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NMR-Spectroscopic Profiling of Arabinan and Galactan Structural Elements Daniel Wefers, and Mirko Bunzel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04232 • Publication Date (Web): 27 Nov 2016 Downloaded from http://pubs.acs.org on December 6, 2016
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Journal of Agricultural and Food Chemistry
NMR-Spectroscopic Profiling of Arabinan and Galactan Structural Elements
Daniel Wefers and Mirko Bunzel*
Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT), Adenauerring 20a, 76131 Karlsruhe, Germany
*Corresponding author (Tel: +49 721 608 42936; Fax: +49 721 608 47255; Email:
[email protected])
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ABSTRACT
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Pectic arabinans and galactans presumably affect the physiological and technological
3
properties of plant cell walls and dietary fiber. Their complex structures are usually analyzed
4
by time-consuming methods, which are based on chemical cleavage to monomers. To gain
5
more detailed insights into the arabinan and galactan structures, a time-efficient approach
6
based on enzymatic cleavage and two-dimensional NMR spectroscopy was developed.
7
Heteronuclear single quantum coherence spectroscopy (HSQC) marker signals were evaluated
8
for various structural elements and relative response factors were determined allowing a
9
semiquantitative estimation of the structural composition. The method was applied to analyze
10
different insoluble plant materials and soluble polysaccharides. It was demonstrated that the
11
developed approach yielded comparable information about various structural elements that
12
can also be detected by using the conventional methylation analysis. However, by using the
13
NMR method additional structural information such as anomeric configuration of the
14
monomers is obtained, demonstrating the value of this novel approach.
15
KEYWORDS
16
NMR spectroscopy, pectins, screening, dietary fiber, polysaccharides.
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INTRODUCTION
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In fruits and vegetables, pectins are often the most abundant plant cell wall polysaccharides
19
besides cellulose.1 These structurally complex polymers are defined as galacturonic acid
20
containing polysaccharides and are composed of several structural subgroups. The
21
quantitatively most important subgroups are homogalacturonan and rhamnogalacturonan I,
22
polymers with comparably conserved, galacturonic acid containing backbone structures.2,
23
Arabinans and galactans are neutral side chains of rhamnogalacturonan I and show, however,
24
highly complex structures. Arabinans are composed of a backbone of α-(1→5)-linked
25
arabinofuranose units, which can be substituted with monomeric or oligomeric arabinose side
26
chains. The arabinan backbone may be ramified at positions O2, O3, and O2 and O3, but a
27
high heterogeneity was reported for different plant materials.4-9 Further complexity results
28
from the occurrence of terminal β-arabinofuranose units in some arabinan chains.10,
29
Galactans consist of a backbone of β-(1→4)-linked galactopyranoses, which is frequently
30
described to be ramified at the positions O3 and O6.2 Recently it has been demonstrated that
31
terminal α-arabinopyranose units may be attached to position O4 of the galactan backbone
32
and that internal α-(1→4)-linked arabinopyranose units are also present.8, 10, 12 The occurrence
33
of various substitution positions and substituents is a challenging task for the structural
34
analysis of arabinans and galactans. Many of the described structural elements were
35
established by preparative isolation and NMR-spectroscopic characterization of diagnostic
36
oligosaccharides after enzymatic hydrolysis. Analytical approaches such as methylation
37
analysis are based on chemical cleavage of all glycosidic bonds, resulting in a loss of
38
information about substituents and the anomeric configuration of the monosaccharides. Most
39
recently, a method for the routine analysis of intact arabinan and galactan oligosaccharides by
40
high-performance anion-exchange chromatography with pulsed amperometric detection
41
(HPAEC-PAD) after enzymatic cleavage with endo-arabinanase and endo-galactanase was
42
described.13 By using this approach it was possible to obtain information about the occurrence 3 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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of common and rare structural elements. However, this chromatographic screening method
44
requires standard compounds for each analyte, and high molecular weight oligosaccharides
45
are not taken into account. In contrast to chromatographic approaches, the application of
46
NMR spectroscopy allows for the detection of all solubilized compounds. In addition, the 1H
47
and
48
environment, which is usually represented by the adjacent sugar units. This results in the
49
simultaneous detection of specific structural elements or substituents in oligosaccharides and
50
polysaccharides of varying sizes. Thus, NMR spectroscopy is a highly suitable detection
51
technique for the routine analysis of different structural elements in carbohydrate mixtures.
52
Both oligosaccharide mixtures and polysaccharides often result in highly complex one-
53
dimensional spectra, preventing the unambiguous identification of specific structural
54
elements. Correlations between NMR active nuclei in two-dimensional NMR experiments
55
such as Heteronuclear Single Quantum Coherence (HSQC), provide an extra gain in
56
resolution by spreading the spectra in a second dimension.
57
Therefore, the aim of this study was to establish a rapid and simple two-dimensional NMR-
58
spectroscopic profiling approach to semiquantitatively determine arabinan and galactan
59
structural elements in insoluble and soluble polysaccharide preparations.
60
MATERIALS AND METHODS
61
Chemicals
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If not stated otherwise, all chemical compounds used were at least of pro-analysi-grade and
63
were purchased from VWR (Radnor, PA), Sigma Aldrich (Schnelldorf, Germany), or Carl
64
Roth (Karlsruhe, Germany). Deuterium oxide (99.9 atom% D) and dimethylsulfoxide-d6
65
(99.9 atom% D) were purchased from Sigma Aldrich. Thermostable α-amylase (Termamyl
66
120 L, EC 3.2.1.1, from B. licheniformis, 120 KNU/g), protease (Alcalase 2.4L, EC 3.4.21.62,
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from B. licheniformis, 2.4 AU/g), and amyloglucosidase (AMG 300L, EC 3.2.1.3, from A.
13
C chemical shifts of different structural elements are based on their direct chemical
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niger, 300 AGU/g) were kindly donated by Novozymes (Bagsvaerd, Denmark). endo-
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Arabinanase (EC 3.2.1.99, from A. niger, 9 U/mg), endo-galactanase (EC 3.2.1.89, from A.
70
niger, 408 U/mg), arabinan from sugar beet, and galactan from potato were purchased from
71
Megazyme (Bray, Ireland). Oligosaccharide standard compounds were isolated from
72
enzymatic digests of various polysaccharide preparations and completely characterized by
73
NMR spectroscopy as described previously.8, 10, 13
74
Materials
75
Apples (Malus domestica cv. Braeburn, grown and harvested 2013 in Germany), quinoa
76
(Chenopodium quinoa Willd., grown and harvested 2012 in Bolivia), amaranth (Amaranthus
77
hypochondriacus, grown and harvested 2012 in Germany), and buckwheat (Fagopyrum
78
esculentum, grown and harvested 2012 in China) were purchased from local suppliers.
79
Soybean extraction meal was kindly provided by ADM Rothensee (Hamburg, Germany), and
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sugar beet pulp was kindly provided by Suedzucker (Mannheim, Germany). Apples were used
81
without peel and apple core and freeze-dried prior to milling. Sugar beet pulp was also freeze-
82
dried prior to milling. All materials were milled to a particle size < 0.5 mm by using an MF10
83
basic mill (IKA-Werke, Staufen, Germany). Amaranth, quinoa, and buckwheat meals were
84
defatted with acetone prior to dietary fiber isolation.
85
Dietary fiber isolation
86
Insoluble dietary fiber was isolated by suspending 20 g of the milled plant material in 200 mL
87
of 0.08 M phosphate buffer (pH 6.2). Thermostable α-amylase (1.5 mL) was added, and the
88
mixture was incubated for 20 min at 95°C. After adjustment of the pH to 7.5 with sodium
89
hydroxide, the suspension was incubated with 700 µL of protease for 30 min at 60°C. The pH
90
was adjusted to 4.5, and 700 µL of amyloglucosidase was added. The suspension was
91
incubated for 30 min at 60°C. Insoluble dietary fiber was obtained by centrifugation and
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washed with water, ethanol (96%, v/v), and acetone. 5 ACS Paragon Plus Environment
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Method development
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NMR spectroscopic analyses were carried out on an Ascend 500 MHz NMR spectrometer
95
(Bruker, Rheinstetten, Germany) equipped with a Prodigy cryoprobe. HSQC spectra were
96
acquired by using the hsqcedetgp pulse sequence with the standard parameter set provided by
97
Bruker. This included a relaxation time of 1.5 s and a 1JCH coupling constant of 145 Hz. 1024
98
points were recorded in the f2 dimension and 256 points were recorded in the f1 dimension.
99
32 scans were acquired for almost all samples, resulting in adequate signal intensities and
100
acceptable analysis times (approximately 3 h 40 min).
101
All HSQC marker signals and relative response factors were evaluated by using several well
102
characterized arabinan and galactan oligosaccharides previously isolated from several plant
103
materials.8,
104
conditions described above. Spectra analysis was performed with Topspin 3.1 software. To
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determine the specificity of the marker signals, the spectrum of each individual
106
oligosaccharide was overlaid with the spectra of all other standard compounds. After
107
evaluation of the marker signals, a (volume) integration mask was created that allowed for the
108
rapid and reproducible analysis of various samples. The relative response factors were
109
determined by analyzing the standard oligosaccharides for the relative intensity of the marker
110
signals to an internal reference signal (signal 1 for arabinans, signal 14 for galactans).
111
Method application
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To analyze insoluble fiber preparations, the sample amount and the incubation volume were
113
adapted to achieve an acceptable, free-flowing sample viscosity during hydrolysis and a
114
sufficient amount of hydrolysate. Generally, 250 – 500 mg of insoluble fiber sample was
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weighed into a 15 mL centrifuge tube and suspended in 10 mL of water. Autoclave extraction
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was performed by autoclaving the tubes for 40 min at 121°C. After cooling, 2 U of endo-
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arabinanase/endo-galactanase per 100 mg insoluble fiber were added to the suspension, and
10, 13
The HSQC spectra of all standard compounds were recorded using the
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samples were incubated for 24 h at 40°C. Appropriate mixing was achieved by rotating the
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tubes for 360 degrees in horizontal position. Following incubation, enzymes were inactivated
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by heating the sample to 100°C for 5 min, and unhydrolyzed material was removed by
121
centrifugation. An aliquot of the clear supernatant (450 µL) was transferred into an NMR
122
tube, and 50 µL of deuterium oxide was added. Acetone (0.5 µL) was used for spectrum
123
calibration (2.22 ppm (1H) / 30.89 ppm (13C)),14 and HSQC spectra were acquired as
124
described above. Soluble polysaccharides were dissolved in 500 µL of D2O, and their spectra
125
were also referenced to acetone. For the calculation of the portions of the structural elements,
126
the intensities of the marker signals were multiplied by their relative response factor, modified
127
as shown in Table 1, and used to calculate the relative abundance of the structural elements.
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All analyses were performed in duplicate.
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Methylation analysis
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Methylation analysis was carried out as described by Nunes et al.15 with minor modifications.
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The samples (5 mg) were dissolved in 2 mL of dimethyl sulfoxide, and ca. 100 mg of freshly
132
ground sodium hydroxide was added. The mixture was incubated for 90 min in an ultrasonic
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bath and 90 min at room temperature. Methyl iodide (1 mL) was added, followed by
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sonication for 30 min and incubation for 30 min at room temperature. The solution was
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extracted with dichloromethane, and the extracted organic phase was washed with 3 mL of
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0.1 M sodium thiosulfate and twice with 3 mL of water. The solvent was evaporated, and
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samples were dried overnight in a vacuum oven at 40°C. The methylated polysaccharides
138
were hydrolyzed by adding 2 mL of 2 M TFA and incubation at 121°C for 90 min. After
139
evaporation of the acid, 20 mg of NaBD4 in 300 µL of an aqueous ammonia solution (2 M)
140
was added. Reduction was carried out at room temperature for 1 h and terminated by addition
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of glacial acetic acid. While cooling with ice, 450 µL of 1-methylimidazole and 3 mL of
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acetic anhydride were added, and the solution was incubated for 30 min at room temperature. 7 ACS Paragon Plus Environment
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After addition of water (3 mL), the solution was extracted with 5 mL of dichloromethane. The
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organic layer was washed three times with water, and residual water was removed by freezing
145
overnight at -18°C. GC-MS analysis of the partially methylated alditol acetates (PMAAs) was
146
performed on GC-2010 Plus and GCMS-QP2010 SE instruments (Shimadzu, Kyoto, Japan)
147
both equipped with a DB-225MS column (30 m x 0.25 mm i.d., 0.25 µm film thickness)
148
(Agilent Technologies, Santa Clara, CA). The following conditions were used: Initial column
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temperature, 140 °C, held for 1 min; ramped at 20 °C/min to 220 °C, held for 25 min. Helium
150
was used as carrier gas at 40 cm/sec. Split injection with a split ratio of 30:1 was used, and the
151
injection temperature was 220°C. The transfer line was held at 220°C, and electron impact
152
mass spectra were recorded at 70 eV. PMAAs were quantitated by analyzing the samples by
153
GC-FID (GC-2010 Plus) (Shimadzu) using the same conditions as described above, but
154
applying a 10:1 split ratio. Nitrogen was used as makeup gas, and the FID temperature was
155
240°C. Molar response factors according to Sweet et al.16 were used for semiquantitative
156
analyses. All analyses were performed in duplicate.
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RESULTS AND DISCUSSION
158
Evaluation of marker signals
159
The two-dimensional HSQC experiment, which provides information about short-range (1J)
160
couplings between protons and
161
galactan structural elements. Application of the HSQC experiment improves virtual resolution
162
by using a second dimension: overlapping proton signals may become distinguishable by
163
carbon shift differences, and overlapping carbon shifts are pulled apart by proton shift
164
differences. Being an inverse experiment, the application of an HSQC experiment is less
165
time-consuming than a one-dimensional carbon experiment, resulting in acceptable spectra
166
acquisition times. Different from one-dimensional experiments, volume integration of the
167
signals is used in two-dimensional experiments to determine the ratios between different
13
C nuclei, was chosen for the analysis of arabinan and
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signals in a spectrum. To unambiguously identify and semiquantitatively estimate the
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numerous arabinan and galactan structural elements, characteristic HSQC marker signals are
170
needed. Ideally, these marker signals are specific for a single structural element and do not
171
overlap with signals derived from the same or other structural elements. To evaluate the
172
suitability of specific signals as marker signals, the HSQC spectra of various arabinan and
173
galactan oligosaccharide standard compounds were compared. These oligosaccharides were
174
isolated from different materials and completely characterized by NMR spectroscopy.8, 10, 13
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The chosen HSQC marker signals and their assignment to structural elements are shown in
176
Figures 1 and 2. Different from the ideal situation, some marker signals represent more than
177
one structural element due to the very similar chemical environment of the corresponding
178
protons/carbons. In these cases the intensities of other marker signals had to be subtracted to
179
get information about a single structural element (Table 1; 5-/1,5-Araf, t-O4-Galp, t-O4-
180
Arap).
181
For example, this approach was used to calculate the intensity of an 1,5-disubstituted
182
backbone α-arabinofuranose unit (1,5-arabinofuranose) from signal 1, which represents the
183
C5/H5 correlation peaks of all arabinofuranose units that are substituted at position 5 (signal 1
184
in Figure 1). In addition, this signal represents two protons and, therefore, needs to be divided
185
by two. It was not possible to use the signal of the anomeric proton/carbon because it showed
186
the same chemical shift as a terminal α-arabinofuranose unit attached to another
187
arabinofuranose unit through a (1→5)-linkage (t-O5-α-arabinofuranose). However, due to the
188
different substitution patterns, all branched backbone arabinose units (additional substitution
189
sites next to position 1 and 5) showed characteristic marker signals. Therefore, it was possible
190
to subtract their intensities from signal 1 to obtain the intensity of 1,5-disubstituted α-
191
arabinose units. However, reducing arabinose units that are only substituted in position 5 (5-
192
arabinofuranose) cannot be distinguished from 1,5-disubstituted α-arabinofuranose units by
193
using the C5/H5 correlation signal. To determine 1,3,5-substituted and 1,2,3,5-substituted α9 ACS Paragon Plus Environment
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arabinofuranose units, the correlation peaks of the anomeric protons/carbons were used
195
(signal 6 and signal 7, respectively, in Figure 1). The C1/H1 HSQC signal of the 1,2,5-
196
substituted α-arabinofuranose also showed a characteristic position, but it was very close to
197
signal 6. This makes the detection of small amounts of 1,2,5-substituted α-arabinofuranose
198
units besides large amounts of 1,3,5-substituted α-arabinofuranose units, a common
199
composition for many pectic materials, difficult. Therefore, the well-separated C2/H2
200
correlation peak was chosen as marker signal for a 1,2,5-substituted α-arabinofuranose unit
201
(signal 5 in Figure 1). The correlation peaks of the anomeric protons/carbons were suitable to
202
identify terminal α-arabinofuranose units that are linked through (1→3)-linkages (t-O3-α-
203
arabinofuranose) or (1→2)-linkages (t-O2-α-arabinofuranose) to the arabinan backbone,
204
respectively (signal 3 and signal 4 in Figure 1). The t-O5-α-arabinofuranose did not show any
205
specific signal, except for the C4/H4 correlation peak (signal 2 in Figure 1). As shown in
206
Figure 3, this peak showed a partial overlap with the C4/H4 signals of t-O2-α-arabinofuranose
207
units. Thus, some inaccuracies may be observed when high amounts of t-O2-α-
208
arabinofuranose and t-O5-α-arabinofuranose units (Figure 3, C) or high amounts of t-O2-α-
209
arabinofuranose, t-O3-α-arabinofuranose, and t-O5-α-arabinofuranose (Figure 3, D) are
210
present (both of which being possible but rather unlikely scenarios). Dimeric, β-(1→3)-linked
211
arabinobiose side chains were recently established as structural elements of quinoa
212
arabinans.10, 13 The analysis of arabinose units in their β-configuration is of particular interest
213
because they may have an impact on the enzymatic degradability of arabinans but cannot be
214
detected by conventional methods such as methylation analysis. By using the NMR
215
spectroscopic approach, terminal β-arabinose units can easily be distinguished from all other
216
structural elements, because the C1/H1 correlation peak shows a unique
217
(signal 8 in Figure 1). Finally, 3-O-(β-arabinofuranosyl)-α-arabinofuranose residues, which
218
were identified as side chains in quinoa arabinans can be identified by their C2/H2 correlation
219
peaks (signal 9 in Figure 1). 10 ACS Paragon Plus Environment
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C chemical shift
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The most common structural elements of galactans are the backbone 1,4-disubstituted β-
221
galactopyranose units (1,4-galactopyranose) and terminal β-galactopyranose units forming the
222
non-reducing end of the backbone by being attached through a (1→4)-linkage (t-O4-β-
223
galactopyranose). The analysis of internal 1,4-disubstituted α-arabinopyranoses (1,4-
224
arabinopyranose) and terminal α-arabinopyranose units attached to the backbone through a
225
(1→4)-linkage (t-O4-β-arabinopyranose), however, might be more important because the role
226
of these recently established structural elements is yet unknown. In addition, these structural
227
elements, which constitute relatively low portions of galactans, are difficult to analyze by
228
using conventional methods. For example, internal 1,4-disubstituted α-arabinopyranose units
229
cannot be detected by methylation analysis because they yield the same PMAA as arabinan
230
derived 1,5-disubstituted α-arabinofuranose units. The anomeric HSQC correlation peaks of
231
β-galactopyranoses and α-arabinopyranose units show minor differences in their 1H chemical
232
shifts and about the same 13C chemical shifts. Although 1,4-disubstituted β-galactopyranose,
233
t-O4-β-galactopyranose, 1,4-disubstituted α-arabinopyranose, and t-O4-α-arabinopyranose
234
can be differentiated through their anomeric signals if standard compounds are measured, this
235
is usually not possible if polysaccharides or enzymatic hydrolysates are analyzed. For most
236
galactans, dominant peaks are obtained representing the galactose units and only low
237
intensities are observed for the arabinopyranose derived signals resulting in signal overlaps.
238
Thus, the anomeric signals of the four structural elements cannot be distinguished in, for
239
example, soluble galactan polysaccharides but are combined in a single signal (signal 14,
240
Figure 4). Therefore, the anomeric signals were not suitable to analyze galactan structural
241
elements in polymeric structures. Instead, the C5/H5 correlation peaks were selected to detect
242
α-arabinopyranose units because these signals were clearly separated from all galactan
243
derived signals. Because of its characteristic 1H downfield shift, the HSQC signal of the H5
244
equatorial
245
arabinopyranose units substituted at position O4 (4-α-arabinopyranose, formed through 11
proton
of
1,4-disubstituted
α-arabinopyranose
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and
reducing
α-
Journal of Agricultural and Food Chemistry
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galactan hydrolysis or being the reducing end of the polymer) can be used to unambiguously
247
identify these structural elements in galactans or galactan hydrolysates (signal 13 in Figure 2).
248
However, the β-anomer of a reducing arabinopyranose substituted at position O4, which is
249
generated due to the endo-galactanase hydrolysis, cannot be detected by using signal 13. To
250
avoid the resulting inaccuracies, the intensity of the β-anomer can be calculated in by
251
multiplying the intensity of the α-anomer with a factor of 1.535. This factor was determined
252
from the ratio between the C5/H5 signals derived from the α- and the β-anomers in the HSQC
253
spectrum of the corresponding oligosaccharide (the ratio between the anomers remains
254
constant in aqueous solution). The HSQC peak of the equatorial H5 proton of a t-O4-α-
255
arabinopyranose was located in the region of signal 1 (representing α-(1→5)-linked
256
arabinofuranose units, Figure 1). Therefore, the signal representing the equatorial H5 proton
257
of t-O4-α-arabinopyranose units will be partially overlapped if arabinans are present in the
258
enzymatic hydrolysates or polysaccharide preparations. Thus, the C5/H5 correlation peak of
259
the axial proton was used as an additional marker signal (signal 12 in Figure 2). Signal 12
260
represents three arabinopyranose units in galactans or galactan oligosaccharides (t-O4-α-
261
arabinopyranose, 4-α-arabinopyranose, 1,4-disubstituted α-arabinopyranose). Therefore,
262
information about t-O4-α-arabinopyranose units will be obtained by subtraction of signal 13
263
from the intensity of signal 12. To characterize the galactose units in galactans or galactan
264
oligosaccharides, the C4/H4 correlation peaks were selected as marker signals for 1,4-
265
disubstituted β-galactopyranose units and reducing galactopyranose units substituted at
266
position O4 (4-galactopyranose units) (signal 10 in Figure 2). Because of the substitution at
267
position O4 and the resulting downfield shift of the corresponding protons/carbons, the
268
chemical shifts are characteristic for this structural element. In endo-galactanase hydrolysates,
269
the main hydrolysis product should be β-(1→4)-linked galactobiose and α-arabinopyranose
270
containing disaccharides.13 Therefore, the signal in the selected part of the anomeric region
271
(4.53-4.67 ppm, 104.9 ppm) of the HSQC spectra obtained from galactan hydrolysates most 12 ACS Paragon Plus Environment
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likely represents t-O4-β-galactopyranose units and t-O4-α-arabinopyranose units (signal 14 in
273
Figure 2). After subtraction of the intensity of t-O4-α-arabinopyranose units (represented by
274
the intensity obtained from signal 12 minus signal 13), this signal can be used to determine t-
275
O4-β-galactopyranose units in endo-galactanase hydrolysates (Table 1).
276
In polymeric galactans, the very intensive anomeric signal represents all of the four important
277
structural elements ((1→4)-linked β-galactopyranose, t-O4-β-galactopyranose), (1→4)-linked
278
α-arabinopyranose, t-O4-α-arabinopyranose). Thus, the C4/H4 correlation peak of t-O4-β-
279
galactopyranose units was used instead as a marker signal for this structural element (signal
280
11 in Figure 2). The intensity of t-O4-α-arabinopyranose units, which showed a very similar
281
chemical shift for their C4/H4 correlation peak, has to be subtracted from this marker signal
282
(Table 1). Signal 11 was not suitable for endo-galactanase hydrolysates, because it showed
283
overlap with the C4/H4 correlation peak of monomeric galactose, which is present due to the
284
enzymatic hydrolysis. All other arabinan and galactan marker signals described in Table 1 can
285
be used for both the analysis of polysaccharides and the analysis of endo-arabinanase/endo-
286
galactanase hydrolysates.
287
Quantitative aspects
288
The protons/carbons that produce marker signals in the HSQC spectra are part of primary
289
alcohol, secondary alcohol, or acetal groups. This results in varying chemical shifts, but also
290
in different 1JCH coupling constants. Because an average value of 145 Hz is applied in the
291
HSQC experiment, different 1JCH coupling constants lead to different signal intensities. To
292
compensate for these differences, relative response factors against an internal reference signal
293
were determined for all marker signals. This approach was favored over an external
294
calibration approach that requires larger amounts of standard compounds and is more time
295
consuming. In addition, the relative response factors can easily be adapted by other users to
296
achieve a semiquantitative estimation of the structural elements. 13 ACS Paragon Plus Environment
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The relative response factors of the HSQC marker signals representing the arabinan and
298
galactan structural elements (Figure 1 and Figure 2) were evaluated separately by determining
299
intensities of these signals that were compared to the reference signal. Signal 1 was chosen as
300
reference signal for arabinan marker signals, whereas signal 14 was used for galactan marker
301
signals. These signals were selected because they appeared in the spectra of all
302
oligosaccharide standard compounds used. If applicable, the relative response factor of a
303
marker signal was determined by measuring multiple oligosaccharide standard compounds
304
and subsequent averaging of the response factors. In addition, the relative response (0.908) of
305
signal 14 to signal 1 was determined by using an equimolar solution of arabinobiose and
306
galactobiose. If both, arabinans and galactans, are present in polysaccharide preparations, this
307
factor can be applied to all corrected signal intensities of the galactan structural elements.
308
Although comparable relative response factors were obtained for the same structural element
309
in different oligosaccharides, this approach can only provide semiquantitative data because
310
minor differences in 1JCH between oligosaccharides are not considered. In addition, varying
311
relaxation times might result in deviating signal intensities. The relative response factors were
312
determined by using low molecular weight compounds, which probably have comparable
313
relaxation times. Because enzymatic hydrolysates mostly contain low molecular weight
314
oligosaccharides, only small deviations should be observed for these samples. However,
315
soluble polysaccharide preparations are supposed to have significantly lower relaxation times.
316
Therefore, these limitations have to be considered when interpreting the obtained
317
semiquantitative data.
318
Sample preparation
319
The only requirement for soluble polysaccharide preparations is their solubility in D2O or
320
H2O/D2O (90/10) in concentrations that can be detected in the HSQC spectrum by using a
321
reasonable number of scans. DMSO-d6 was not used because it resulted in a limited solubility 14 ACS Paragon Plus Environment
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of some polysaccharides and solutions with a higher viscosity. In general, larger sample
323
concentrations are preferred because they reduce analysis time and/or increase sensitivity.
324
However, dissolving very high amounts of polysaccharides largely increases viscosity, which
325
in turn decreases spectroscopic resolution and sensitivity due to reduced T2 relaxation times.
326
Therefore, concentration versus viscosity needs to be balanced. Finding an optimum is highly
327
dependent on the polysaccharide preparation and its structural composition. For example, the
328
commercially available polysaccharides used in this study showed very different
329
physicochemical properties. Whereas it was possible to use soluble arabinans in
330
concentrations up to 50 mg/mL, a 10 mg/mL solution of soluble galactans already showed a
331
significant increase in viscosity. Therefore, both sample amount and number of scans have to
332
be adapted for each individual sample.
333
For the NMR spectroscopic analysis of insoluble polysaccharide preparations, solubilization
334
of the arabinan and galactan structural elements is necessary. This was achieved by using an
335
endo-arabinanase/endo-galactanase catalyzed hydrolysis, which was already successfully
336
applied for the isolation of the standard compounds and as sample preparation procedure in a
337
chromatographic approach to profile arabino- and galacto-oligosaccharides.13 Applying endo-
338
acting enzymes specifically hydrolyzes the arabinan and galactan backbones but preserves
339
side chains and other structural elements. Following addition of D2O for the lock signal (final
340
concentration 10 %), the hydrolysates can be directly analyzed by NMR.
341
To obtain suitable concentrations of the hydrolysis products, a relatively high substrate
342
concentration of 50 mg insoluble fiber/ mL was used. For some materials, this concentration
343
results in highly viscous suspensions, which may affect the enzymatic hydrolysis and the
344
isolation of the supernatant. In these cases, lower concentrations were used. To maximize the
345
amount of liberated oligosaccharides, 2 U of endo-arabinanase/endo-galactanase per 100 mg
346
insoluble fiber and an incubation time of 24 h at 40°C were applied. These conditions were 15 ACS Paragon Plus Environment
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347
demonstrated earlier to extensively hydrolyze arabinans and galactans.13 However, only low
348
amounts of oligosaccharides were obtained from the hydrolysis of some materials such as
349
sugar beet pulp or soybean extraction meal. Therefore, autoclave assisted extraction was
350
tested to get larger concentrations of oligosaccharides in the enzymatic hydrolysates. This
351
approach can be performed in water and was already successfully applied for the extraction of
352
pectic polysaccharides from sugar beet pulp and as a sample preparation procedure for the
353
chromatographic analysis of arabinan and galactan oligosaccharides.13,
354
suitability of the autoclave assisted extraction for the NMR spectroscopic profiling, amaranth,
355
quinoa, and buckwheat insoluble fiber were analyzed with and without autoclave extraction
356
prior to the enzymatic hydrolysis. These materials were chosen because they also yielded
357
sufficient oligosaccharide amounts without prior autoclave extraction. A preferential
358
degradation of specific structural elements or negative effects on the spectroscopic analysis
359
and precision were not detected after autoclave extraction. Thus, this pretreatment was
360
deemed suitable for the NMR spectroscopic profiling method, too. Uncertainties determined
361
by using the half range method were generally between 1 – 15 %. In some cases higher
362
uncertainties up to 25 % were observed, mostly for signals of very low intensity. The
363
comparably high uncertainties are potentially due to inaccurate integration of partially
364
overlapping signals such as signal 2. Generally, however, the precision of the method was
365
deemed acceptable for a semiquantitative estimation of arabinan and galactan structural
366
elements.
367
Although high quality HSQC spectra were achieved in H2O/D2O (90/10), a solvent exchange
368
after enzymatic hydrolysis was considered. Switching to D2O reduces disturbances caused by
369
the large water signal, while choosing DMSO-d6 might reveal other marker signals due to
370
changing chemical shifts, especially those of protons. In addition, the solvent exchange can be
371
used to concentrate the hydrolysates, too. To evaluate the influence of this approach on the
372
spectroscopic analysis, it was applied to an endo-arabinanase hydrolysate of amaranth 16 ACS Paragon Plus Environment
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To evaluate the
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373
insoluble fiber. As shown in Figure 5 the spectra in D2O and DMSO-d6 are characterized by
374
both decreased resolution and lower peak intensities. This might be due to incomplete
375
resolubilization of the oligosaccharides, which was observed for both solvents. Therefore, all
376
oligosaccharide hydrolysates were directly measured in H2O/D2O (90/10), and autoclave
377
extraction was applied to increase oligosaccharide concentrations.
378
Method application
379
To demonstrate the applicability of the developed method, various insoluble fiber samples
380
were analyzed. All samples allowed for a semiquantitative estimation of the structural
381
elements represented by the oligosaccharides in the hydrolysates. The main products of the
382
endo-galactanase degradation, t-O4-β-galactopyranose and 4-substituted galactopyranose
383
units, were detected in almost all hydrolysates, which provides clear evidence for the presence
384
of (1→4)-linked galactans. However, the ratio of these two structural elements [a) terminal
385
units represented by t-O4-β-galactopyranose and b) (1→4)-linked β-galactopyranose units
386
represented by 4-galactopyranose] is of limited value, because no information about the
387
original content of terminal galactopyranoses is obtained. From the chromatographic analysis
388
of endo-galactanase hydrolysates it was suggested that t-O4-α-arabinopyranose and 4-
389
substituted arabinopyranose containing oligosaccharides can be liberated from various
390
insoluble fiber samples.13 However, due to the low abundance of these structural elements,
391
they were only detected in soybean extraction meal insoluble fiber by using the NMR
392
approach. The semiquantitative estimation of the portions of terminal α-arabinopyranose units
393
(which are mainly considered to be derived from α-arabinopyranoses located at the chain ends
394
of the galactans) (5.7 %) and 1,4-disubstituted α-arabinopyranoses (represented by 4-α-
395
arabinopyranose containing oligosaccharides) (5.8 %) were comparable with the results from
396
the chromatographic approach. This demonstrates that NMR spectroscopy can be used to
397
prove the presence of these structural elements, provided suitable concentrations are present. 17 ACS Paragon Plus Environment
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398
Methylation analysis is unable to unambiguously detect these structural elements; therefore, a
399
comparison of the two methods is not possible.
400
For the endo-arabinanase hydrolysates, the portions of t-O2-α-arabinofuranose and t-O3-α-
401
arabinofuranose units and the corresponding branched backbone units (1,2,5-/1,2,3,5-
402
substituted or 1,3,5-/1,2,3,5-substituted α-arabinofuranose units) were mostly comparable.
403
Slight differences might occur due to unknown structural elements such as dimeric side
404
chains or the integration of small and partially overlapped signals. The consistency among
405
these structural units demonstrates that the semiquantitative estimation is applicable to
406
analyze different hydrolysates. To evaluate the information obtained from the NMR
407
spectroscopic approach, the results were compared with methylation analysis data (Table 2).
408
Similar results were obtained for all materials. Thus, they are exemplarily discussed for apple,
409
quinoa, and soybean extraction meal insoluble fiber. For a better comparison of the two
410
approaches, the portions of the arabinose-derived PMAAs were calculated from the
411
methylation analysis data. Because no information about the linkage position of terminal
412
arabinofuranose units can be derived from methylation analysis, the portions of t-O2-α-
413
arabinofuranose and t-O3-α-arabinofuranose units were summarized for the NMR
414
spectroscopic approach (t-β-arabinofuranose units were not taken into account). Due to the
415
endo-arabinanase catalyzed cleavage of the (1→5)-linked arabinan backbone, the portions of
416
the t-O5-α-arabinofuranose yield no information about the chain length of the arabinan
417
backbone. Therefore, the portions of this structural element were combined with the portions
418
of 5-arabinofuranose and 1,5-disubstituted α-arabinofuranose units. This approach was based
419
on the assumption that most of the t-O5-α-arabinofuranose units are derived from enzymatic
420
cleavage. This approach provides a better comparison with the methylation analysis, although
421
the amount of originally present chain ends are neglected.
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422
Despite these limitations and the fact that insufficient enzymatic cleavage might lead to an
423
underestimation of some structural units, comparable ratios are obtained for the different
424
structural elements. From both methods it can be concluded that mostly linear, 1,5-
425
disubstituted α-arabinofuranose units are present in all three materials with quinoa insoluble
426
fiber having the largest portion of 1,5-disubstituted arabinofuranose units, followed by
427
soybean extraction meal insoluble fiber, and apple insoluble fiber. In addition, both methods
428
reveal the same trends about the occurrence of branched backbone arabinose residues and for
429
the ratios between 1,3,5- and 1,2,5-substituted α-arabinofuranose units. The portions of
430
1,2,3,5-substituted α-arabinofuranose units as obtained by both methods are in good
431
agreement, too, demonstrating that apple and soybean extraction meal insoluble fiber contain
432
higher portions of this structural unit than quinoa insoluble fiber. However, the unambiguous
433
detection of this structural element is only possible with the NMR spectroscopic approach,
434
because the corresponding PMAA can also be derived from undermethylation. In addition, the
435
NMR spectroscopic approach allows for the detection of 1,3-disubstituted α-arabinofuranose
436
units as well as terminal β-arabinose units. Thus, the NMR approach gives similar
437
conclusions regarding common structural elements but provides additional information about
438
less common structural elements.
439
To evaluate the suitability of the developed approach for polysaccharide preparations, soluble
440
arabinan and galactan polysaccharides were analyzed and compared to methylation analysis
441
(Table 3). For soluble polysaccharides, the NMR spectroscopic approach allows for the
442
analysis of the unmodified structural elements. Therefore, estimations about the backbone
443
length can be deduced from the amount of t-O5-α-arabinofuranose units and t-O4-β-
444
galactopyranose units. Also, all terminal units determined by the NMR spectroscopic
445
approach were summarized for a better comparison with the methylation analysis data. For
446
the soluble arabinan polysaccharides a higher portion of terminal arabinofuranose units and a
447
lower portion of 1,5-disubstituted arabinofuranose units is obtained if compared to 19 ACS Paragon Plus Environment
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448
methylation analysis. This might be due to relaxation time differences of these structural
449
elements. It is possible that the arabinose units that are incorporated into the arabinan
450
backbone show a shorter relaxation time than the terminal arabinose units being associated
451
with a lower relative signal intensity of the backbone arabinose units. However, the portions
452
of the differently substituted backbone arabinose units are in good agreement between the two
453
methods. Also, small amounts of galactans were detected with both approaches in this
454
arabinan polysaccharide preparation. In addition, small portions of terminal β-
455
arabinofuranoses and 1,3-disubstituted α-arabinofuranose units were detected by NMR
456
spectroscopy, demonstrating again the advantages of this approach. Linear arabinan chains
457
were detected with both approaches in commercially available, soluble galactans. However,
458
significant differences between the two methods were observed if applied to the galactan
459
structural elements. NMR spectroscopy indicates a high amount of 1,4-disubstituted β-
460
galactopyranose residues, whereas a rather low portion of 1,4-disubstituted galactopyranose
461
units was detected by methylation analysis. Because of these discrepancies, the
462
polysaccharides were also analyzed for their monosaccharide composition after methanolysis
463
and TFA hydrolysis. The high galactose to arabinose (87:13) ratio indicated that 1,4-
464
disubstituted galactopyranose units are underestimated by methylation analysis under the
465
conditions used. Therefore, only the NMR spectroscopic approach allows for an estimation of
466
the approximate average chain length of the galactans, although different relaxation times
467
have to be considered. Furthermore, NMR spectroscopy provides information about 1,4-
468
disubstituted α-arabinopyranose units in addition to information about terminal α-
469
arabinopyranose units that can be obtained from both methods.
470
These results demonstrate that the developed NMR spectroscopic approach is well suitable for
471
the analysis of arabinan and galactan structural elements. The comparison with methylation
472
analysis data showed that similar information about the common structural elements are
473
obtained from both methods. While methylation analysis leads to a more complete cleavage 20 ACS Paragon Plus Environment
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474
of the polysaccharides and a higher precision, the NMR-spectroscopic approach provides
475
information about the anomeric configuration and the type of terminal units. In addition,
476
structural elements such as β-arabinofuranoses units and internal α-arabinopyranose units can
477
be detected by NMR spectroscopy, which is not possible by using conventional methods.
478
ABBREVIATIONS USED
479
HPAEC-PAD, High-performance anion-exchange chromatography with pulsed amperometric
480
detection; HSQC, Heteronuclear Single Quantum Coherence; PMAA, partially methylated
481
alditol acetate.
482
ACKNOWLEDGMENT
483
SUPPORTING INFORMATION DESCRIPTION
484
Supporting Information Available: Arabinan composition of amaranth, buckwheat, and sugar
485
beet pulp insoluble fiber and galactan composition of apple, quinoa, soybean extraction meal,
486
amaranth, buckwheat, and sugar beet pulp insoluble fiber. HSQC spectra of the endo-
487
arabinanase hydrolysates of apple, quinoa, soybean extraction meal, amaranth, buckwheat,
488
and sugar beet pulp insoluble fiber. This material is available free of charge via the Internet at
489
http://pubs.acs.org.
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REFERENCES
491 492 493 494 495 496 497 498 499 500 501 502 503 504
1. Harris, P. J.; Smith, B. G., Plant cell walls and cell-wall polysaccharides: Structures, properties and uses in food products. Int. J. Food Sci. Technol. 2006, 41, 129-143. 2. Voragen, A. G. J.; Coenen, G. J.; Verhoef, R. P.; Schols, H. A., Pectin, a versatile polysaccharide present in plant cell walls. Struct. Chem. 2009, 20, 263-275. 3. Renard, C. M. G. C.; Crepeau, M. J.; Thibault, J. F., Structure of the repeating units in the rhamnogalacturonic backbone of apple, beet and citrus. Carbohydr. Res. 1995, 275, 155165. 4. Navarro, D. A.; Cerezo, A. S.; Stortz, C. A., NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos. Carbohydr. Res. 2002, 337, 255-263.
505 506 507 508
5. Pustjens, A. M.; Schols, H. A.; Kabel, M. A.; Gruppen, H., Characterisation of cell wall polysaccharides from rapeseed (Brassica napus) meal. Carbohydr. Polym. 2013, 98, 1650-1656.
509 510 511 512
6. Wefers, D.; Bunzel, M., Characterization of dietary fiber polysaccharides from dehulled, common buckwheat (Fagopyrum esculentum) seeds. Cereal Chem. 2015, 92, 598603.
513 514 515 516
7. Wefers, D.; Gmeiner, B. M.; Tyl, C. E.; Bunzel, M., Characterization of diferuloylated pectic polysaccharides from quinoa (Chenopodium quinoa WILLD.). Phytochemistry 2015, 116, 320-328.
517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534
8. Wefers, D.; Tyl, C. E.; Bunzel, M., Neutral pectin side chains of amaranth (Amaranthus hypochondriacus) contain long, partially branched arabinans and short galactans, both with terminal arabinopyranoses. J. Agric. Food Chem. 2015, 63, 707-715. 9. Westphal, Y.; Kühnel, S.; de Waard, P.; Hinz, S. W. A.; Schols, H. A.; Voragen, A. G. J.; Gruppen, H., Branched arabino-oligosaccharides isolated from sugar beet arabinan. Carbohydr. Res. 2010, 345, 1180-1189. 10. Wefers, D.; Tyl, C. E.; Bunzel, M., Novel arabinan and galactan oligosaccharides from dicotyledonous plants. Front. Chem. 2014, 2, 100. 11. Cardoso, S. M.; Silva, A. M. S.; Coimbra, M. A., Structural characterisation of the olive pomace pectic polysaccharide arabinan side chains. Carbohydr. Res. 2002, 337, 917924. 12. Huisman, M. M. H.; Brull, L. P.; Thomas-Oates, J. E.; Haverkamp, J.; Schols, H. A.; Voragen, A. G. J., The occurrence of internal (1→5)-linked arabinofuranose and 22 ACS Paragon Plus Environment
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arabinopyranose residues in arabinogalactan side chains from soybean pectic substances. Carbohydr. Res. 2001, 330, 103-114. 13. Wefers, D.; Bunzel, M., Arabinan and galactan oligosaccharide profiling by highperformance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD). J. Agric. Food Chem. 2016, 64, 4656-4664. 14. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A., NMR chemical shifts of common laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512-7515.
544 545 546 547 548 549 550 551
16. Sweet, D. P.; Shapiro, R. H.; Albersheim, P., Quantitative-analysis by various GLC response-factor theories for partially methylated and partially ethylated alditol acetates. Carbohydr. Res. 1975, 40, 217-225.
552 553 554
17. Oosterveld, A.; Beldman, G.; Schols, H. A., Arabinose and ferulic acid rich pectic polysaccharides extracted from sugar beet pulp. Carbohydr. Res. 1996, 288, 143-153.
15. Nunes, F. M.; Reis, A.; Silva, A. M. S.; Domingues, M. R. M.; Coimbra, M. A., Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans. Phytochemistry 2008, 69, 1573-1585.
555 556 557 558
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FIGURE CAPTIONS
560 561
Figure 1:
562
1
563
the NMR-spectroscopic analysis of arabinan structural elements.
564
Figure 2:
565
1
566
the NMR-spectroscopic analysis of galactan structural elements.
567
Figure 3:
568
Integrated C4/H4 correlation peaks of t-O5-α-arabinofuranose units (signal 2) in the HSQC
569
spectra of arabinotriose (A), and differently branched arabino oligosaccharides constituted of
570
an arabinotriose substituted at the middle arabinose at positions O3 (B), O2 (C), and O2/O3
571
(D). The neighboring signals are derived from the t-O2-α-arabinofuranose and t-O3-α-
572
arabinofuranose units.
573
Figure 4:
574
HSQC-spectrum of soluble potato galactan polysaccharides in D2O.
575
Figure 5:
576
Anomeric region of the HSQC-spectrum of an amaranth insoluble dietary fiber endo-
577
arabinanase hydrolysate in H2O/D2O (90/10, A), D2O after freeze drying (B), and DMSO-d6
578
after freeze drying (C).
H/13C chemical shifts and relative response factors (RRF) of the HSQC marker signals for
H/13C chemical shifts and relative response factors (RRF) of the HSQC marker signals for
579 580
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Journal of Agricultural and Food Chemistry
TABLES Table 1: Calculation of the signal intensities of the arabinan and galactan structural elements in endoarabinanase and endo-galactanase hydrolysates and soluble polysaccharides. Structural element
Calculation in enzymatic Calculation in hydrolysates polysaccharides 5-/1,5-Araf S1-(S5+S6+S7)* S1-(S5+S6+S7)* t-O5-Araf S2 S2 t-O2-Araf S3 S3 t-O3-Araf S4 S4 1,2,5-Araf S5 S5 1,3,5-Araf S6 S6 1,2,3,5-Araf S7 S7 t-β-Araf S8 S8 1,3-Araf S9 S9 4-/1,4-Galp S10 S10 t-O4-Galp S14-(S12-S13) S11-(S12-S13) t-O4-Arap S12-S13 S12-S13 4-/1,4-Arap S13** S13 t= terminal, Ara = arabinose, Gal = galactose, p = pyranose, f = furanose, S = signal. Numbers indicate the substituted positions of a sugar unit, O2/O3/O4/O5 indicate the position to which a terminal residue is attached. * The intensity of signal 1 has to be divided by 2 ** Inaccuracies due to the underestimation of the β-anomer of 4-Arap can be corrected by multiplying by 1.535
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Table 2: Arabinan composition of apple, quinoa, and soybean extraction meal insoluble fiber determined by NMR spectroscopy after autoclave extraction and endo-arabinanase hydrolysis and by methylation analysis. The portions (in %) of some structural elements are listed summarized and individually. All analyses were performed in duplicate, half range uncertainties were mostly < 2 % for methylation analysis and < 15 % for the NMR spectroscopic profiling. Apple IDF
Quinoa IDF
5-/1,5-Araf t-O5-Araf t-O2-Araf t-O3-Araf
19.7 15.6 12.8 24.8
29.7 24.2 5.7 15.9
Soybean extraction meal IDF 19.8 23.5 15.8 18.3
Σ 5-/1,5-Araf/ t-O5-Araf
35.3
53.8
43.3
Σ t-O3/O2-Araf
37.6
21.5
34.1
1,2,5-Araf 1,3,5-Araf 1,2,3,5-Araf t-β-Araf 1,3-Araf
2.0 18.0 7.2 -
1.3 16.7 0.7 2.8 3.1
4.7 11.6 6.2 -
29.9 37.0 1.5 22.3 9.4
58.3 20.2 1.4 18.7 1.5
37.4 38.4 5.2 10.5 8.4
Structural element
Linkage type from PMAA) 1,5-Araf t-Araf 1,2,5-Araf 1,3,5-Araf 1,2,3,5-Araf
(derived
IDF = insoluble dietary fiber, t = terminal, Ara = arabinose, f = furanose, PMAA = partially methylated alditol acetate. Numbers indicate the substituted positions of a sugar unit, O2/O3/O4/O5 indicate the position to which a terminal residue is attached.
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Journal of Agricultural and Food Chemistry
Table 3: Arabinan and galactan composition of soluble sugar beet arabinans and soluble potato galactans determined by NMR and by methylation analysis. Portions are given in %. All analyses were performed in duplicate, half range uncertainties were mostly < 2 % for methylation analysis and < 15 % for the NMR spectroscopic profiling. Structural element t-O5-Araf t-O2-Araf t-O3-Araf
Soluble arabinans 8.9 5.5 27.6
Soluble galactans 1.1 -
Σ t-Araf 1,5-Araf 1,2,5-Araf 1,3,5-Araf 1,2,3,5-Araf t-β-Araf 1,3-Araf 1,4-Galp t-Galp t-Arap 1,4-Arap
42.0 23.2 1.1 23.8 3.8 0.4 0.7 3.9 1.1 -
15.0 79.3 2.0 0.6 2.0
Linkage type (derived from PMAA) t-Araf 1,5-Araf 1,2,5-Araf 1,3,5-Araf 1,2,3,5-Araf t-Arap t-Galp 1,4-Galp
36.9 28.2 1.6 24.6 4.7 2.5 1.6
4.2 35.6 1.9 13.2 45.1
IDF = insoluble dietary fiber, t = terminal, Ara = arabinose, Gal = galactose, p = pyranose, f = furanose, PMAA = partially methylated alditol acetate. Numbers indicate the substituted positions of a sugar unit, O2/O3/O4/O5 indicate the position to which a terminal residue is attached.
27 ACS Paragon Plus Environment
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Page 28 of 33
FIGURES Figure 1:
Signal 1 3.70 - 3.98 ppm 66.7 ppm RRF: 1.000 O
Ara
O
OH
Signal 3
4.09 ppm 84.4 ppm
5.18 ppm 107.5 ppm
RRF: 1.052
O Ara
Ara
Ara
Ara
O
O
HO
O
O O
O OH
OH
OH
OH
O
Ara O OH HO
OH
Ara
Ara
Signal 4
Signal 5
Signal 6
5.14 ppm 107.5 ppm
4.15 ppm 87.5 ppm
5.11 ppm 107.9 ppm
RRF: 1.209
RRF: 1.056
OH
HO OH O
O
Ara
O
O O
O OH
OH
Ara
O OH
O
OH
Ara
HO
OH
OH
Signal 7
Signal 8
Signal 9
5.24 ppm 106.8 ppm
5.07 ppm 102.0 ppm
4.38 ppm 80.1 ppm
RRF: 1.494
RRF: 1.145
RRF: 0.868
O OH
OH
HO
Ara
OH O
HO
O
O OH OH
O
O
O
O
O HO
HO
HO
+
OH
O OH
Ara
O O
Ara
O
O
HO
OH
O
O OH
O
Ara
OH
HO
O O
Ara O
O OH
Ara
O
O O
O HO
Ara
OH O O
Ara O
O
HO
OH
HO
O
O O
+
O
O
O OH
Ara
OH O
O
O O
RRF: 1.456
Ara
OH
HO Ara
O
O
O
HO
O
O
Ara
OH O
+
OH
O
O
O
O OH
Ara
O
O
O
O
O OH
O
OH
HO
Ara
O
O OH
OH OH
RRF: 1.338 Ara
OH Ara O O
Ara
O
O OH
O OH
Signal 2
OH
Ara
OH
28 ACS Paragon Plus Environment
HO
OH
Page 29 of 33
Journal of Agricultural and Food Chemistry
Figure 2:
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Figure 3:
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Journal of Agricultural and Food Chemistry
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31 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 5:
32 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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33 ACS Paragon Plus Environment