IJCB 53B(3) 332-338

Indian Journal of Chemistry
Vol. 53B, March 2014, pp.332-338
Generation and characterization of complex bioactive oligosaccharides from flax
meal by a combination of enzyme hydrolysis, HPAEC and MALDI-TOF-MS
Sayani Ray, Utpal Adhikari & Bimalendu Ray*
Natural Products Laboratory, Department of Chemistry, The University of Burdwan, Burdwan 713 104, India
E-mail: [email protected]
Received 17 December 2012; accepted (revised) 20 September 2013
This study aimed at analyzing complex neutral and acidic oligosaccharides generated from the hemicellulosic
polysaccharides (4A and 4B) of flax meal. A 86 kDa xyloglucan rich population (4Baq) purified from soluble 4B fraction by
anion exchange chromatography was digested with endo-(1→4)-β-D-cellulase. Analysis of the resulting fragments by
chemical methods as well as high performance anion exchange chromatography (HPAEC) and matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) showed the presence of penta(1)-, hexa(2)-,
hepta(3)-, octa(4)-, nona(5,6)-and deca(7)-saccharides as the building sub-units. Hemicellulose 4A is composed of a linear
chain of β-D-xylopyranosyl units, bonded together by (1→4)-glycosidic links, containing a single D-xylopyranosyl, 4-Omethyl-D-glucopyranuronosyl, D-glucopyranuronosyl residues joined by glycosidic links to position 2 of the xylose units of
the main chain, in proportions of one branch to every eight units of xylose.
Keywords: Flax meal, enzyme hydrolysis, oligosaccharides, GC-MS, HPAEC, MALDI-TOF-MS
In a preceding paper1 it was shown that 30% of the
flax meal (FM) could be solubilised during sequential
extraction with water at 30-35°C (WE),
1,2-cyclohexanediaminetetraacetic acid (CT), water at
100°C (fraction named HWE) and 1M KOH (B).
Partial
chemical
characterization
of
the
polysaccharides present in WE, CT, HWE and B
fractions revealed the presence of high molar-mass
mucilage-like
polysaccharides,
arabinogalactan
protein,
glucans,
xyloglucan
and
xylan.
Hemicelluloses constitute a complex group of
heterogeneous polysaccharides and represent one of
the major sources of renewable organic matter in
nature2. Since flax is an important commercial crop,
and as hemicellulosic polysaccharides have
considerable potential for application as precursor for
microbe-fermented ethanol or other molecules3,4,
health-related value-added products5-7, and in foods8,9,
pharmaceuticals10,11, paper and cotton12,13 industries
further studies on purified polymers will be of interest
from scientific as well as industrial purposes.
Oligosaccharides often posses some unique properties
which is of special value to the biological entity14,15.
Xylo-oligosaccharides, for example, are reported to
enhance growth of bifidobacteria and are frequently
defined as prebiotics16. Because of its anti-cancer
property xylitol, has already been used in food
applications, e.g., chewing gum and tooth paste17.
Several oligosaccharins regulate plant growth,
organogenesis, and defense against pathogens15.
Oligosaccharides possess antimicrobial effects against
pathogenic bacteria or fungi18 and hence might be
useful to substitute chemical additives19 for food
preservations. Therefore, the oligosaccharides
generated from flax meal will be potential raw
material for various industries. The present study
reports on the structural features of hemicellulosic
polysaccharides present in 4M KOH extracted
fractions (4A and 4B). In particular, isolation of a
xyloglucan rich pool (4Baq) by anion exchange
chromatography of 4B fraction and structural
elucidation of oligosaccharides generated by
endo-glucanase digestion of this fraction is described.
Analysis of the resulting oligosaccharides (Scheme I)
was carried out by combination of chemical,
chromatographic and matrix-assisted laser desorption
ionisation-time
of
flight-mass
spectrometric
techniques. In addition, structural features of xylan
present in 4A fraction as obtained by endo-xylanase
digestion and structural characterization of the
generated oligosaccharides have also been reported.
Results and Discussion
Isolation
and
sugar
composition
of
hemicelluloses. Hemicellulosic polysaccharides were
isolated from the depectinated cell wall1 of Linum
RAY et al.: COMPLEX BIOACTIVE OLIGOSACCHARIDES
333
Scheme I
usitatissimum meal by extraction with 4M KOH. This
extract, which forms precipitate during neutralisation,
was then separated into two fractions: the
hemicellulose
fraction
4A
(yield,
4.5%)
corresponding to the precipitate and the hemicellulose
fraction 4B (yield, 5.5%) corresponding to the soluble
polymers. Sugar analysis of the latter fraction mainly
revealed the presence of xylose, glucose and galactose
suggesting the presence of xyloglucan (Table I). In
contrast, xylose was the main monosaccharide of the
hemicellulose 4A fraction indicating the presence of
xylan. Other monosaccharides, such as arabinose,
galacturonic acid and rhamnose, probably arose from
pectic material co-extracted with hemicelluloses.
FT-IR and molecular mass of xyloglucan rich
fraction. FT-IR spectrum of fraction 4B showed
absorption bands at 1644, 1420 and 1055 cm-1, which
are indicative of hemicelluloses20. The broad band
between 3600 and 3000 cm-1, corresponding to
vibrations of the hydroxylic band as well as the
methyl and methylene group vibrations around 2931
cm-1 were present in this spectrum. The small band at
895 cm-1 characteristics of β-glycosidic linkages
between the sugar units21 was also observed. Sugar
composition of 4B fraction indicates the probable
presence of xyloglucan type polysaccharides. But
composition analysis by simple acid hydrolysis may
yield ambiguous information22. So, attempt has been
made to purify the xyloglucan present in fraction 4B,
by passing it through DEAE-Sepharose FF column.
On total sugar basis the recovery yield from the anion
exchanger was 95%. Moreover, 39% of the recovered
sugar material was eluted with water (designated as
4Baq) and the bound material (61%) was eluted from
the column by using salt gradient. Sugar
compositional analysis of the non-retained fraction
(4Baq) shows the presence of glucose and xylose as
the major constituent sugars together with smaller
amount of other sugars (Table I). Therefore, 4Baq
fraction contains xyloglucan type polysaccharide.
This macromolecule gave a single narrow band on
size exclusion chromatography, having an apparent
molecular weight of 86 000 Da and a specific rotation
of [α]25D +9.2° (c 1.02, 1 M sodium hydroxide). The
INDIAN J. CHEM., SEC B. MARCH 2014
334
assignments of the D-, L-configuration to the different
sugar moieties are based on the literature precedents
for other hemicelluloses23.
Generation and sugar composition of xyloglucan
oligosaccharides (XGO). It is well known that
endo-(1→4)-β-D-glucanase
cleaves
(1→4)-β-Dglucosidic linkages of xyloglucan next to an
unbranched glucose residue22, without damaging side
chains. The xyloglucan rich population (4Baq) on
treatment with endo-(1→4)-β-D-xylanase generates a
water soluble xyloglucan-oligosaccharide rich
fraction (designated as XGO) containing Xyl
(ca. 32%), Glc (ca. 47%), Gal (ca. 13%), and Man
(ca. 7%) residues as major sugars (Table I). The
sugar composition is thus consistent with the presence
of galactoxyloglucan. The mannose present in 4Baq
was probably originated by hydrolysis of the mannan
with endo-mannanase present as contaminant in the
Table I — Sugar composition (mol %) of hemicellulosic
polysaccharides of Flax meal (4A and 4B) and of fractions
generated there from by digestion with endo-xylanase (fraction
XO from 4A) and endo-glucanase (fractions XGO and FMXGO
from 4B and FM, respectively), and by anion exchange
chromatography (fraction 4Baq from 4B)
Rha Fuc Ara Xyl Man Gal Glc GlcA GalA
4B
3
4A
tr
4Baq
tr
XGO
nd
FMXGO tr
XO
tr
tr: trace
nd: not detected
1
tr
2
1
tr
tr
9
1
5
tr
tr
tr
27
97
30
32
34
98
2
nd
4
7
6
tr
11
tr
13
13
14
tr
36
1
40
47
46
tr
tr
1
nd
nd
nd
1
11
tr
6
nd
nd
Tr
commercial endo-glucanase preparation used in this
study.
Glycosyl linkage composition of xyloglucan
derived oligosaccharides (XGO). Methylation of
XGO produced a yellow, solid product that was
hydrolyzed, and the resulting sugars were converted
into their corresponding partially methylated alditol
acetates and analyzed by GC and GC-MS. The results
obtained are shown in Table II. The presence of
(1→4,6)-and (1→6)-linked glucopyranosyl residues,
typical for the cellulosic backbone of xyloglucan is
revealed. Terminal fucose, galactose and xylose (all)
and (1→2)-linked xylose and (1→2)-linked galactose
are also present. Together, these results demonstrate
the presence of xyloglucan thus confirming sugar
compositional data. Similarly, terminal mannose and
(1→4)-Manp residues are likely due to result from the
permethylation of the contaminating mannan
fragments. These data confirm the results of sugar
compositional analysis where degradation of mannan
by commercial endoglucanase preparation has been
inferred.
Matrix-Assisted Laser Desorption IonizationTime of Flight-mass spectrometry (MALDI-TOF
MS). MALDI-TOF mass spectrum of XGO revealed
the presence of numerous oligosaccharide fragments.
Taking into consideration the mode of action of the
endo-(1→4)-β-D-glucanase, sugar composition and
linkage analysis data of fragments present in XGO
fraction and on the basis of the molecular masses of
the known xyloglucan oligosaccharides24-29, tentative
structures for the xyloglucan-derived oligomers are
proposed. For example, m/z value of 791 corresponds
to Hex3Pent1 and it is, therefore, assigned as XGGG
Table II — Methylation analysis of native xylan (4A), reduced xylan (4AR) and of xyloglucan
oligosaccharides derived from Linum usitatissimum meal
Methylation products
m/z values
(4A)
2,3,5-Arab
2,3,4-Xyl
2,3-Xyl
3,4-Xyl
3-Xyl
2,3,4,6-Gal
3,4,6-Gal
2,3,4-Fuc
2,3,4-Glc
2,3,6-Glc
2,3-Glc
a
43, 45, 102, 118, 129, 161 and 205
43, 101, 102, 117, 118, 161 and 162
43, 87, 102, 118, 129, 189 and 233
43, 88, 101, 117, 130 and 190
43, 45, 87, 102, 118, 129, 145, 161, 162 and 205
43, 45, 71, 88,101, 129, 130, 145, 161, 190 and 205
43, 72, 88,102, 1115, 118, 131, 162 and 175
43, 87, 102, 118, 129, 162, 189 and 233
43, 45, 87, 102, 113, 118, 129, 162, 173 and 233
43, 102, 118, 127, 162, 201, 261 and 305
Percentage of total area of the identified peaks
2,3,5-Ara denotes 1,4-di-O-acetyl-2,3,5-tri-O-methylarabinitol, etc.
c
nd: not detected
b
3
2
82
nd
13
nd
nd
nd
nd
nd
nd
Peak areaa
(4AR) (XGO)
2
2
76
13
13
nd
nd
nd
8
nd
nd
ndc
13
1
22
nd
14
2
2
15
2
29
RAY et al.: COMPLEX BIOACTIVE OLIGOSACCHARIDES
(1) named according to Fry et al., 1993 (Ref 30).
Similarly, 953 assigned as XXGG (2),1085 as XXXG
(3), 1247 as XXLG (4), 1393 as XXFG (5), 1440 as
XLLG (6) and 1555 as XLFG (7) (Scheme I).
Although mass spectroscopy cannot distinguish
stereoisomers, but sugar compositional and glycosidic
compositional analysis indicates the presence of
xyloglucan derived oligosaccharides. Xyloglucans,
based upon the types of oligosaccharides released
after hydrolysis by endo-glucanase, were classified as
XXXG and XXGG type31. A XXXG-type in which
side chains is substituted with Xyl-Gal-Fuc residues
and a XXGG type whose side chains contain terminal
Gal residues such as in tobacco and tomato cell walls.
The fragments generated from flax meal xyloglucan
are classical fragments of XXXG type xyloglucan
characteristics of many dicots31,32. Additional
oligomers XGGG (1) and XXGG (2) specific to flax
were generated from XXGG-type of xyloglucans.
Such a XXGG-type of branching pattern is not
common in seeds but has also been previously found
in the cell walls of tobacco leaves and tomato
suspension cultures, two solanaceous species33,34.
Besides, a series of ions having a mass difference of
162 Da has also been observed in the mass spectrum
with degrees of polymerisation ranging from four to
as much as seven. Based on the data obtained from
sugar composition and methylation analysis of XGO
fraction these pseudomolecular ions, which lack
pentosyl units, probably originated from mannan
335
derived oligomers. Hence, the presence of mannan in
the hemicellulosic polysaccharides of flax meal has
been confirmed. MALDI mass analysis of the
xyloglucan oligosaccharides (FMXGO) generated
from flax meal, FM, reveals similar structures of
xyloglucan and indicates that O-acetyl substituents
are present on XXLG (4a), XXFG (5a, 6a) and XLFG
(7a)-type building subunits (Scheme I). Interestingly,
all oligomers that have O-acetyl group also contain
galactose residues. Such an occurrence of O-acetyl
group specifically on galactose unit of xyloglucan
oligosaccharide has been previously reported12,22.
Remarkably, abundance of all oligomers containing
fucose residues and O-acetyl group are higher than
the non-acetylated subunits.
High Performance Anion Exchange-Pulse
Amperometric
Detection
(HPAE-PAD)chromatography. HPAE-PAD chromatographic
analysis of the XGO fraction corroborates the results
obtained from MALDI-TOF-mass spectrometry.
Indeed, the HPAEC-PAD chromatography elution
profile (Figure 1) of XGO fraction shows the
presence of several peaks having different intensity.
Retention times of four of these peaks are similar to
xyloglucan oligosaccharides XXXG (3), XXFG (5),
XXLG (4) + XLFG (7) and XLLG (6), respectively
generated from Arabidopsis thaliana25, Argania
spinosa24,26,
Benincasa
hispida28,
Brassica
29
campestris and Sesamum indicum27 xyloglucan by
endo-glucanase digestion.
Figure 1 — HPAE-PAD chromatographic elution profile of heptasaccharide 2, nonasaccharide 4, mixture of octasaccharide 3 and
decasaccharide 6, and nonasaccharide 5 generated from Linum usitatissimum meal using endo-β-(1→4)-D-glucanase degradation. See
Scheme I for identification of fractions
336
INDIAN J. CHEM., SEC B. MARCH 2014
Glycosyl linkage composition of xylan.
Methylation of 4A produced a yellow, solid product
with a specific rotation of [α]25D-22.3° (c 1.12,
chloroform), indicative of the existence of β-Dglycosidic linkages. The methylated polysaccharide
was reduced (AR), hydrolyzed, and the resulting
sugars were converted into their corresponding
partially methylated alditol acetates and analyzed by
GC and GC-MS. The results obtained as shown in
Table II revealed that hemicellulose 4A is a linear
acidic xylan having a backbone of β-D-xylopyranosyl
residues bonded together by (1→4)-glycosidic links.
This linear chain has single unit branches of
4-O-methyl-D-glucopyranuronosyl and/or D-glucopyranuronosyl, and D-xylopyranosyl residues attached
at C-2. The relative proportions of 2,3,4-Me3-Glc,
2,3,4-Me3-Xyl and 3-Me-Xyl indicate that,
approximately, there is one branch point for every
eight units of xylose in the main chain.
Generation and analysis xylan oligosaccharides
(XO). Further information on the structure of xylan
present in 4A fraction was obtained by treating this
fraction with endo-(1→4)-β-D-xylanase, an enzyme
specific for β-D-xylan. Sugar compositional analysis
of the xylan-derived oligomers (XO) showed the
presence of xylose residues (98 mol%) together with
smaller amount of arabinose, and glucuronic acid
residues (Table I). HPAE-PAD analysis of this
fraction (XO) indicated the presence of D-Xylose
monomer together with β-(1→4)-D-xylobiose, as well
as peaks arising from xylan-derived acidic
oligosaccharides eluted with high concentration of
sodium acetate.
MALDI-TOF-mass spectrum (Figure 2) of XO
fraction showed one major peak at m/z 759, which
corresponds to one 4-O-MeGlcA linked to five xylose
residues 8 (Scheme I). In the same way, ions at
m/z = 891 and 1023 were assigned to [M+Na] + of
Xyl5-4-O-MeGlcA1 (9) and Xyl6-4-O-MeGlcA1 (10).
Pseudomolecular ions at 775 and 797 corresponding
to the potasiated and di-sodiated pseudomolecular
ions of the said oligosaccharide were also present.
Ions at 745, 877 and 1009 would correspond to
Xyl4-GlcA1(11), Xyl5-GlcA1 (12) and Xyl6-GlcA1
(13), respectively, indicating the presence of another
set of fragments with substitution of a 4-O-MeGlcA
by a GlcA residue. Pseudomolecular ions [M+Na]+ at
173 and 305 corresponding to xylose and xylobiose
have also been detected (not shown). Therefore, a
series of neutral and acidic oligosaccharides were
obtained from flax meal by treatment of
hemicellulosic 4A fraction with an endo-(1→4)-β-Dxylanase. Based on the results of chemical,
chromatographic, and spectroscopic analysis, it
appeared that flax xylan is formed by a main chain of
β-D-xylopyranosyl units joined by (1→4) glycosidic
links, which show branch units of containing a single
D-xylopyranosyl, 4-O-methyl-D-glucopyranuronosyl,
D-glucopyranuronosyl residues joined at position 2 of
the xylose units of the main chain, in proportions of
one branch to every eight units of xylose.
Experimental Section
Chemicals used were analytical grade or best
available. All determinations were done at least in
duplicate. Evaporations were performed under
Figure 2 — MALDI-TOF mass spectrum of oligosaccharides generated from 4M KOH soluble fraction (4B) of Linum usitatissimum
meal after degradation by endo-β-(1→4)-D-xylanase. See Scheme I for identification of fractions
RAY et al.: COMPLEX BIOACTIVE OLIGOSACCHARIDES
diminished pressure at 45-50°C (bath) and small
volume of aqueous solutions was lyophilized. Gas
liquid chromatography (GC; Shimadzu GC-17A,
Shimadzu, Kyoto, Japan) and gas liquid
chromatography
mass
spectrometry
(GCMS;
Shimadzu QP 5050 A, Shimadzu) as described35.
Recording of IR spectra and optical rotation
measurements were carried out as described
previously36.
Isolation of polysaccharides. The depectinated
material from Linum usitatissimum meal was obtained
as described previously1. Ten grams of this residue
was then extracted twice with 500 mL of 4 M KOH
solutions containing 20 mM NaBH4 for 14 hr.
Combined extract was acidified with AcOH to pH 6
and dialysed extensively against water. The
precipitate was removed by centrifugation to yield 4A
fraction (450 mg). The soluble fraction has been
designated as 4B (550 mg).
Anion-exchange chromatography. Fraction (4B)
was submitted to anion-exchange chromatography on
DEAE-Sepharose FF column equilibrated previously
with water. After loading with sample, the column
was eluted with the same solvent at a flow rate of 30
mL h-1 to obtain the non-retained fraction (named
4Baq). Bound materials were eluted from the column
by using salt gradient.
Size Exclusion Chromatography (SEC). SEC of
the 4Baq fraction was done on a Sephacryl S-400
column (90 × 2.6 cm, BioRad) calibrated with
standard dextrans (molecular-weight range of 10,000
to 1,000,000 kDa) using 500 mmol sodium acetate
(pH 5.0) at a flow rate of 20 mL h-1 as described26.
Preparation of xyloglucan oligosaccharides.
Fraction 4Baq (10 mg) was dissolved in 2 mL of
50 mmolar NaOAc (pH 5.0) and the mixture
incubated with 5 units of endo-glucanase (Megazyme
International, Ireland) for 24 hr at 30-37°C.
Glucanase-resistant materials were removed by
diluting the digest with EtOH to a final concentration
of 80%. The EtOH soluble oligosaccharides were
concentrated under a stream of nitrogen to yield
xyloglucan oligosaccharide XGO. In a similar way,
another xyloglucan oligosaccharides containing
fraction (FMXGO) was generated from flax meal
(FM).
Preparation
of
xylan
oligosaccharides.
Hydrolysis of the 10 mg xylan rich fraction (4B) was
performed in 4 mL of 10 mmolar NaOAc
(pH 5.0) using 40 units of endo-xylanase (Megazyme
International, Ireland) at 30-37°C for 24 hr. To
337
remove enzyme resistant polymeric material, the
digest was treated with 4 volumes of cold ethanol, the
suspension was stored overnight at 4°C and then
centrifuged. Xylan oligomers (XO) were recovered by
concentrating the supernatant under a stream of
nitrogen at 40°C and lyophilising the concentrated
solution.
Sugar analysis. Total sugars were determined by
the phenol-sulfuric acid assay using glucose as
standard37. The neutral sugar compositions of
fractions were determined after hydrolysis with
sulfuric acid (2M, 100°C, 2hr), reduction and
acetylation38. Alternatively, the polysaccharides in the
samples were hydrolyzed using trifluoro acetic acid
(2M, 2 hr at 110°C), followed by an 18 hr
methanolysis at 80°C with dry 2M methanolic-HCl.
The generated methyl glycosides were converted into
their TMS-derivatives and separated by gas
chromatograph (GC) with H2 as carrier gas as
described26.
Methylation analysis. The pool of oligosaccharides (XGO) generated from Linum usitatissimum
meal xyloglucan was permethylated according to
Blakeney39. Permethylated material was extracted,
dried, hydrolysed, converted into its partially
methylated alditol acetates (PMAA) and was analysed
by GC and GC-MS as described previously26-29.
A quantity (23.5 mg) of 4A was also methylated by
the method of Blakeney et al.39 The methylated
product was purified by precipitation from benzene
with light petroleum (b.p. 30-60°C). To a solution of
the methylated polysaccharide (11.5 mg) in dry
tetrahydrofuran (10 mL) was added LiAlH4 (200 mg)
(Ref 40). The mixture was refluxed in an atmosphere
of N2 for 24 hr, after which time the reaction was
stopped by addition of 2 mL of acetone and then
filtered through Whatman No. 1 paper. The product of
the reaction was then extracted in CHCl3 and
vacuum-dried for 48 hr over P2O5. The permethylated
xylan (4A) and its reduction product (4AR) were
hydrolyzed in separate vials, and the resulting sugars
were converted into PMAA and analyzed by GC and
GC-MS.
HPAE-PAD chromatography. Fragments present
in XGO fraction was analysed on a Dionex DX 500
system equipped with a GP 50 gradient pump, an
eluent degas module, a CarboPac PA-1 column and a
pulse amperometric detector (PAD). Samples
(10-100 µL) were injected and eluted (1 mL min-1)
with NaOAc gradient in 100 mmolar NaOH as
described26.
338
INDIAN J. CHEM., SEC B. MARCH 2014
MALDI-TOF mass spectrometry. MALDI TOF
mass spectrometry in reflectron mode was performed
using a Bruker Daltonics flexAnalysis MALDI-TOF
mass spectrometer. 2,5-Dihydroxybenzoic acid
(10 mg mL-1) was used as matrix.
IR Spectroscopy. Infrared spectrum was recorded
on a JASCO FTIR 420 spectrophotometer using a
KBr disc. The sample was dried at 35-44°C in
vacuum over P2O5 for 72 hr prior to analysis.
Conclusions
The findings of this study highlight several unique
and significant aspects of the Linum usitatissimum
meal derived hemicellulosic polysaccharides with
regard to their structures: (i) hemicellulosic
polysaccharides of flax meal contained xyloglucan,
mannan and xylan type polymers, (ii) the isolated
xylan is branched, and has a backbone of β-(1→ 4)linked xylosyl residues with some zones containing a
single D-xylopyranosyl, 4-O-methyl-D-glucopyranuronosyl, D-glucopyranuronosyl residues joined at
position 2 of the xylose units of the main chain,
(iii) the 86 kDa xyloglucan is branched and contained
penta(1)-, hexa(2)-, hepta(3)-, octa(4)-, nona(5,6)-and
deca(7)-saccharides
as
building
sub-units,
(iv) xyloglucan derived oligomers that have O-acetyl
group also contain galactose residues and
(v) altogether theses polysaccharides represented
about 10% of the FM and might be individually used
in various industries, either as polymers or as
oligosaccharides. Finally, enzymatic digestion of the
hemicellulosic fractions coupled to chromatographic
separation and mass spectrometric analysis of
generated oligosaccharides, as well as monosaccharide compositional and linkage analysis of the
associated fractions, proved a powerful approach to
probe for detailed structural information.
Acknowledgement
This work was supported by DST (project number
SR/S1/OC-38/2012), New Delhi, India, to B. Ray.
References
1 Ray S, Paynel F, Morvan C, Lerouge P, Driouich A & Ray
B, Carbohydr Polym, 93, 2013, 651.
2 Scheller H V & Ulvskov P, Annu Rev Plant Biol, 61, 2010,
263.
3 Liu S, Biotechnol Adv, 28, 2010, 563.
4 Dhawan S & Kaur J, Crit Rev Biotechnol, 27, 2007,197.
5 Prisenžzáková L, Nosál'ová G, Hromádková Z &
Ebringerová A, Fitoterapia, 81, 2010, 1037.
6 Faber T, Hopkins C, Middelbos I S, Price N P & Fahey G C,
J Anim Sci, 2010, 103.
7 Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y,
Yoshimura K, Tobe T, Clarke J M, Topping D L, Suzuki T,
Taylor T D, Itoh K, Kikuchi J, Morita H, Hattori M & Ohno
H, Nature, 469, 2011, 543.
8 Kabel M A, Carvalheiro F, Garrote G, Avgerinos E, Koukios
E, Pajaro J C, Girio F M, Schols H A & Voragen A G J,
Carbohydr Polym, 50, 2002, 47.
9 Kato Y, Uchida J, Ito S & Mitsuishi Y, Int Cong Ser, 1223,
2001, 161.
10 Miyazaki S, Kawasaki N, Endo K & Attwood D, J Pharm
Pharmacol, 53, 2001, 1185.
11 Kato Y, Uchida J, Ito S & Mitsuishi Y, Int Cong Ser, 1223,
2001, 161.
12 Sims I M, Munro S L A, Currie G, Craik D & Bacic A,
Carbohydr Res, 293, 1996, 147.
13 Glicksman M, in Food Hydrocolloids, Vol III, edited by M
Glicksman (CRC Press, Boca Raton, Florida, USA), 1986,
191.
14 Pazur J H, The Carbohydrate-Chemistry/Biochemistry Vol.
2A (Academic Press, New York), 1970, 69.
15 Albersheim P, Darvil A, Augur C, Cheong J J, Eberhard S,
Hahn M G, Maarfa V, Mohenm D, O’Neil M A, Spiro M D
& York W, Acc Chem Res, 25, 1992, 77.
16 Ebringerova A & Heinze T, Macromol Rapid Commun, 21,
2000, 542.
17 Pepper T & Olinger P M, Food Technol, 42, 1988, 98.
18 Christakopoulos P, Katapodes P, Kalogeris E, Kekos D,
Macris B J, Stamatis H & Skaltsa H, Int J Biol Macromol,
31, 2003, 171.
19 Guilloux K, Gaillard I, Courtois J, Courtoix B & Petit E,
J Agric Food Chem, 57, 2009, 11308.
20 Kacurakova M, Capek P, Sasinkova V, Wellner N &
Ebringerova A, Carbohydr Polym, 43, 2000, 195.
21 Gupta S, Madan R S & Bansal M C, Tappi J, 70, 1987, 113.
22 Fry S C, J Exp Bot, 40, 1989, 1.
23 Olson A, Gray G M & Chiu M C, Food Technnol, 41, 1987, 71.
24 Aboughe-Angone S, Nguema-Ona E, Ghosh P, Lerouge P,
Ishii T, Ray B & Driouich A, Carbohydr Res, 343, 2008, 67.
25 Lerouxel O, Choo T S, Seveno M, Usadel B, Faye L,
Lerouge P & Pauly M, Plant Physiol, 130, 2002, 1754.
26 Ray B, Loutelier-Bourhis C, Lange C, Condamine E,
Driouich A & Lerouge P, Carbohydr Res, 339, 2004, 201.
27 Ghosh P, Ghosal P, Thakur S, Lerouge P, Loutelier-Bourhis
C, Driouich A & Ray B, Food Chem, 90, 2005, 719.
28 Mazumder S, Lerouge P, Loutelier-Bourhis C, Driouich A &
Ray B, Carbohydr Polym, 59, 2005, 231.
29 Ghosh P, Ghosal P, Thakur S, Lerouge P, Loutelier-Bourhis
C, Driouich A & Ray B, Carbohydr Polym, 57, 2004, 7.
30 Fry S C, York W S, Albersheim P, Darvill A, Hayashi T,
Joseleau J P, Kato Y, Lorences E P, Maclachlan G A,
McNeil M, Mort A J, Reid J S G, Seitz H U, Selvendran R R,
Voragen A G J & White A R, Physiol Plant, 89, 1993, 1.
31 Vincken J P, York W S, Beldman G & Voragen A G J, Plant
Physiol, 114, 1997, 9.
32 Hayashi T, Annu Rev Plant Biol, 40, 1989, 139.
33 Hoffman M, Jia Z, Peña M J, Cash M, Harper A &
Blackburn A R, Carbohydr Res, 340, 2005, 1826.
34 Jia Z, Qin Q, Darvill A G & York W S, Carbohydr Res, 338,
2005, 1197.
35 Bandyopadhyay S S, Astani A, Ghosh T, Schnitzler P & Ray
B, Phytochemistry, 72, 2011, 276.
36 Ray B, Carbohydr Polym, 66, 2006, 408.
37 Dubois M, Gilles K A, Hamilton J K, Rebers P A & Smith F,
Anal Chem, 28, 1956, 350.
38 Blakeney A B, Harris P, Henry R J & Bruce A B, Carbohydr
Res, 113, 1983, 291.
39 Blakeney A B & Stone B A, Carbohydr Res, 140, 1985, 319.
40 Asensio A, Can J Chem, 66, 1988, 449.

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