Flaxseed gum from axseed hulls: Extraction, fractionation, and characterization

K.Y. Qian
a, b
, S.W. Cui
b,
*
, Y. Wu
b
, H.D. Goff
a
a
University of Guelph, Department of Food Science, Guelph, ON, Canada N1G 2W1
b
Agriculture and Agri-Food Canada, Guelph Food Research Centre, 93 Stone Rd. W., Guelph, ON, Canada N1G 5C9
a r t i c l e i n f o
Article history:
Received 24 September 2011
Accepted 21 December 2011
Keywords:
Flaxseed gum
Fractionation
Rheology
Intrinsic viscosity
Critical concentration
Emulsifying properties
a b s t r a c t
Soluble dietary bre with low viscosity has the potential to deliver acceptable mouthfeel and texture
when included in the diet in a signicant amount to show health benets. Soluble axseed gum (SFG)
was reported to have low viscosity, thus has potential in applications as a bre fortier. In the present
work, SFG extracted from axseed hulls was fractionated into a neutral fraction gum (NFG) and an acidic
fraction gum (AFG) using ion exchange chromatography. The protein content in SFG (11.8%) and AFG
(8.1%) were completely removed by protease to obtain two more protein-free fractions, SFGnP and
AFGnP; NFG contained no protein. The uronic acid content in NFG (1.8%) was mostly eliminated, whereas
in AFC increased to 38.7%. NFG consisted of high molecular weight (MW) (1470 kDa) arabinoxylans and
exhibited pseudoplastic ow behaviour; whereas AFG was mainly composed of rhamnogalacturonans
with a higher MW fraction (1510 kDa) and a lower MW fraction (341 kDa) and showed Newtonian ow
behaviour. The ranking of intrinsic viscosities (mL g
1
) in decreasing order was: SFG (446.0) > NFG
(377.5) > AFG (332.5). AFG was expected to have higher chain exibility for its lower value of Huggins
constant (0.16) compared to that of NFG (0.54) and SFG (0.48). In comparison with SFGnP, AFGnP and
NFG, SFG and AFG showed better surface activities and emulsifying stabilities due to the presence of
protein in both fractions.
Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Flax (Linum usitatissiumum L.) was rst introduced to North
America as a crop for structural bres, but its value and importance
as an oil source quickly became apparent (Cunnane & Thompson,
1995). Canada is the leading country in producing and exporting
oil-type axseed. Western Canadian axseed is composed of 45%
oil and 23% protein (1990e2008 means) (www.grainscanada.gc.ca).
The renewed interest in axseed as a food source is due to its health
benets attributed to its components including lignans (secoiso-
lariciresinol diglucoside (SDG) being the predominant form), a-
linolenic acid, and soluble axseed gum(SFG) (Hall Iii, Tulbek, & Xu,
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 1000 S
1
(Mazza &
Biliaderis, 1989). Its low viscosity is favoured in dietary bre forti-
cation in food without leading to an over-texturization, when
a signicant concentration of bre is required to show health
benets.
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 ax
meal, wheat andrye bran, due toits signicantly higher soluble bre
content and solubility (Fodje, Chang, & Leterme, 2009). Inclusion of
axseed in the diet of broiler chickens signicantly 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, Barbara, &Dominique, 2007; Theuwissen &Mensink, 2008).
The high short-chain fatty acids (acetate, propionate and butyrate)
productivity and selective stimulation of growth and/or activity of
probiotics (Gibson, Probert, Van Loo, Rastall, & Roberfroid, 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 andthe Firmicutes, were foundto have positive and
negative correlation with percentage loss of body weight (Ley,
Turnbaugh, Klein, & Gordon, 2006).
Flax mucilage (soluble axseed gum, SFG) occurs mainly at the
outermost layer of hull. This bre-rich hull is able to release muci-
laginous material (soluble gum) easily when soaked in water. In
earlier research analyses were based on the gum extracted from
the whole seed (Cui, Mazza, & Biliaderis, 1994; Diederichsen, Raney,
&Duguid, 2006; MazzaandBiliaderis, 1989; Muralikrishna, Salimath,
* Corresponding author. Tel.: 1 519 780 8028; fax: 1 519 829 2600.
E-mail addresses: kqian@uoguelph.ca (K.Y. Qian), cuis@agr.gc.ca (S.W. Cui),
Wuy@agr.gc.ca (Y. Wu), dgoff@uoguelph.ca (H.D. Goff).
Contents lists available at SciVerse ScienceDirect
Food Hydrocolloids
j ournal homepage: www. el sevi er. com/ l ocat e/ f oodhyd
0268-005X/$ e see front matter Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2011.12.019
Food Hydrocolloids 28 (2012) 275e283
& Tharanathan, 1987; Naran, Chen, & Carpita, 2008; Oomah,
Kenaschuk, Cui, & Mazza, 1995; Warrand et al., 2003, 2005a,
2005b) or ax meal (Fedeniuk & Biliaderis, 1994; Mueller, Eisner,
Yoshie-Stark, Nakada, & Kirchhoff, 2010). With the success of
a patented dehulling process (Cui & Han, 2006), the whole axseed
couldbeefcientlyseparatedintoa kernel (w63%) fractionanda hull
(w37%) fraction (Cui, 2000) in large scale. Investigating the compo-
sition, structure, physicochemical and rheological properties of SFG
from hulls will assist in exploring its potential for the food industry.
Inthis current study, the gum(SFG) extracted fromaxseed hulls
was further separated into neutral (NFG) and acidic (AFG) fraction
gums using ion exchange chromatography (IEC). The chemical
(neutral monosaccharide composition, uronic acid and protein
content), physical (molecular weight distributionandheat stability),
rheological (viscosity, viscoelasticity, intrinsic viscosity and critical
concentration,) and functional (surface tension and emulsication)
characteristics of SFG and its two fractions are reported.
2. Methodology
2.1. Materials
Flaxseed hull (variety Bethune) was supplied by Natunola
Health Biosciences (Winchester, Ontario, Canada).
2.2. Extraction of gum
The extraction of the gum was conducted at room temperature
as shown on the owchart in Fig. 1. A batch of 500 g of axseed hull
was soaked in 4 L of distilled water overnight under gentle stirring.
A coarse ltration followed using cheesecloth to separate the hull.
The ltered 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 poly-
saccharide. The precipitate was recovered by centrifugation of the
mixture at 3000 g for 10 min and redissolved in distilled water at
approximately 4e5 (w/v) % before lyophilization.
2.3. Fractionation and purication of gum
2.3.1. Fractionation
The procedure of fractionation followed that of Warrand et al.
(2003) with minor modication: the washing solution of 0.1 M
NaOH was replaced by 2 M NaCl and 1 M NaOH. A owchart of the
fractionation procedure is also shown in Fig. 1. For each fraction-
ation, 0.35 g of SFG was dissolved in 1 L of buffer 1 (20 mMTris/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-Sephrose fast
ow as matrix) at a ow rate of 10 mL min
1
for 100 min and
ushed 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 w90% of NFG was eluted out. The column
was successively washed with 1 L of buffer 2 (20 mMTris/HCl 1 M
NaCl, pH 8) at the same ow rate (10 mL 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 ushed 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 2e3 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 Scientic) against distilled water at 25

C for 72 h,
and freeze-dried.
2.3.2. Purication
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 SFGor 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 puried protease (Mega-
zyme 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 dia-
lyzed (MW cut-off 3.5 kDa, Fisher Scientic) against distilled water
at 25

C for 72 h, and freeze-dried.
2.4. Pysico-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, Wood, Blackwell, &
Nikiforuk, 2000). The hydrolysis procedure followed that of Wu,
Cui, Eskin, and Goff (2009). Uronic acid analysis was conducted
Fig. 1. Flow chart for extraction, fractionation and characterization of soluble axseed gum (SFG: soluble axseed gum; NFG and AFG: neutral and acidic fraction gum; IEC: ion
exchange chromatography; Buffer 1: 20 mM Tris/HCl, pH 8).
K.Y. Qian et al. / Food Hydrocolloids 28 (2012) 275e283 276
using the m-hydroxyphenyl colorimetric method (Blumenkrantz &
Asboe-Hansen, 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 con-
sisting of an HPLC systemwith 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 ltered through
0.2 mmlters (Millipore, Fisher Scientic). 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
ltered through disposable 0.45 mm lters (Millipore, Fisher
Scientic) before injection, and aliquots (100 mL) of samples were
injected into the column (Shodex OHpak SB-806M HQ, 7.8 mm
ID 300 mm, column temperature at 40

C) and eluted at a ow
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
22,800) were used as MW markers to calibrate the column.
To investigate the association of protein and polysaccharides,
aliquots (20 mL) of 1 g 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

C. Separation was carried out at a ow rate of
1.0 mL/min in the same mobile phase (100 mM sodium
nitrate 5 mM Sodium azide).
Both steady shear and oscillation measurement of axseed 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 at shear rates ranging from 0.01 to100 s
1
. Visco-
elastic properties of samples were measured at 20 g L
1
and 25

C.
Intrinsic viscosity and critical concentration measurement fol-
lowed that of Doyle, Lyons, and Morris (2009). Gum dispersions
were heated at 80

C for 1 h and cooled down. Salt solutions (0.2 M)

were prepared individually before mixing with gum solutions
under constant stirring at ambient temperature, and salt concen-
tration in each nal 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 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 ([h]) was calculated using the following rela-
tionships (Harding, 1997).
h
rel
= h=h
s
(1)
h
sp
= h
rel
1 (2)
h
red
= h
sp
=c = [h[ K
H
[h[
2
$c (3)
h
inh
= (lnh
rel
)=c = [h[ K
K
[h[
2
$c (4)
where h and h
s
are the zero-shear viscosities (the constant apparent
viscosities in a limited Newtonian region at very lowshear rates) of
the solution and solvent (MilliQ H
2
O); h
rel
and h
sp
are two dimen-
sionless parameters of relative and specic viscosity; and h
red
and
h
inh
represent reduced viscosity and inherent viscosity, respec-
tively. K
H
(generally positive) and K
K
(generally negative) are
Huggins and Kraemer constants in Huggins and Kraemer equations
(Eqs. (3) and (4)). Both equations are valid only for solution
viscosity up to twice of the solvent viscosity (i.e. h
rel
_ 2). Beyond
this region, with concentration increase polymerepolymer inter-
actions will progressively become signicant, thus higher-order
terms (c
2
, c
3
, etc.) will no longer be negligible. A lower limit of
h
rel
_ 1.2 is also required to eliminate the increasing errors in h
sp
as
h
sp
approaches 0. Concentrations of each gum fraction adopted
were conned to this region (1.2 _ h
rel
_ 2). The value of intrinsic
viscosity ([h]) was reported as the mean of both intercepts in linear
functions (3) and (4) by plotting h
sp
/c and lnh
rel
/c against c and
extrapolating each linear trendline to zero concentration.
2.5. Functional properties
2.5.1. Surface activity
The surface tension of the airewater interface was measured by
the Du Nouy ring method using the Fisher Surface Tensiomat
(model 21, Fisher Scientic, Nepean, ON) (Izydorczyk, Biliaderis, &
Bushuk, 1991). SFG and its fractions were dissolved in 20 mL of
distilled water at various concentrations, heated at 80

C for 1 h
under gentle stirring and then left unperturbed for 2 h before
measurements. The surface tension (dyn cm
1
) of distilled water
and gum solutions were measured every 5 min for 6 times at 25

C.
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, nal gum
concentrations were adjusted to 0.44% wt) using Polyton (Kine-
matica 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 5000 psi.
The particle size distribution of the emulsions was measured
using integrated light scattering (Mastersizer X, Malvern South-
borough, MA) (Khallou et al., 2008). The measurements were
performed within one day after emulsion preparation as phase
separation occurred in all the emulsions within 24 h. The emulsi-
fying stability was determined by comparing the shifts of the oil
droplet size distributions.
3. Results and discussions
3.1. Extraction, fractionation and chemical composition
The composition and yield of axseed gumhas been reported to
vary with extraction conditions, as well as culture environment and
genotype. Gum yield from axseed increased from 4 to 9.4 % with
extraction temperature increased from 25 to 80

C (Fedeniuk &
Biliaderis, 1994). Broad variations of 3.6e8.0% among 109
cultivars (80

C, 2 h) and 5.4e7.9% among 12 genotypes (85

C,
3 h) were also found by Cui, Kenaschuk, and Mazza (1996) and
Oomah et al. (1995), respectively. Soaking axseed with a water:
seed ratio of 13 at 85e95

C and pHfrom6.5 to 7.0 for 3 h was found

to be the optimumgumextraction condition to achieve higher yield
and quality, using response surface methodology (Cui, Mazza,
Oomah, & Biliaderis, 1994). However, higher extraction tempera-
ture induced higher protein content and browning in colour
(Fedeniuk & Biliaderis, 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.
K.Y. Qian et al. / Food Hydrocolloids 28 (2012) 275e283 277
The yield and chemical composition of SFG, NFG and AFG are
shown in Table 1. 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 insufcient
unbinding process while washing AFG out. The uronic acid content
in AFG was about 20 fold of that in NFG, suggesting an efcient
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 2. No signicant differences were found
between SFG and SFGnP, implying the removal of protein via
enzymatic hydrolysis from SFG fraction did not modify the 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 signicant, since AFG occupied only w27%
of SFG. The neutral monosaccharide composition of SFG, NFG and
AFGwas comparable with previous results (Cui, Mazza, & Biliaderis,
1994; Fedeniuk and Biliaderis, 1994). NFG was mainly composed of
xylose (68.2%) and arabinose (20.2%) therefore being identied 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 amount of xylose (8.9%) and arabinose (2.9%) in AFG might be
due to the residual fraction of NFG(Cui, Mazza, &Biliaderis, 1994), or
might be derived from side chain components covalently linked to
its backbone (Naran et al., 2008).
3.2. Physical characterization
The molecular weight (MW) distribution of SFG, NFG and AFG
are shown in Fig. 2(a). NFG consisted of one fraction with high MW
of approximately 1470 kDa. By contrast, AFG consisted mainly of 3
portions of MW: 1510, 341 and 6.6 kDa, respectively. When sub-
jected to heat at 80

C from1 to 3 h, the MWof NFG decreased from

1470 to 850 kDa. Similarly, a signicant reduction of intermediate
MW (340 kDa) polymers and the appearance of additional small
MW molecules (2.6 kDa) also implied the degradation of AFG.
The UV and DRI response in Fig. 2(b) depicts the association of
protein and polysaccharides. No protein was detected in NFG. The
major peak of high-MW protein was eluted out at around 32 min,
signicantly later than two major high-MW polysaccharide peaks
(at w26 and w30 min, respectively) in both SFG and AFG. The
differences in elution time between these portions 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 w37min, making it
difcult to reveal their association. That the protein could be
completely removed by protease hydrolysis (in Section 3.1) also
supported this conclusion, as it was reported that built-in protein
could be very difcult to remove (Garti & Reichman, 1994).
3.3. Rheological properties
3.3.1. Viscosity and viscoelasticity
Flaxseed gum exhibited relatively low viscosity. At a concentra-
tion of 0.3% (w/v), the viscosity of axseed gum was only around
half of that of guar gumand locust bean gum(Mazza and Biliaderis,
1989). The steady shear ow curves and dynamic rheological
properties of SFG and its fraction solutions at 25

C from this study
are shown in Fig. 3. SFG and NFG exhibited pseudoplastic ow
behaviour at concentrations above 0.5 and 1.0%, respectively, over
a wide range of shear rate; whereas the steady shear owcurves of
AFG were more Newtonian-like at all concentrations examined.
This was in good agreement with those reported earlier by Cui,
Mazza, and Biliaderis (1994). Mazza and Biliaderis (1989) also
found that axseed gum solutions exhibited pseudoplastic ow
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. 3b). In previous work by Cui,
Mazza, and Biliaderis (1994), dispersions of crude, dialyzed ax-
seed gums and neutral fractions all exhibited weak gel properties.
The discrepancy may be due to the different sources of raw mate-
rials and extraction procedures.
3.3.2. Intrinsic viscosity
Intrinsic viscosity, denoted as [h], is a parameter reecting the
hydrodynamic volume occupied by the polymer. The value of [h]
primarily depends on the molecular size and chain rigidity of the
polymer, as well as the solution quality (Lapasin & Pricl, 1995).
Intrinsic viscosity increases with molecular weight (MW) according
to the MarkeHouwink relationship,
[h[ = KM
a
(5)
where K is a constant, M is the average MW and, a is an exponent
constant related to the chain exibility. For polyelectrolytes, the
presence of counterions in the solution will affect their intrinsic
viscosity primarily through the reduction of intramolecular repul-
sion among groups with the same charges. With increasing
concentration of salts (i.e. increasing ionic strength), the [h] of both
SFG and AFG will decrease signicantly as they contain acidic
groups (see uronic acid content inTable 1). The relation between [h]
Table 1
Yield and chemical components of axseed 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 axseed gum; NFG and AFG: neutral and acidic fraction gum; nd: not
determined; all data were calculated on a dry basis.
a
yield was based on axseed hull mass.
b
yield was based on SFG mass.
Table 2
Neutral monosaccharide in axseed gum and its fractions (%).
SFG SFGnP NFG AFG AFGnP
Fucose 7.0 0.2
c
7.0 0.4
c
nd 14.7 0.3
b
16.5 0.1
a
Rhamnose 16.5 0.6
c
14.5 0.3
c
nd 38.3 2.3
a
32.8 0.7
b
Arabinose 12.7 0.1
b
12.6 0.5
b
20.2 0.1
a
2.9 0.4
c
3.0 0.1
c
Galactose 22.4 1.0
c
22.5 0.7
c
7.9 0.1
d
35.2 0.4
b
39.7 0.4
a
Glucose 2.7 0.1
b
3.1 0.3
ab
3.7 0.4
a
nd nd
Xylose 38.6 1.2
b
40.9 2.1
b
68.2 0.6
a
8.9 1.3
c
7.9 0.3
c
SFG: soluble axseed 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 signicantly different by Duncans multiple-range test; nd:
not determined.
K.Y. Qian et al. / Food Hydrocolloids 28 (2012) 275e283 278
and ionic strength has been proposed by Pals and Hermans as
follows (Harding, 1997):
[h[ = [h[
N
SI
0:5
(6)
where [h[
N
is the intrinsic viscosity at innite 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 param-
eter, B, is dened by
S = B$

[h[
I=0:1

y
(7)
where [h[
I=0:1
is the intrinsic viscosity at 0.1 M ionic strength, and
y = 1:3 0:1. This modied parameter B could be used to compare
the relative stiffness of polymers even without knowing their MW.
It has been reported that axseed polysaccharide was between
a exible and semi-exible polymer with a B value of w0.018,
which lies between w0.005 (for xanthan gum with stiff confor-
mation) and w0.045e0.065 (for carboxymethylcellulose with
a exible chain) (Goh, Pinder, Hall, & Hemar, 2006). As a rough
guide, Huggins constant K
H
, also reveals the general conformation
of a polymer. The value of K
H
can be as high as w2 for uncharged
spheres. Lower value is expected for more extended biopolymer,
such as w0.35 for exible biomolecules (Harding, 1997). Goh et al.
(2006) reported the K
H
value for ax meal polysaccharide equal to
0.34 0.05, which also implied its exible conformation. The
values of K
H
for SFG, NFG and AFG are shown in Table 3. K
H;NFG
(0.54) was slightly higher than K
H;SFG
(0.48) and both were much
higher than K
H;AFG
(0.16), implying the chain exibility of SFG was
between that of NFG and AFG, as SFG is composed of both fractions.
The Huggins and Kraemer plots of SFG, NFG and AFG are shown
in Fig. 4(a) and the intrinsic viscosities ([h], in 0.1 M NaCl) of SFG,
NFG and AFG are 446.0, 377.5 and 332.5 mL g
1
, respectively
(Table 3). NFG had a larger intrinsic viscosity than AFG, probably
due to its higher molecular rigidity and average MW (see Fig. 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 pres-
ence of the synergistic interaction between NFG and AFG. Signi-
cantly higher intrinsic viscosities of deacetylated xanthan-guar
mixtures have been reported in comparisonwith those expected on
the assumption of no synergistic interactions (Khouryieh, Herald,
Aramouni, & Alavi, 2007). Goh et al. (2006) reported a linear rela-
tion between [h] of axseed gum and I
0:5
as follows:
[h[ = [h[
N
SI
0:5
= (289 4) (52:5 0:9)$I
0:5
, according to
which the value of [h] in 0.1 and 1 M NaCl would be approximately
455 and 342 mL g
1
, respectively. The former value was comparable
to the [h] of SFG (446.0 mL g
1
in 0.1 M NaCl), but higher values of
[h] (in 1 M NaCl) ranged from 434.0 to 657.8 mL g
1
for ve
genotypes of ax were also reported (Cui & Mazza, 1996).
Fig. 2. Molecular weight (DRI) distributions of polysaccharides, column temperature 40

C (a); and their protein (UV) fractions, column temperature, 40

C (b) (SFG: soluble
axseed gum; NFG and AFG: neutral and acidic fraction gum; the unit of numbers in (a) is KDa).
Fig. 3. The steady shear ow curves (a) and dynamic rheological properties (b) of SFG, NFG and AFG solutions (SFG: soluble axseed gum; NFG and AFG: neutral and acidic fraction
gum).
K.Y. Qian et al. / Food Hydrocolloids 28 (2012) 275e283 279
3.3.3. Critical concentrations
The dependence of viscosity on concentration and intrinsic
viscosity could be expressed by a power law relationship (Doublier
& Launay, 1981; Morris, Cutler, Ross-Murphy, Rees, & Price, 1981;
Ross-Murphy, 1994):
h
sp
= k$(c$[h[)
n
(8)
where h
sp
is the zero-shear specic viscosity and can be obtained
by Eqs. (1) and (2) (see Section 2.4); the degree of space occupancy
(c[h]) is also called the coil overlap parameter, in which c is the
concentration and [h] is the intrinsic viscosity; the value of (lg k)
and n equal to the extrapolated interception and the slope of each
linear region, respectively, in the double logarithmic plots
(Fig. 4(bed)) for SFG, NFG and AFG. A drastic change of slope was
found in each of the three fractions (Fig. 4(bed)) at a specic
degree of space occupancy ((c[h])
cr
). The corresponding concen-
tration at this point is called the critical concentration (c
cr
). Solu-
tions with concentrations below and above c
cr
are referred to as
dilute and semi-dilute solutions, respectively. In dilute solution,
the increase in viscosity is caused by interfering the owof 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 polymerepolymer interaction, as the overlap and
interpenetration of neighbouring polymer chains are no longer
negligible (Morris & Ross-Murphy, 1981). 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 on each trendline. Therefore critical concen-
tration is an approximate measure for the onset of overlap and
entanglement between polymer chains. The critical concentra-
tions (c
cr
, mL g
1
) for SFG (w6.61), NFG (w6.68) and AFG (w6.43)
were similar to each other, irrespective of their signicant differ-
ences in intrinsic viscosities (Table 3), and the corresponding
values of (c[h])
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., 1981).
The values of n
1
were conned to 1.08e1.21 (Fig. 4) and were
comparable with the value (1.1e1.4) reported (Doyle et al., 2009;
Morris et al., 1981; Wang & Cui, 2005). The n
2
for SFG (3.28) and
NFG (3.51) were close to the typical value (w3.3) for various
disordered polysaccharides (Morris et al., 1981), though higher
value (n
2
= w5) was also found in the chain of several gal-
actomannans (Doyle et al., 2009) due to the hyperentanglement
of unsubstituted mannan regions. Compared with SFG and AFG,
a much lower value of n
2
(2.17) detected for AFG may probably also
be due to its relatively higher exibility and thus be less likely to
form an entanglement network, just as implied from its lower
value of K
H;AFG
(0.16).
3.4. Functional properties
Most water soluble polysaccharides are known to act as stabi-
lizers in oil-in-water emulsions, only a few can act as emulsiers
mainly due to the presence of hydrophobic groups, such as protein.
Gum Arabic is mostly used as an emulsier for the built-in
proteinaceous moieties, which have hydrophobic groups that can
adsorb onto the oil phase (Garti & Leser, 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 signicant role in their stabilizing action (Garti and
Reichman, 1994). The adsorption mechanism, however, for either
protein or the gums with trace protein left are still not clear.
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 (in
dyn cm
1
) of dispersions decreased slightly with the addition of
SFGnP, AFGnP and NFG (0.01e0.5%, w/v) (Fig. 5). SFG and AFG
reduced the surface tension to 55.0 0.5 and 55.6 0.3 dyn cm
1
,
respectively at a concentration of 0.5%, which is comparable with
the value of the solution containing 0.5% of guar gum (Huang,
Kakuda, & Cui, 2001). After protein removal, SFGnP (62.0 0.5)
and AFGnP (65.4 1.7) reduced the surface tension to the same
level as NFG (62.8 2.5) did at an equivalent concentration (0.5%),
implying that the protein fractions in both SFG and AFG were
responsible for their higher surface activity.
Fig. 4. The intrinsic viscosity (a) and the dependence of viscosity on concentration (bed) of SFG, NFG and AFG (SFG: soluble axseed gum; NFG and AFG: neutral and acidic fraction
gum; viscosity was measured in 0.1 M NaCl (aed). Reduced viscosity (h
red
= h
sp
/c) and inherent viscosity (h
rel
= (lnh
rel
)/c) versus concentration are plotted with empty and solid
symbols (a), respectively; the common intercept gives [h], and the positive and negative slopes are K
H
[h]
2
and K
K
[h]
2
, where K
H
and K
K
are Huggins and kraemer constant,
respectively (a). n
1
and n
2
represent the slope of each region; the gum concentration for the circles within the squares in gure (bed) equals to 5 g L
1
).
Table 3
The intrinsic viscosity ([h]), Huggins constant (K
H
), critical concentrations (c
cr
) and
coil overlap parameter values ((c[h])
cr
) at critical concentrations of SFG, NFG and
AFG.
SFG NFG AFG
[h] (mL g
1
) 446.0 3.4 377.5 5.7 332.5 6.8
K
H
/ 0.48 0.54 0.16
c
cr
(g L
1
) 6.61 6.68 6.43
(c[h])
cr
/ 2.95 2.52 2.14
SFG: soluble axseed gum; NFG and AFG: neutral and acidic fraction gum.
K.Y. Qian et al. / Food Hydrocolloids 28 (2012) 275e283 280
3.4.2. Emulsifying stability
The volumeelength diameter, D[4,3], is sensitive to the presence
of large particles (i.e. sensitive to occulation 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 sumof the volume ratio of droplets in each size-class multiplied
by the mid-point diameter of the size-class (McClements, 2005):
D[4; 3[ =
X
i =1
n
i
d
4
i
=
X
i =1
n
i
d
3
i
=
X
i =1
f
i
d
i
(9)
where D[4,3] is the volume fraction-length mean diamerter, n
i
, d
i
and f
i
represent the number, mid-point diameter, and the volume
fraction of each size-class.
The top ve factors affecting the rate of creaming, thus the
stability of an emulsion, 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 mm) 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 mm) was found, phase separation
occurred within 4 days after emulsication, indicating the higher
viscosity by addition of 1% SFG retarded the rate of occulation
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
(6.4e6.7 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
50
55
60
65
70
75
0 0.1 0.2 0.3 0.4 0.5
S
u
r
f
a
c
e

t
e
n
s
i
o
n

(
d
y
n
e
s
/
c
m
)
NFG
AFGnP
SFGnP
AFG
SFG
Concentration (%, w/v)
Fig. 5. Reduction of surface tension of dispersions by SFG and its fractions (SFG and
SFGnP: soluble axseed gum with and without protein; NFG: neutral fraction gum;
AFG and AFGnP: acidic fraction gum with and without protein; surface tension of
distilled water: 72.3 0.1 dyn cm
1
).
Fig. 6. The shifts of D[4,3] and particle size distribution proles of emulsions stabilized by SFG and its fractions within 24 h after emulsication with 2% wt of canola oil (SFG and
SFGnP: soluble axseed gum with and without protein; NFG: neutral fraction gum; AFG and AFGnP: acidic fraction gum with and without protein).
K.Y. Qian et al. / Food Hydrocolloids 28 (2012) 275e283 281
distributions and D[4,3] values of emulsions with the above ve
fractions are shown in Fig. 6. Monodispersed droplet size distri-
bution and stable D[4,3] values were found in emulsions containing
SFG and AFG from 3 to 24 h after emulsication, whereas the
volume fraction (%) of the second peak (larger size particles), as
well as the D[4,3] values, increased signicantly within 24 h for all
three protein-free fractions (SFGnP, NFG and AFGnP), which again
conrmed 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.
4. Conclusions
Flaxseed gum may become a signicant source of soluble bre
for both its availability and low viscosity. Two fractions, neutral
(NFG) and acidic (AFG), of soluble axseed 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 and had associated 8% of protein, but
not covalently linked. Both fractions were stable at 80

C for 1 h,
however, extended heating resulted in degradations of the poly-
mers. 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 signicant contribution of the
protein fractions to their emulsication, regardless of their
distinctions in molecular conformation (molecular mass and chain
exibility) and rheological properties. However, the differences
between these two fractions in MWdistribution and chain stiffness
signicantly affected their intrinsic viscosity and viscosity depen-
dence on the concentration in both dilute and semi-dilute
solutions.
Acknowledgements
The authors would like to thank Edita Verespej, Cathy Wang and
Ben Huang for their technical assistance. Special thanks are also
due to Prof. Shao-Ping Nie and Qing-Bin Guo for their helpful
suggestions and support.
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