A 5 mm inverse geometry 1H/13C/15N probe was used.
Chemical shifts are reported relative to external standards Trimethylsilyl propionate (TSP
in D2O, 4.76 ppm, for 1H) and 1,4-Dioxane (in D2O, 66.5 ppm, for 13C). Homonuclear
1H/1H correlation spectroscopy (COSY, TOCSY) and nuclear overhauser effect
spectroscopy (NOESY), heteronuclear 1H /13C multiple-quantum coherence spectroscopy
(HMQC) experiments, and heteronuclear multiple bond correlation spectroscopy (HMBC)
were run using the standard Bruker pulse sequence at 70 oC.
43
4.3 Results and discussion
4.3.1 Methylation and GC-MS of partially methylated alditol acetate (PMAA) of the
acidic fraction gum
Methylation-GC-MS analysis of the acidic fraction gum (AFG) showed that this
fraction was composed of four hexosyl residues (rhamnose (38.1 %), galactouronic acid
(24.8 %), galactose (20.8 %), and fucose (4.5 %)) and two pentosyl residues (xylose
(7.7 %) and arabinose (2.6 %)) as listed in Table 4.1. Four major linkage patterns
constituted 78.5 % of the total sugar residues include: →2,3) -L-Rhap-(1→ (19.5 %),
→2)-L-Rhap-(1→ (16.5 %), →4)-D-GalpA-(1→ (22.9 %) and D-Galp-(1→ (19.6 %).
The molar ratio of total non-reducing terminal residues and total branching points were
29.2 and 25.8 %, respectively.
The degree of branching (DB) of AFG was equal to 0.55 as calculated according to
the equation below (Hawker et al., 1991; Tao et al., 2007):
DB=(NT+NB)/(NT+NB+NL)=(29.2+24.5)/(29.2+24.5+44.8)=0.55 (4.1)
where NT, NB and NL are the molar percentage of the terminal, branched and linear
residues, respectively. The DB value of a linear chain equals to 0, whereas that of a fully
branched polymer is 1. The DB value (0.55) of AFG indicated it was highly branched.
44
Table 4.1. Partially Methylated Aditol Acetate (PMAA) sugar residue derivatives of
the acidic fraction gum
Deduced Residue RT* Mol%** PMAA
R3 →2,3)-L-Rhap-(1→ 1.307 19.5 4-Me Rhap
R →2)-L-Rhap-(1→ 0.957 16.5 3,4-Me2 Rhap
RT L-Rhap-(1→ 0.603 1.2 2,3,4-Me3 Rhap
R34/4 →2,3,4)/→2,4)-L-Rhap-(1→ 1.379 0.9 Rhap-(OAC)5 /3-Me Rhap
Rhamnose 38.1
GA →4)-D-GalpA-(1→ 1.543 22.9 2,3,6-Me3 GalpA
GA2 →2,4)-D-GalpA-(1→ 1.900 1.1 3,6-Me2 GalpA
GA3 →3,4)-D-GalpA-(1→ 1.764 0.8 2,6-Me2 GalpA
Galacturonic Acid 24.8
GT D-Galp-(1→ 1.117 19.6 2,3,4,6-Me4 Galp
G' →6)-D-Galp-(1→ 1.703 1.2 2,3,4-Me3 Galp
Galactose 20.8
FT L-Fucp-(1→ 0.754 4.5 2,3,4-Me3 Fucp
Fucose 4.5
XT D-Xylp-(1→ 0.784 3.1 2,3,4-Me3 Xylp
X →4)-D-Xylp-(1→ 1.250 2.4 2,3,-Me2 Xylp
X34 →2,3,4)-D-Xylp-(1→ 2.054 1.3 Xylp-(OAC)5
X2/3 →2,4)/3,4)-D-Xylp-(1→ 1.646 0.9 3/2-Me Xylp
Xylose 7.7
A →3)-L-Araf-(1→ 1.054 1.8 2,5-Me2 Araf
AT L-Araf-(1→ 0.642 0.8 2,3,5-Me3 Araf
Arabinose 2.6
(RT*: Retention time is relative to 2,3,4,6-Me4 Glc (14.591 min); Mol%**: molar ratio of each sugar residue
is based on the percentage of its peak area; Letter labeled by a superscript T refers to a terminal residue;
The superscript numbers follow the letters indicate the branching sites in these residues; Data for sugar
residues less than 0.6 % are not shown; Linkages below 2% were not included in the proposed repeating
unit for AFG in Fig. 4.7 )
45
4.3.2 1D and 2D NMR analysis of the acidic fraction gum
More than six peaks are shown in the anomeric region (4.3~5.2 ppm) of the 1H NMR
spectrum (Fig. 4.1a). Two peaks at 1.08 and 1.11 ppm arose from the proton resonances
of methyl groups in fucose and rhamnose, respectively; their corresponding resonances in
the 13C spectrum (Figure 4.1b) are at 16.2 and 17.6 ppm, respectively.
Fig. 4.1. 1H (a) and 13
(R3: →2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→; βGT:β-D-Galp-(1→; αGT:
α-D-Galp-(1→; FT: α-L-Fucp-(1→; XT: β-D-Xylp-(1→; X: →4)-β-D-Xylp-(1→.)
C (b) NMR spectra of the acidic fraction gum (ppm)
46
Fig. 4.2. Key fragments of COSY spectrum of the acidic fraction gum
(Intra-ring COSY correlations are labeled by residue abbreviations and two unseparareted numbers of correlating
protons; R3: →2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→; α/βGT: α/β-D-Galp-(1→; FT:
α-L-Fucp-(1→; XT: β-D-Xylp-(1→; X: →4)-β-D-Xylp-(1→; A: →3)-α-Araf-(1→.)
47
Fig. 4.3. Key fragments of TOCSY spectrum of the acidic fraction gum
(Intra-ring TOCSY correlations are labeled by residue abbreviations and two unseparated numbers of correlating
protons; R3: →2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→; α/βGT: α/β-D-Galp-(1→; FT:
α-L-Fucp-(1→.)
48
Fig. 4.4. Key fragments of HMQC spectrum of the acidic fraction gum
(Intra-ring H/C correlations are labeled by residue abbreviation and the number of correlating proton/carbon; R3:
→2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→; α/βGT: α/β-D-Galp-(1→; FT:
α-L-Fucp-(1→.)
49
Fig. 4.5. Key fragment of HMBC spectrum of the acidic fraction gum
(Inter-ring H/C correlations are labeled by correlating proton or carbon and its number followed by the residue
abbreviation; R*: R & R3; R3: →2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→; α/βGT:
α/β-D-Galp-(1→; FT: α-L-Fucp-(1→; XT: β-D-Xylp-(1→.)
50
Fig. 4.6. Key fragment of NOESY spectrum of the acidic fraction gum
(Inter-ring H/H correlations are labeled by the residue abbreviation/its proton number; R*: R & R3; R3:
→2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→; α/βGT: α/β-D-Galp-(1→; FT:
α-L-Fucp-(1→; XT: β-D-Xylp-(1→; X: →4)-β-D-Xylp-(1→.)
51
The spin system for each sugar residue of AFG was assigned according to the COSY
spectrum (Fig. 4.2), with assistant/confirmative information from the TOCSY (Fig. 4.3)
and HMQC spectra (Fig. 4.4). The sequence of the linkages of sugar residues were
inferred from the HMBC (Fig. 4.5) and NOESY spectra (Fig 4.6). The assignment of
each sugar residue and their relative sequences are discussed in the following sections
before a possible structure of the repeating unit of AFG is proposed.
4.3.2.1 Rhamnose residues
Rhamnosyl residues (38.2 %) in AFG include four linkages as listed in decreasing
order of predominance in Table 4.1: →2,3) -α-L-Rhap-(1→ (R3, 19.5 %),
→2)-α-L-Rhap-(1→ (R, 16.5 %), and minor amount of T-α-Rhap-(1→ (RT, 1.2 %) and
→2,3,4)/→2,4)-α-L-Rhap-(1→ (R34/4, 0.9 %). The α-configuration of all Rhap units was
inferred from their common chemical shifts of anomeric carbon (99.6 ppm) and proton
(5.11 ppm) as shown in the anomeric region of HMQC spectrum (Fig. 4.4a).
The spin units for residue →2) -α-L-Rhap-(1→ (R) and →2,3)-α-L-Rhap-(1→ (R3)
are fully assigned in COSY, TOCSY and HMQC spectra (Fig. 4.2~4.4 & Table 4.2). The
H-2 (4.05 ppm), H-3 (3.83/3.85 ppm) and H-4 (3.50 ppm) chemical shifts of the R3
moved to downfield by 0.1~0.2 ppm, due to the glycosylation at O-3 position (Colquhoun
et al., 1990; Pawan, 1992). The substitution at O-3 positions of rhamnosyl residues
instead of the typical O-4 positions, as found in pectin and soluble soybean
polysaccharides, was also reported based on methylation analysis of flaxseed mucilage in
earlier studies (Muralikrishna et al., 1987; Naran et al., 2008). Both cross-peaks at
52
3.83/72.2 and 3.85/77.9 ppm in HMQC (Fig. 4.4) were tentatively assigned to H-3/C-3
correlation of R3. This splitting may be due to the substitution by different sugar units,
according to HMBC (Fig. 4.5) and NOESY (Fig. 4.6) spectra. The chemical shifts of
glycosylated carbon (C-3) moved downfield by 1.7 and 7.5 ppm (Table 4.2), respectively.
The former value was far smaller than the value of 7~10 ppm reported for rhamosyl
residues branched at O-3 position(Choma et al., 2009; Fedonenko et al., 2008; MacLean
et al., 2010; Ojha et al., 2008); other than this, the provisional assignment was in good
agreement with literature data(Cui et al., 1996a; Mikshina et al., 2012; Ojha et al., 2008;
Sengkhamparn et al., 2009).
4.3.2.2 Galactouronic acid residue
The presence of three peaks at 173.6-174.5 ppm from carboxyl group in 13C 1D NMR
spectrum (Fig. 4.1b) is the evidence for the existence of galactouronic acid (GA, 24.8 %).
Linear →4) -D-GalpA-(1→ (GA, 22.9 %) accounted for 92 % of this residue, with the
rest singly substituted at O-2 or O-3 position (Table 4.1). A full assignment for
→4)-α-D-GalpA-(1→ (Table 4.2) was in good agreement with previous reports
(Fedonenko et al., 2008; MacLean et al., 2010; Mikshina et al., 2012; Ojha et al., 2008).
53
Table 4.2. 1H/13C NMR chemical shifts for the acidic fraction gum in D2O at 70o
Deduced Residues
C
(ppm)
H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6/C-6
R3 →2,3)-α-L-Rhap-(1→ 5.11/99.6 4.05/78.0 3.83/72.2 3.50/74.1 3.74/70.5 1.16/17.5
3.85/77.9
R →2)-α-L-Rhap-(1→ 5.12/99.6 3.97/77.5 3.73/70.5 3.30/73.1 3.62/70.5 1.13/17.5
GA →4)-α-D-GalpA-(1→ 4.89/99.0 3.76/69.0 3.97/71.1 4.32/78.3 4.63/72.1 /173.6-174.5
αGt α-D-Galp-(1→ 5.12/102.1 3.67/69.5 3.69/69.6 3.87/70.5 3.77/70.5 3.59/62.5
βGt β-D-Galp-(1→ 4.40/97.4 3.32/- 3.48/- 3.80/- 3.59/- -/-
4.44/97.2 3.38/- 3.48/- 3.80/- 3.59/- -/-
FT α-Fucp-(1→ 5.02/102.3 3.64/- 3.60/- -/- 3.90/- 1.08/16.2
XT β-D-Xylp-(1→ 4.44/97.2 3.20/- -/- -/- -/- -/-
X →4)-β-D-Xylp-(1→ 4.31/97.4 3.20/- -/- -/- -/- -/-
A →3)-Araf-(→ 5.12/- 4.17/- -/- -/- -/- -/-
54
Table 4.3 Heteronuclear (HMBC) and homonuclear (TOCSY & NOESY)
connectivities in the acidic fraction gum (ppm)
Inter-residue connectivities Intra-residue connectivities
HMBC NOESY HMBC
Atom δ Atom δ Atom δ Atom δ Atom δ Atom δ
H-1 R* 5.12 C-4 GA 78.3 H-1 GA 4.90 H-2 R 3.97 H-1 R* 5.12 C-2 78.0
C-2 R* 77.5-78.0 H-2 R3 4.05 H-1 R 5.12 C-3 70.9
C-1 R* 99.7 H-2 R 3.99 H-1 FT 5.03 H-2 R 3.97 H-1 R3 5.12 C-3 72.2
99.9 H-4 GA 4.32 H-1 R* 5.12 H-4 GA 4.32 H-4 GA 4.32 C-2 69.4
C-1 GA 99.0 GA H-4 4.32 H-1 GA 4.90 H-4 GA C-3 71.3
99.0 H-2 R 3.97 H-1 αGT 5.12 C-2/3 69.8
(Appendants linked to C-3 R3 ) (Appendants linked to C-3 R3 ) TOCSY
Atom δ Atom
H-1 αGT 5.12 C-3 R3 72.1/77.9 H-1 αGT 5.12 H-3 R3 3.83 H-1 αGT 5.12 H-2,3,4,5,6
H-1 βGT 4.44 C-3 R3 72.2 H-1 βGT 4.46 H-3 R3 3.85 H-1 βGT 4.44 H-3,4
H-1 XT 4.44 C-3 R3 72.2 H-1 XT 4.46 H-3 R3 3.85 H-1 R* 5.12 H-2,3,4,5
H-1 R 5.12 C-3 R3 72.1 H-1 R 5.12 H-3 R3 3.85 H-6 R* 1.11 H-1,2,3,4,5
H-1 GA 4.90 C-3 R3 71.7 H-1 GA 4.90 H-3 R3 3.85 H-1 GA 4.89 H-2,3,4
H-1 FT 5.03 C-3 R3 70.8 H-1 FT 5.03 H-3 R3 3.83 H-6 FT 1.08 H-5,6
(R*: R and R3; R3: →2,3)-α-L-Rhap-(1→; R: →2)-α-L-Rhap -(1→; GA: →4)-α-D-GalpA -(1→;
βGT:β-D-Galp-(1→; αGT: α-D-Galp-(1→; FT: α-L-Fucp-(1→; XT: β-D-Xylp-(1→.)
4.3.2.3 Galactose residues
The second most abundant neutral sugar residue is Galactose (20.8 %, Table 4.1),
94 % of which is (GT, 19.6 %). The cross-peak at 5.12/102.1 ppm in HMQC (Fig. 4.4a)
55
arose from H-1/C-1 correlation of α-D-Galp-(1→ (αGT), and its full assignment
completed was in accordance with literature data (Ahrazem et al., 2002; Galbraith et al.,
1999; Sengkhamparn et al., 2009). Two cross-peaks at 4.40/97.4 and 4.44/97.2 ppm in
HMQC (Fig. 4.4a) may arise from H-1/C-1 connectivity of β-D-Galp-(1→ (βGT)(Deng et
al., 2006; Sengkhamparn et al., 2009; Vidal et al., 2000) though both could be only
partially assigned according to COSY and TOCSY spectra (Fig. 4.2 & 4.3) due to the low
intensity in the rest of spectra. The α-configuration was predominant, however, the ratio
of both configurations could not be estimated due to the overplayed anomeric proton
chemical shifts of α-D-Galp-(1→ and Rhap (5.12 ppm), or of β-D-Galp-(1→ and
β-D-Xylp-(1→(4.44 ppm) as shown in Fig. 4.1.
4.3.2.4 Fucose residue
Terminal α-L-Fucp-(1→ (FT, 4.5 %) was the only linkage for Fucosyl residue.
Correlations of H-1/H-2 and H-5/H-6 (Fig. 4.2 & 4.3) could be assigned according to
literature data (Shashkov et al., 1993).
4.3.2.5 Minor residues
Xylose (7.7 %) was constituted of 3.1 % β-D-Xylp-(1→ (XT) and 2.4 % of
→4)-β-D-Xylp-(1→ (X). The rest were →4)-β-D-Xylp-(1→ branched at O-2, O-3 or
both. Two group of cross-peaks at 4.44-4.46/3.20 and 4.31-4.33/3.20 ppm in COSY (Fig.
4.2) may arise from XT and X, respectively (Höije et al., 2006; Pastell et al., 2008; Sun et
56
al., 2011). However, full assignment was impossible due to the low intensity of signals.
A small portion (2.6 %) of Arabinofuranose existed in two linkages:
→3)-α-L-Araf-(1→ (A, 1.8 %) or α-L-Araf-(1→ (AT, 0.8 %). The chemical shift for
H-1/H-2 correlation of α-L-Araf-(1→ was reported to be close to that of rhamnose (Höije
et al., 2006; Pastell et al., 2008; Sun et al., 2011), thus could not be assigned. The
cross-peak at 5.12/4.17 ppm in COSY (Fig. 4.2) may arise from H-1/H-2 correlation of
→3)-α-L-Araf-(1→ as the H-2 chemical shift moved to downfield by 0.1-0.2 ppm due to
the glycosylation at O-3(Westphal et al., 2010).
Two cross-peaks at 4.79/3.42 and 4.98/3.42 may be from the H-1/H-2 correlation of
→4)-α-D-Glcp-(1→ and →6)-α-D-Glcp-(1→, respectively. However, the glucose content
in this acidic fraction was too low to be detected as indicated in either methylation (Table
4.1) or monosaccharide analysis (Qian et al., 2012b).
4.3.2.6 Linkage sequence of AFG
Heteronuclear multiple bond correlation (HMBC) spectrum shows correlation
between protons and carbons 2-3 bonds away. Nuclear overhauser effect spectroscopy
(NOESY) correlates nuclei through space (distance smaller than 5Å). Both methods are
helpful to reveal the glycosylic linkages between sugar residues although intra-residue
connectivities also included. The inter-residue and intra-residue correlations observed are
listed in Table 4.3.
The most intense inter-residue connectivities assigned in Fig 4.5 were C-1R*/H-4GA
57
(99.9/4.32 ppm, R*: R or R3) and H-1R*/C-4GA (5.12/78.3 ppm), indicating the
abundance of diglycosyl repeating unit →2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→. Links
between →4) -α-D-GalpA-(1→ itself, i.e. homogalacturonan (HG) regions, was evident
by H-1/H-4 correlation of →4) -α-D-GalpA-(1→ (4.89/4.32 ppm, Fig. 4.6), though its
evidence in HMBC (C-1/H-4GA, 99.0/4.32 ppm, Fig. 4.5) overplayed with
C-1R*/H-4GA, (99.9/4.32 ppm). However, the amount of HG should be limited due to
the low GalpA content and its predominant occurrence in repeating unit
→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→. Strong correlations of H-1/C-2R*
(5.12/77.5-78.0 ppm) and C-1/H-2R (99.7/3.99 ppm) observed in Fig. 4.5 may derive
from both intra- and inter-residue connectivites between rhamnosyl residues. However,
the existence of homorhamnan (HR) region could still be confirmed due to the abundance
of rhamnosyl residues. Given the above evidence, a backbone consisting of RG-I possibly
intervened by small amount of HG and HR could be drawn.
The branching site of R3 (19.5 %) at O-3 were mostly substituted by monosaccharides,
e.g., α/β-D-Galp-(1→ (α/βGT, 19.6 %), α-L-Fucp-(1→ (FT, 4.5 %) and β-D-Xylp-(1→ (XT
,
3.1 %) (Table 4.1). The C-3 chemical shifts of R3 varied with substitution by different
sugar units (Table 4.3, Fig. 4.5 & 4.6). However, when it was substituted with
→2)-α-L-Rhap-(1→ or →4)-α-D-GalpA-(1→, a longer side chain consisting of more
than two residues may induced.
58
Fig. 4.7. Schematic representations of the conventional (a) and recently proposed
alternative (b) structures of pectin
(RG-I: rhamnogalacturonan-I; RG-II: rhamnogalacturonan-II; HG: homogalacturonan;
schematics are adapted from Willats & Knox et al. 2006.)
[→2)-α-L-Rhap-(1→]m[→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→]n[→4)-α-D-GalpA-(1→]i
Fig. 4.8. Proposed repeating unit of the acidic fraction gum
(HR, RG-I and HG refer to homorhamnan, rhamnogalacturonan-I and homogalacturonan, respectively.
The locations of HR, RG-I and HG are interchangeable; (m+n)/(n+i)≈1.5. The substitution rate of R1 is
~54 %. R1 is mostly monosaccharide (α/β-D-Galp-(1→, α-L-Fucp-(1→ or β-D-Xylp-(1→). R1 may also
occasionally be a longer side chain with more than two residues beginning with →4) -α-GalpA-(1→ or
→2)-α-L-Rhap-(1→, wherein the side-chain structure may be similar to part of the main chain.)
3
↑
R1
3
↑
R1
HR RG-I HG
HG
(a)
(b)
RG-II
b
a
RG-I
59
The fine structure of pectin is still in dispute due to its complexity and heterogeneity
between plants and tissues (Willats et al., 2006). The controversy between conventional
and recently proposed alternative structural models of pectin is whether the HG domain is
inserted into its backbone or linked as a side chain (Fig. 4.7). A similar question also
exists in revealing the structure of AFG. A combination of both models is tentatively
adopted. Given the above evidence, a possible repeating unit of AFG (Fig. 4.8) is
proposed.
4.4 Conclusions
The acidic fraction gum (AFG) from flaxseed hulls was a pectic polysaccharide as
investigated by methylation analysis and 2D NMR spectroscopy. AFG contained a
backbone consisting of rhamnogalacturonan-I which might be intervened by small
amount of homorhamnan or homogalacturonan. However, AFG differed from pectin in a
much higher rhamnose (38.2 %) and much lower galacturonic acid content (25.4 %), thus
had limited amount of HG region. This fraction also featured its high amount of
mono-galactosyl branches 3-linked to half of 1,2-linked rhamnosyl residues. However
longer side chains with more than two residues also existed when substitution with
→4)-D-GalAp-(1→ or →2)-α-L-Rhap-(1→ was 3-linked to 1,2-linked rhamnosyl
residues.
60
Chapter 5. Structure Elucidation of the Neutral Fraction Gum
5.1 Introduction
In chapter 3, soluble flaxseed gum extracted from flaxseed hulls was separated into a
neutral and an acidic fraction using anion-exchange chromatography. The neutral fraction
gum (NFG) was referred to as arabinoxylans for its high xylose (70 %) and arabinose
(20 %) content (Qian et al., 2012b). Earlier results of methylation analysis showed the
main linkages of the NFG included T-α-D-Xylp, 1,4-linked α-D-Xylp unsubstituted or
substituted at O-2 or O-3 or both positions and 1,3-linked or 1,5-linked α-L-Araf (Cui et
al., 1994a; Naran et al., 2008). Arabinoxylan is one of the most abundant components in
plant cell wall polysaccharide, along with cellulose and pectins. The structural features of
arabinoxylans including the ratio of arabinose to xylose residues, the relative amount and
sequence of mono-, di- and unsubstituted Xylp, and the possible presence of other
substituents (Izydorczyk et al, 2008). The structural diversity and complexity are likely
related to their functionality and the source of material, the genotype and cellular origin,
as well as the growth stage of the plants (Izydorczyk, 2009).
Revealing the fine structure of arabinoxylans provides the basis for further tracing
their potential functional and/or physiological properties. Detailed structure of NFG has
not been reported. This study focused on the structure of NFG using methylation-GC-MS
analysis and 1D/2D NMR spectroscopy including homonuclear 1H/1H correlations
spectroscopy (COSY, TOCSY), heteronuclear 13C/1H multiple-quantum coherence
61
spectroscopy (HMQC), heteronuclear multiple bond correlation spectroscopy (HMBC)
and nuclear overhauser effect spectroscopy (NOESY).
5.2 Experimental
5.2.1 Materials
Soluble flaxseed gum was isolated by aqueous extraction from flaxseed hulls
(variety Bethune) supplied by Natunola Health Biosciences (Winchester, Ontario,
Canada). The protein-free neutral fraction gum (NFG) was separated from soluble
flaxseed gum using ion exchange chromatography (Qian et al., 2012b). All chemicals
were of reagent grade unless otherwise specified.
5.2.2 Methylation analysis
The methylation procedure of neutral fraction (NFG) followed that of previous work
(Kang, 2011). The resultant partially methylated alditol acetates (PMAAs) were injected
into a GC-MS system (ThermoQuest Finnigan, San Diego, CA) with an SP-2330
(Supelco, Bellefonte, Pa) column (30 m×0.25 mm, 0.2 mm film thickness, 160-210 oC at
2 oC/min, and then 210-240 oC at 5 oC /min) equipped with an ion trap MS detector.
5.2.3 NMR analysis
Sample was dissolved in deuterium oxide (D2O, 3 %, 80 oC, 1 h) and lyophilized for
three times to replace the exchangeable protons with deuterons before being finally
redissolved in D2O (2 %) for NMR analysis. High-resolution 1H and 13C NMR spectra
were recorded in D2O at 500.13 and 125.78 MHz, respectively, on a Bruker ARX500
62
NMR spectrometer operating at 70 oC. A 5 mm inverse geometry 1H/13C/15N probe was
used. Chemical shifts are reported relative to external standards Trimethylsilyl propionate
(TSP in D2O, 4.76 ppm, for 1H) and 1,4-Dioxane (in D2O, 66.5 ppm, for 13C).
Homonuclear 1H/1H correlation spectroscopy (COSY, TOCSY and nuclear overhauser
effect spectroscopy (NOESY)), heteronuclear 1H /13C multiple-quantum coherence
spectroscopy (HMQC) and heteronuclear multiple bond correlation spectroscopy (HMBC)
experiments were conducted using the standard Bruker pulse sequence. To increase the
signal-to-noise ratio(s/n), all experiments were run at 70 oC.
5.3 Results and discussion
5.3.1 Methylation and GC-MS of partially methylated alditol acetate (PMAA) of the
neutral fraction gum
Methylation-GC-MS analysis of the neutral fraction gum (NFG) showed this fraction
was composed of 65.1 % of Xylp and 24.8 % of Araf，and the rest (9.5 %) originated
from Galp or Glcp (Table 5.1). Four major linkage patterns constituted 77.4 % of the total
sugar residues and they are: linear →4) -D-Xylp-(1→ (25.0 %) or double substituted at
O-2 and O-3 (19.6 %), T-D-Xylp-(1→ (15.5 %), and →1)-L-Araf-(5→ (17.3 %).
63
Table 5.1. Partially Methylated Aditol Acetate (PMAA) sugar residue derivatives of
the neutral fraction gum
Abbr. Deduced Linkage RT* Mol%** PMAA
X4 →4)-β-D-Xylp-(→ 1.255 25.0 2,3-Me2 Xylp
X234 →2,3,4)-β-D-Xylp-(1→ 2.066 19.6 Xylp-(OAc)5
XT T-β-D-Xylp-(1→ 0.790 15.5 2,3,4-Me3 Xylp
X24/34 →2/3,4)-β-D-Xylp-(1→ 1.648 5.1 3/2-Me Xylp
Xylose 65.1
A5 →5)-α-Araf-(l→ 1.158 17.3 2,3-Me2 -Araf
A3 →3)-α-Araf-(l→ 1.045 4.9 2,5-Me2 Araf
AT T-α-Araf-(l→ 0.646 2.6 2,3,5-Me3 Araf
Arabinose 24.8
Pentose 89.9
GT T-α-D-Galp-(1→ 1.114 4.5 2,3,4,6-Me4 Galp
G4/C4 →4)-α-D-Galp/Glcp -(1→ 1.576 3.5 2,3,6-Me3 Galp/Glcp
C6 →6)-α-D-Glcp-(1→ 1.517 1.4 2,3,4-Me3 Glcp
Hextose 9.4
(RT*: Retention time is relative to 2,3,4,6-Me4 Glc (14.591 min); Mol%**: molar ratio of each sugar residue
is based on the percentage of its peak area; Letter labeled by a superscript T refers to a terminal residue;
The superscript numbers follow the letters indicate the branching sites in these residues; Data for sugar
residues less than 0.3 % are not shown.)
64
Molar ratio of total non-reducing terminal residues and total branching points were
22.6 % and 44.3 %, respectively. The degradation of T-Araf-(1→ may be the main reason
causing the low estimation of terminal residues. The degree of branching (DB) of NFG
was equal to 0.55 as calculated according to the equation below:
DB=(NT+NB)/(NT+NB+NL)=(22.6+24.7)/(22.6+24.7+52.1)=0.48 (5.1)
where NT, NB and NL are the molar percentage of the terminal, branched and linear
residues, respectively. The DB value varies from 0 (a linear chain) to 1 (a fully branched
polymer). The DB value (0.48) of NFG indicated it was highly branched.
5.3.2 1D and 2D NMR analysis of the neutral fraction gum
As shown from the 1H NMR spectrum (Fig. 5.1a), eleven peaks in the anomeric
region (δ 4.3~5.3 ppm) can be divided into three groups of sugar residues: three peaks
from α-Araf (5.17-5.26 ppm), three peaks from α-Glcp or α-Galp (4.77-4.97 ppm) and
five peaks from β-Xylp (4.29-4.55 ppm). Their corresponding 13C peaks were also
assigned (Fig. 5.1b). The spin system for each sugar residue of NFG was assigned
according to the COSY spectrum (Fig. 5.2), with the assistant/confirmative information
from the TOCSY (Fig. 5.3) and HMQC spectra (Fig. 5.4). The linkage sites and sequence
of sugar residues were inferred from HMBC (Fig. 5.5) and NOESY spectra (Fig. 5.6).
The assignment of each sugar residue and their relative sequences are discussed in the
following sections before the repeating unit structure (Fig. 5.7) of NFG is proposed.
65
Fig. 5.1. 1H (a) and 13
C (b) spectra of the neutral fraction gum
(X4: →4)-β-D-Xylp-(1→; X234: →2,3,4)-β-D-Xylp-(1→; XT: T-β-D-Xylp-(1→; X24:
→2,4)-β-D-Xylp-(1→; X34: →3,4)-β-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-α-Glcp-(1→; C6: →6)-D-α-Glcp-(1→; GT: T-D-α-Galp-(1→.)
66
Fig. 5.2. COSY spectrum of the neutral fraction gum
(X4: →4)-â-D-Xylp-(1→; X234: →2,3,4)-â-D-Xylp-(1→; XT: T-â-D-Xylp-(1→; X24:
→2,4)-â-D-Xylp-(1→; X34: →3,4)-â-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-Glcp-(1→; C6: →6)-D-Glcp-(1→; GT: T-D-Galp-(1→.)
67
Fig. 5.3. TOCSY spectrum of the neutral fraction gum
(X4: →4)-β-D-Xylp-(1→; X234: →2,3,4)-β-D-Xylp-(1→; XT: T-β-D-Xylp-(1→; X24:
→2,4)-β-D-Xylp-(1→; X34: →3,4)-β-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-α-Glcp-(1→; C6: →6)-D-α-Glcp-(1→; GT: T-D-α-Galp-(1→.)
68
Fig. 5.4. HMQC spectrum of the neutral fraction gum
(X4: →4)-β-D-Xylp-(1→; X234: →2,3,4)-β-D-Xylp-(1→; XT: T-β-D-Xylp-(1→; X24:
→2,4)-β-D-Xylp-(1→; X34: →3,4)-β-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-α-Glcp-(1→; C6: →6)-D-α-Glcp-(1→; GT: T-D-α-Galp-(1→.)
69
Fig. 5.5. HMBC spectrum of the neutral fraction gum
(X4: →4)-β-D-Xylp-(1→; X234: →2,3,4)-β-D-Xylp-(1→; XT: T-β-D-Xylp-(1→; X24:
→2,4)-β-D-Xylp-(1→; X34: →3,4)-β-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-α-Glcp-(1→; C6: →6)-D-α-Glcp-(1→; GT: T-D-α-Galp-(1→.)
70
Fig. 5.6. NOESY spectrum of the neutral fraction gum
(X4: →4)-β-D-Xylp-(1→; X234: →2,3,4)-β-D-Xylp-(1→; XT: T-β-D-Xylp-(1→; X24:
→2,4)-β-D-Xylp-(1→; X34: →3,4)-β-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-α-Glcp-(1→; C6: →6)-D-α-Glcp-(1→; GT: T-D-α-Galp-(1→.)
71
Fig. 5.7. Proposed repeating unit of the neutral fraction gum
5.3.2.1 Xylose residues
Xylose (65.1 %) is the most abundant component in NFG including five linkage
patterns: linear →4) -β-D-Xylp-(1→ (X, 25.0 %) and substituted at either (X24/34, 5.1 %)
both of O-2 and O-3 (19.6 %), as well as T-β-D-Xylp-(1→ (XT, 15.5 %). The integration
labeled under each anomeric proton peak (Fig 5.1a) also showed the single substituted
→4)-β-D-Xylp-(1→ at O-2 or O-3 was the minority compared with the other three
linkages. However, there is no good correlation between the relative ratio of each linkage
from integration calculation and its percentage from methylation analysis. All five
linkage patterns were fully assigned according to the literature data (Fischer et al., 2004;
Höije et al., 2006; Mazumder et al., 2010; Pastell et al., 2008; Pastell et al., 2009; Skendi
et al., 2011; Sun et al., 2011). The chemical shift of C-2 shifted downfield by 4-6 ppm in
the C13 NMR spectrum due to the glycosylation at O-2 (Table 5.2). The chemical shift of
72
C-4 moved downfield by ~4 or 7-8 ppm depending on whether its neighboring C-3 was
also glycosylated or not.
Table 5.2. 1H/13
Sugar Residues
C NMR Chemical Shifts for the neutral fraction gum (ppm)
H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6/C-6
X4 →4)-β-D-Xylp-(0→ 4.30/102.1 3.15/74.4 3.40/74.4 3.58/76.8 3.92/63.5 3.20/63.5
X234 →2,3,4)-β-D-Xylp-(1→ 4.54/100.7 3.55/80.1 3.73/77.5 3.74/73.7 4.00/62.7 3.36/62.7
Xt T-β-D-Xylp-(1→ 4.43/104.0 3.19/73.8 3.29/76.5 3.48/70.1 3.83/65.9 3.14/65.9
X24 →2,4)-β-D-Xylp-(1→ 4.40/103.9 3.48/78.3 3.65/75.2 3.78/77.8 -/- 3.37/62.8
X34 →3,4)-β-D-Xylp-(1→ 4.46/103.8 3.23/73.8 3.52/78.3 3.54/74.1 3.98/64.8 3.3/64.8
A5/T →5)-α-Araf-(l→ 5.26/108.7 4.02/81.7 3.88/78.3 4.24/83.8 3.70/70.3 -/-
A3 →3)-α-Araf-(l→ 5.16/108.7 4.17/80.9 4.02/85.2 4.19/83.6 3.61/62.0 3.67/61.3
AT T-α-Araf-(l→ 5.20/108.6 4.19/80.9 4.00/81.2 4.06/85.0 3.62/61.3 3.58/61.3
C4 →4)-α-D-Glcp-(1→ 4.78/99.6 3.39/74.5 3.48/74.1 3.59/76.8 -/- -/-
C6 →6)-α-D-Glcp-(1→ 4.97/100.5 3.37/72.4 3.48/74.1 3.48/70.2 3.56/70.2 3.76/68.8
GT T-α-D-Galp-(1→ 4.89/99.9 3.70/69.1 3.87/70.1 -/- -/- 3.63/61.9
5.3.2.2 Arabinose residues
The second abundant component in NFG, arabinose (24.8 %), existed in three linkage
patterns: →1) -L-Araf-(5→ (17.3 %), →1)-L-Araf-(3→ (4.9 %) and T-L-Araf-(1→
(2.6 %). The percentages of these three anomeric protons from methylation analysis are
in the same decreasing order as the relative ratios from integration calculation (Fig. 5.1a).
73
Table 5.3. Heteronuclear (HMBC) and homonuclear (NOESY) connectivities in the
neutral fraction gum (ppm)
Inter-residue connectivities
HMBC NOESY
Atom δ Atom δ Atom δ Atom δ
A5H-1 5.26 X234C-3 77.4 A5H-1 5.26 X234H-3 3.73
X34C-3 78.3 X34H-3 3.52
X24C-2 78.3 X24H-2 3.48
GTH-1 4.88 A5C-5 70.3 GTH-1 4.88 A5H-5 3.7
C6H-1 4.97 X34C-3 78.3 C6H-1 4.97 X34H-3 3.52
X24C-2 78.3 X24H-2 3.48
C4C-1 99.6 C4H-4 3.59 C4C-1 4.78 C6H-4 3.76
X34H-3 3.52 X34 H-3 3.52
X234H-2 3.55 X234H-2 /
Additional Inter-residue connectivities in NOESY
Atom δ Atom δ Atom δ Atom δ
XTH-1 4.43 A5H-5 3.70 ATH-1 5.20 X234H-3 3.73
A3H-3 4.02 A5H-1 5.26 X234H-2 3.55
X234H-3 3.73 A3H-1 5.16 X234H-2 3.55
X234H-2 3.55 X234H-3 3.73
X34H-3 3.52 X34H-3 3.52
X24H-2 3.48
(X4: →4)-β-D-Xylp-(1→; X234: →2,3,4)-β-D-Xylp-(1→; XT: T-β-D-Xylp-(1→; X24:
→2,4)-β-D-Xylp-(1→; X34: →3,4)-β-D-Xylp-(1→; A5: →5)-α-Araf-(l→; AT: T-α-Araf-(l→; A3:
→3)-α-Araf-(l→; C4: →4)-D-α-Glcp-(1→; C6: →6)-D-α-Glcp-(1→; GT: T-D-α-Galp-(1→.)
74
A full assignment for each linkage was in good agreement with previous data (Ferreira et
al., 2006; Habibi et al., 2004; Westphal et al., 2010). The chemical shifts in the C13 NMR
spectrum of glycosylated C-5 of →1) -L-Araf-(5→ and C-3 of→1) -L-Araf-(3→ moved
downfield by 4-7 and 8-10 ppm, respectively.
5.3.2.3 Minor residues
A minor portion of glucose residue in NFG mainly existed as →1) -D-Glcp-(6→
(1.4 %) and →1) -D-Glcp-(4→ (~3.5 % or less). The α-configurations of
→1)-D-Glcp-(6→ (De Castro et al., 2005; Leone et al., 2006; Muldoon et al., 2002) and
→1)-D-Glcp-(4→ (Chakraborty et al., 2004; Ghosh et al., 2008; Perepelov et al., 2011)
were evident by the cross-peaks at 4.78/99.6 and 4.97/100.5 ppm in the anomeric region
of HMQC, respectively.
Galactose residues were appended to NFG mainly in forms of T-α-D-Galp-(1→
(4.5 %) as assigned in COSY (Fig. 5.2) and TOCSY (Fig. 5.3) spectra according to the
literature (Ahrazem et al., 2002; Galbraith et al., 1999; Sengkhamparn et al., 2009).
5.3.2.4 Sequence of sugar residues
The β-1,4-linked xylose backbone was evident by the cross-peaks confined in the
dashed square in the NOESY spectrum (Fig. 5.6), where the H-1/H-4 connectivities of
linear or branched →4) -β-D-Xylp-(1→ could be observed. The evidence in HMBC (Fig.
5.5) was listed in Table 5.3: 1) the →5)-L-Araf-(1→ and →4)-D-Glcp-(1→ was appended
75
to the backbone at O-3 and O-2, respectively; 2) mono-substitution with →5)-L-Araf-(l→
or →6)-D-Glcp-(1 →at either O-2 or O-3, or with →4)-D-Glcp-(1→ at O-2 were also
evident; 3) the appendence of T-D-Galp-(1→ to →5)-L-Araf-(l→ and links between
→4)-D-Glcp-(1→ also existed. All above links found in HMBC were also confirmed in
NOESY, except that links between →4) -D-Glcp-(1→ was replaced by
→6)-D-Glcp-(1→4)-D-Glcp-(1→ and that the appendence of →4) -D-Glcp-(1→
1.2-linked to →2,3,4)-D-Xylp-(1→ was missing. More evidence shown in NOESY was
listed in Table 5.1. Given the above evidence, a proposed structure of the repeating unit
of NFG is given (Fig. 5.7).
5.4 Conclusions
The neutral fraction gum (NFG) from flaxseed hulls was an arabinoxylan as indicated
by methylation analysis and 1D/2D NMR spectroscopy. NFG contained a β-1,4-linked
xylose backbone being mono-, di- or unsubstituted at O-2 and/or O-3 positions by short
branches mostly consisting of one to three sugar residues. These branches are appended
to the backbone through linear linkages like →5) -L-Araf-(1→ (17.3 %),
→3)-L-Araf-(1→ (4.9 %), →4)-D-Glcp/Galp-(1→ (3.5 %) and →6)-D-Glcp-(1→(1.4 %),
and ended with three terminals: T-D-Glcp-(1→(15.5 %), T-L-Araf-(1→(2.6 %) and
T-D-Galp-(1→(4.5%).
76
Chapter 6. Conformation of Neutral and Acidic Fraction Gums
6.1. Introduction
In our previous studies, soluble flaxseed gum extracted from flaxseed hulls was
separated into a neutral and an acidic fraction using anion-exchange chromatography
(Qian et al., 2012b). Both neutral (DB=0.48) and acidic (DB=0.55) fractions were highly
branched as indicated by their high degree of branching (DB) (Qian et al., 2012a). The
neutral fraction (NFG) was an arabinoxylan and contained a β-1,4-linked xylose
backbone being mono-, di- or unsubstituted at O-2 and/or O-3 positions by short branches
consisting mainly of one to three sugar residues. In contrast, the acidic fraction (AFG)
consisted of a rhamnogalacturonan-I (RG-I) backbone intervened by limited amount of
homogalacturonan (HG) or homorhamnan (HR). Half of its most abundant neutral
residues (→2) -α-L-Rhap-(1→, 38.2%) were monosubstitued at O-3 mostly by
monosaccharides and occasionally by longer side chains consisting of HR, or RG-I, or a
blend of both.
Molecular conformation varies with the linkage type in the backbone, as well as the
appendance of side chains. Soybean soluble polysaccharide (SSPS) is a pectin-like
polysaccharide and contains a backbone consisting of rhamnogalacturanon-I (RG-I)
intervened by homogalacturonan (HG) (Akihiro et al., 2001). This backbone was highly
branched by long side chains of α-(1,3/5)-arabinans and β-(1,4)-galactans, which
constituted 21 and 50 % of total sugar respectively. With 80 % of galactan side chains
77
removed by β-(1,4)-D-galactosidase (GPase), SSPS changed from an overall globular
shape (ρ=1.1) to a more linear random coil conformation (ρ=1.3), as indicated by the
increase of structure parameter ρ, but still differed from a linear random coil (ρ=1.7-2.0)
(Wang et al., 2005c). Knowledge of the conformation of both neutral and acidic fractions
from flaxseed hulls may assist in confirming their characteristic structure, and in
correlating the functions with their structures and shapes.
Light scattering is one of few techniques bringing a deep insight into molecular
structure by directly measuring the molecular parameters (Burchard, 2005). There are two
types of light scattering measurements, static and dynamic scattering. Static light
scattering measures the average total scattering intensity over a relatively long period
(1~2 s) and provides information on weight average molecular weight (Mw), the radius
of gyration (Rg), and the inter-particle interaction (the second viral coefficient, A2).
Dynamic light scattering measures the fluctuation of the intensity over time by detecting
the time-dependent autocorrelation function, and derives the hydrodynamic radius (Rh).
The objectives of the present study were to determine the molecular characteristics of
both neutral and acidic fraction gums from flaxseed hulls in aqueous solution, using static
and dynamic light scattering, and to correlate their conformational features with the
corresponding structural characteristics and functions.
78
6.2. Experimental
6.2.1. Materials
A neutral (NFG) and an acidic fraction gum (AFG) were separated from soluble gum
extracted from flaxseed hulls, using anion-exchange chromatography (Qian et al., 2012b).
6.2.2. Solution preparation
Dust should be first removed from sample solutions for all light scattering
measurements by consecutive filtration four times through a 0.2 μm (for solvents) or a
0.45 μm (for sample solutions) nylon filter to prepare dust-free solutions. Consecutive
filtration was also reported to be capable of eliminating macromolecular aggregates in
solutions (Wang et al., 2005c). Stock solutions of NFG and AFG were separately
prepared by dissolving each freeze dried fraction into MilliQ water at 80℃ for 1 h under
constant stirring. After cooling down to room temperature, each stock solution was
filtered through a 0.45 μm nylon filter 4 times to remove dust and diluted by MilliQ
water and/or 1 M NaOH, or by MilliQ water and/or 1M NaCl, into various concentrations
of solutions for both static and dynamic light scattering measurements. Each sample
solution was filtered directly into a cylindrical quartz cell (25 mm in diameter), which
was immersed in a decalin bath for each measurement. Samples were fully recovered
after filtration as indicated by the unreduced concentration of total sugar content in NFG
solutions or uronic acid content in AFG solutions.
79
6.2.3. Light scattering measurements
Both static and dynamic light scattering measurements were carried out on a
BI-200SM Brookhaven light scattering instrument (Brookhaven Instruments, Holtsvile,
New York, USA) with a He-Ne laser (637nm) as the monochromatic light source. This
instrument includes a precision Goniometer, a photomultiplier, and a 128-channel
BI-9000AT digital autocorrelator. Instrument alignment was conducted before every use
with well-filtered dust-free toluene. All measurements were carried out over the angular
range of 40°-150° at a room temperature of 25°C.
For static light scattering measurements, toluene (Rayleigh ratio: 1.40×10-5cm -1) was
also used as a reference. Data were presented by the Zimm plot method.
The results of dynamic light scattering measurement or the particle size distributions
were calculated by either the constrained regularization (CONTIN) method or
Non-Negatively Constrained Least Squares (NNLS) method using Brookhaven dynamic
light scattering software.
6.3. Results and discussion
6.3.1 The presence and elimination of aggregates
Various non-covalent interactions, such as van der Waals attraction, hydrogen
bonding, ionic and hydrophobic interactions (Wang et al., 2005a), may contribute
molecular aggregates to obtain a generally lower energy in solution systems. Elimination
of aggregates is critical to guarantee a precise measurement of molecular size and
80
conformation.
Molecular distribution was pre-measured by dynamic light scattering (at 90o) to check
the presence or elimination of aggregates in sample solutions. NaCl or NaOH solutions (0.
1 M and 0.5 M) were applied instead of water as solvents to help reduce or retard
aggregates by interrupting the hydrogen bonding and/or charge interactions (Li et al.,
2006). The molecular size distributions of AFG (0.5 M) in both solvents are shown in Fig.
6.1.
Fig. 6.1. Molecular size distribution of acidic fraction gums in different solvents
(a: 0.1 M NaCl; b: 0.5 M NaCl; c: 0.1 M NaOH; d: 0.5 M NaOH)
Fairly broad molecular distribution ranging from 20 to 600 nm was found (Fig. 6.1 a
& b) in either 0.1M or 0.5 M NaCl solutions and the mean hydrodynamic radius Rh
reduced from 77.3 to 55.6 nm with the concentration of NaCl increased from 0.1 M to 0.5
Diameter (nm)
a
b
c
d
81
M. The molecular distribution was narrowed to a range from 20 to 120 nm by 0.1 M
NaOH solutions, which was similar as in 0.5 M NaOH (20-88nm). However, the
molecular size turned to a broader distribution and the mean value of Rh also increased
with time gradually. Even in 0.5 M NaOH solution (Fig. 6.1 c & d), gradual increases in
the mean value of Rh from 29 to 63 nm were also detected due to a higher concentration
(0.8 mg/ml) of AFG within 6 h after filtration, indicating that aggregates were prone to
form gradually with time. The interval to form detectable aggregates reduced to 3 h for
1.0 mg/ml AFG in 0.5 M NaOH. Similarly, although NFG in both 0.1 and 0.5 M NaOH
solution had shown stable mean value of Rh at the beginning after filtration, yet 0.5 M
NaOH exhibited better prevention of aggregates with time. Therefore, both AFG and
NFG samples were detected for static and dynamic light scattering measurements
immediately after filtration before an aggregate initialization. Within a measuring period
of 24 h, no obvious degradation of NFG or AFG was detected.
6.3.2 Static light scattering
Static light scattering (SLS) determined weight average molecular weight (Mw),
radius of gyration (Rg), and the second virial coefficient (A2) using the Debye equation
(Debye, 1947):
cAqMRMRKc wgw 222 2)/(3/1/1/ ++=θ (6.1)
Where K is an optical contrast factor, c the polymer concentration, Rθ is the Rayleith
ration, and the scattering vector q is defined as
82
00 /)2/sin(4 λθπnq = (6.2)
With n0 is the refractive index and λ0 is the wavelength in a vacuum.
The SLS data of NFG and AFG are presented with the Zimm plot (Zimm, 1948) by
plotting Kc/∆Rθ against q2+kc (Fig. 6.2), where the k is a freely chosen constant. The
slope of angular dependence at c=0 referred to the z-average mean square radius of
gyration (Rg)2 . The second virial coefficient, A2, was obtained from the slope of
concentration dependence at θ=0. Weight average molecular weight Mw equaled the value
at the interception of two extrapolated lines at θ=0 and c=0.
Fig. 6.2. Static light scattering data presented in Zimm plot obtained from the
neutral (NFG, a) and acidic (AFG, b) fraction gums in 0.5 M NaOH at 25 °C
(mol/g)
0.0 1.0
7.339e-0
Sin2(θ/2)+32C
Kc/∆Rθ
3.126e-0
b C (mg/mL) 1.3 1.0 0.6 0.3 0.1
Kc/∆Rθ
1.322e-0
(mol/g)
4.932e-0
a C (mg/mL) 1.0 0.8
0.6 0.4 0.2
0.0 1.0 Sin2(θ/2)+56C
83
The parameters from obtained SLS, including Mw, Rg and A2, are summarized in Table.
6.1. The positive value of A2 for both fractions indicated the molecular-solvent
interactions were more favorable than molecular interactions, indicating that 0.5 M
NaOH was a good solvent.
Table 6.1. Molecular characteristics of the neutral (NFG) and acidic (AFG) fractions
obtained from static and dynamic light scattering
Mw
/kDa
Rg
/nm
A2
/(cm3mol/g2)
Rh
/nm ρ
NFG 616±19 49.2±3.4 9.8×10-4 27.1±1.5 1.8
AFG 285±25 38.2±2.7 9.3×10-4 29.6±2.3 1.3
(Where Mw: weight average molecular weight; Rg: the z-average radius of gyration; A2: the second
virial coefficient; Rh: the hydrodynamic radius; ρ: structural parameter.)
6.3.3 Dynamic light scattering
The dynamic light scattering measurements were applied for various concentrations
of both NFG and AFG in 0.5 M NaOH at different detecting angles (Fig. 6.3). The
dynamic radius Rh of both NFG and AFG in 0.5 M NaOH were independent of either
detection angles or sample concentrations, as no obvious correlation between Rh and
detection angle or sample concentration was found (Fig. 6.3).
The structural parameter ρ, which is equal to the ratio of Rg/Rh, is an indicator of
84
macromolecular conformation and depends on the molecular structure and polydispersity,
regardless of molecular mass (Burchard, 2004): a ρ value between 1.5-2.05 indicates a
typical random coil conformation, whereas lower ρ values between 1.0-1.3 suggest a
star-like conformation.
Fig. 6.3. The angular dependence (a: NFG, 0.2 g/L; c: AFG, 0.2 g/L) and
concentration dependence (at 90o, b: NFG; d: AFG) of the hydrodynamic radius (Rh
The structural parameter ρ of NFG was 1.8 (Table 6.1), indicating a random coil
conformation (Fig. 6.4c). This was in good agreement with the presence of longer side
chains in AFG (
)
of neutral (NFG) and acidic (AFG) fractions determined in 0.5 M NaOH.
Qian et al., 2012a) which increased the segment density. Whereas NFG
has a β-1,4-xylan backbone mono-, di- or un-substituted at O-2 and/or O-3 by short
branches mostly consisting of one to three sugar residues.
a b
c d
85
Fig. 6.4. Schematic representation of molecular structure (a & b) and conformation
(c & d) of neutral (NFG) and acidic (AFG) fraction gums
Lower value of ρ (1.3) for AFG (Table 6.1) suggested its star- like conformation (Fig.
6.3d) and relatively higher segment density. AFG, like soybean soluble polysaccharide
(SSPS) (Akihiro et al., 2001), was a pectin-like polysaccharide due to its
rhamnogalacturanon-I (RG-I) backbone. Thanks to the presence of high amount of
α-1,3/5-arabinans (21% of total sugar) and β-1,4-galactans (50% of total sugar) as side
chains, SSPS features a star-like conformation with a typical ρ value (1.1) (Wang et al.,
2005c). With removal of 80 % galactan branches, β-1,4-D-galactosidase treated SSPS
(GPase/SSPS) was changed into to a more linear conformation (ρ=1.3), but still varied
from a linear random coil (ρ=1.7-2.0). As the ρ value indicated, AFG seemed to exhibit a
more linear shape than natural SSPS, and to be more like GPase/SSPS. This could be
(a) NFG (b) AFG
(d) AFG
Xylose Glucose Arabinose Galactose
(c) NFG
86
traced to its high amount (19.6 %) of mono-galactosyl branches (Qian et al., 2012a),
which overall shortened its average side chain length compared to that of SSPs. Yet, its
non-linear-random-coil conformation was supposed to be attributed to the existence of
longer side chains, which imparted more substantial steric hindrance than
monosaccharide side chains.
The fine structure of pectin is still in dispute due to its complexity and heterogeneity
between plants and tissues (Willats et al., 2006). The controversy between conventional
and recently proposed alternative structural models of pectin is whether the HG domain is
inserted into its backbone or linked as a side chain. A similar question also exists in
revealing the structure of AFG. Although a small portion of HG exists in AFG, we
propose its presence in both backbone and side chains. A schematic of AFG representing
its proposed structure and conformation properties is proposed (Fig. 6.4b).
6.4. Conclusions
Sodium hydroxide (0.5 M) was capable of eliminating aggregates from both neutral
and acidic fraction solutions. To retard aggregates initialization with time at higher
concentrations (0.8-1.0 mg/mL), filtration through 0.45μm filter was carried out before
each measurement. The neutral fraction exhibited random coil conformation with a
molecular weight of 616±19 kDa. Whereas acidic fraction had a star-like conformation
with a molecular weight of 285±25 kDa due to the presence of longer side chains along
87
its backbone. The structural and conformational features of both fractions may account
for the distinctions in their flow behavior.
88
Chapter 7. Concluding Discussion
Soluble dietary fibre with low viscosity may be a solution to increase daily dietary
fiber consumption, as it can be included in the diet in a significant amount without
over-texturization. Research interests in low viscous polysaccharide have boosted the
potential use of soluble flaxseed gum in both academic research and industry
applications.
Flaxseed hulls, as a byproduct from the dehulling process of flaxseeds, are available
in large amount in Canada since it is the leading country in producing and exporting
oil-type flaxseed. The patented separation method and equipment applied to fractionate
hulls (occupy ~37 % in whole seed mass) from flaxseed kernels facilitated the aqueous
extraction of soluble flaxseed gum (SFG) from the gum-enriched hull fraction rather than
from the whole seed by reducing the costs and raising the extraction yields.
Two fractions, a neutral (NFG) and an acidic (AFG) fraction gums, distinct in
molecular structure, conformation and flow behavior, were obtained by efficient
fractionation of SFG using ion-exchange chromatography. For the first time, the detailed
primary structure of each fraction was revealed using methylation analysis and 1D/2D
NMR, and its corresponding conformation was also investigated with static and dynamic
light scattering.
Correlating macromolecular structure and conformation with its function is one of the
most challenging topics in polysaccharide studies. The flow behavior of each fraction
89
may be traced back to its primary structure and conformation. The NFG had β-1,4-linked
xylose backbone, which was mono-, di- or unsubstituted at O-2 and/or O-3 positions by
short side chains composed of one to three linearly linked sugar residues (24.8 %
arabinose, 15.5 % xylose and 9.4 % galactose/glucose). This primary structure of NFG
coincided with its random coil conformation. The limited flexibility of this random coil
chain was evident by a higher intrinsic viscosity (377.5 mL g-1), a higher Huggins
constant (0.54), and its shear-thinning behavior and relatively higher viscosity compared
with those of AFG. AFG had a highly-branched rhamnogalacturonan-I backbone
substituted at O-3 of 1,2-Rhap mostly by monomeric sugar units (total terminal sugar
units: 27.2 %) or occasionally by longer side chains with a similar structure as its
backbone. The attachment of longer side chains along the backbone of AFG was
confirmed by its star like conformation. The Newtonian flow behavior and low viscosity
arose from its low molecular weight (285±25 kDa) and star like conformation, and the
high flexibility indicated by a fairly low value of Huggins constant (0.16), as well as a
lower intrinsic viscosity (332.5 mL g-1).
With the knowledge of the flow behavior distinction between NFG and AFG,
adjusting the viscosity of soluble flaxseed gum to favored levels may be obtained by
targeting the mass ratio of NFG/AFG, which varies with flaxseed breeds. With
acknowledgement of the correlation between primary structure and flow behavior of each
fraction, targeting the mass ratio of NFG/AFG can be simplified into a detection of
monosaccharide composition.
90
Functionality analyses of soluble flaxseed gum were mainly restricted to its viscosity,
viscoelastisity, surface properties and emulsification in our research. For possible food
and nonfood application of soluble flaxseed gum, other functionalities like gel forming
ability, and potential physiological functions in the gastro-intestinal tract associated with
decreasing glycemic and insulemic response, reducing serum cholesterol, and
fermentability also deserve further investigations in the near future. When the gelling
potential and mechanism of soluble flaxseed gum is considered, the possible presence of
ferulic acid and its association with the neutral fraction needs to be examined, due to its
significant contribution to the gelation of various arabinoxylans from cereal cell wall
materials. The heterogeneity of the acidic fraction in soluble gums as indicated by the
heterogeneous Mw distribution by high performance size exclusion chromatography
(HPSEC), also need some attention especially when the molecular structure is concerned
with possible mechanisms related to its potential physiological properties.
91
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