NMR Spectroscopic Profiling of Arabinan and Galactan Structural

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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

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properties of plant cell walls and dietary fiber. Their complex structures are usually analyzed

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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

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based on enzymatic cleavage and two-dimensional NMR spectroscopy was developed.

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Heteronuclear single quantum coherence spectroscopy (HSQC) marker signals were evaluated

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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

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different insoluble plant materials and soluble polysaccharides. It was demonstrated that the

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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

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NMR method additional structural information such as anomeric configuration of the

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monomers is obtained, demonstrating the value of this novel approach.

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KEYWORDS

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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

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besides cellulose.1 These structurally complex polymers are defined as galacturonic acid

20

containing polysaccharides and are composed of several structural subgroups. The

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quantitatively most important subgroups are homogalacturonan and rhamnogalacturonan I,

22

polymers with comparably conserved, galacturonic acid containing backbone structures.2,

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Arabinans and galactans are neutral side chains of rhamnogalacturonan I and show, however,

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highly complex structures. Arabinans are composed of a backbone of α-(1→5)-linked

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arabinofuranose units, which can be substituted with monomeric or oligomeric arabinose side

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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

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from the occurrence of terminal β-arabinofuranose units in some arabinan chains.10,

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Galactans consist of a backbone of β-(1→4)-linked galactopyranoses, which is frequently

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described to be ramified at the positions O3 and O6.2 Recently it has been demonstrated that

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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

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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

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recently, a method for the routine analysis of intact arabinan and galactan oligosaccharides by

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high-performance anion-exchange chromatography with pulsed amperometric detection

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(HPAEC-PAD) after enzymatic cleavage with endo-arabinanase and endo-galactanase was

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described.13 By using this approach it was possible to obtain information about the occurrence 3 ACS Paragon Plus Environment

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of common and rare structural elements. However, this chromatographic screening method

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requires standard compounds for each analyte, and high molecular weight oligosaccharides

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are not taken into account. In contrast to chromatographic approaches, the application of

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NMR spectroscopy allows for the detection of all solubilized compounds. In addition, the 1H

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and

48

environment, which is usually represented by the adjacent sugar units. This results in the

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simultaneous detection of specific structural elements or substituents in oligosaccharides and

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polysaccharides of varying sizes. Thus, NMR spectroscopy is a highly suitable detection

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technique for the routine analysis of different structural elements in carbohydrate mixtures.

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Both oligosaccharide mixtures and polysaccharides often result in highly complex one-

53

dimensional spectra, preventing the unambiguous identification of specific structural

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elements. Correlations between NMR active nuclei in two-dimensional NMR experiments

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such as Heteronuclear Single Quantum Coherence (HSQC), provide an extra gain in

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resolution by spreading the spectra in a second dimension.

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Therefore, the aim of this study was to establish a rapid and simple two-dimensional NMR-

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spectroscopic profiling approach to semiquantitatively determine arabinan and galactan

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structural elements in insoluble and soluble polysaccharide preparations.

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MATERIALS AND METHODS

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Chemicals

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If not stated otherwise, all chemical compounds used were at least of pro-analysi-grade and

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were purchased from VWR (Radnor, PA), Sigma Aldrich (Schnelldorf, Germany), or Carl

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Roth (Karlsruhe, Germany). Deuterium oxide (99.9 atom% D) and dimethylsulfoxide-d6

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(99.9 atom% D) were purchased from Sigma Aldrich. Thermostable α-amylase (Termamyl

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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.

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niger, 408 U/mg), arabinan from sugar beet, and galactan from potato were purchased from

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Megazyme (Bray, Ireland). Oligosaccharide standard compounds were isolated from

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enzymatic digests of various polysaccharide preparations and completely characterized by

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NMR spectroscopy as described previously.8, 10, 13

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Materials

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Apples (Malus domestica cv. Braeburn, grown and harvested 2013 in Germany), quinoa

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(Chenopodium quinoa Willd., grown and harvested 2012 in Bolivia), amaranth (Amaranthus

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hypochondriacus, grown and harvested 2012 in Germany), and buckwheat (Fagopyrum

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esculentum, grown and harvested 2012 in China) were purchased from local suppliers.

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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

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without peel and apple core and freeze-dried prior to milling. Sugar beet pulp was also freeze-

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dried prior to milling. All materials were milled to a particle size < 0.5 mm by using an MF10

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basic mill (IKA-Werke, Staufen, Germany). Amaranth, quinoa, and buckwheat meals were

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defatted with acetone prior to dietary fiber isolation.

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Dietary fiber isolation

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Insoluble dietary fiber was isolated by suspending 20 g of the milled plant material in 200 mL

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of 0.08 M phosphate buffer (pH 6.2). Thermostable α-amylase (1.5 mL) was added, and the

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mixture was incubated for 20 min at 95°C. After adjustment of the pH to 7.5 with sodium

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hydroxide, the suspension was incubated with 700 µL of protease for 30 min at 60°C. The pH

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was adjusted to 4.5, and 700 µL of amyloglucosidase was added. The suspension was

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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

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(Bruker, Rheinstetten, Germany) equipped with a Prodigy cryoprobe. HSQC spectra were

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acquired by using the hsqcedetgp pulse sequence with the standard parameter set provided by

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Bruker. This included a relaxation time of 1.5 s and a 1JCH coupling constant of 145 Hz. 1024

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points were recorded in the f2 dimension and 256 points were recorded in the f1 dimension.

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32 scans were acquired for almost all samples, resulting in adequate signal intensities and

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acceptable analysis times (approximately 3 h 40 min).

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All HSQC marker signals and relative response factors were evaluated by using several well

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characterized arabinan and galactan oligosaccharides previously isolated from several plant

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materials.8,

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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

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oligosaccharide was overlaid with the spectra of all other standard compounds. After

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evaluation of the marker signals, a (volume) integration mask was created that allowed for the

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rapid and reproducible analysis of various samples. The relative response factors were

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determined by analyzing the standard oligosaccharides for the relative intensity of the marker

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signals to an internal reference signal (signal 1 for arabinans, signal 14 for galactans).

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Method application

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To analyze insoluble fiber preparations, the sample amount and the incubation volume were

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adapted to achieve an acceptable, free-flowing sample viscosity during hydrolysis and a

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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

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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

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centrifugation. An aliquot of the clear supernatant (450 µL) was transferred into an NMR

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tube, and 50 µL of deuterium oxide was added. Acetone (0.5 µL) was used for spectrum

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calibration (2.22 ppm (1H) / 30.89 ppm (13C)),14 and HSQC spectra were acquired as

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described above. Soluble polysaccharides were dissolved in 500 µL of D2O, and their spectra

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were also referenced to acetone. For the calculation of the portions of the structural elements,

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the intensities of the marker signals were multiplied by their relative response factor, modified

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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

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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

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were hydrolyzed by adding 2 mL of 2 M TFA and incubation at 121°C for 90 min. After

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evaporation of the acid, 20 mg of NaBD4 in 300 µL of an aqueous ammonia solution (2 M)

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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

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overnight at -18°C. GC-MS analysis of the partially methylated alditol acetates (PMAAs) was

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performed on GC-2010 Plus and GCMS-QP2010 SE instruments (Shimadzu, Kyoto, Japan)

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both equipped with a DB-225MS column (30 m x 0.25 mm i.d., 0.25 µm film thickness)

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(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

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was used as carrier gas at 40 cm/sec. Split injection with a split ratio of 30:1 was used, and the

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injection temperature was 220°C. The transfer line was held at 220°C, and electron impact

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mass spectra were recorded at 70 eV. PMAAs were quantitated by analyzing the samples by

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GC-FID (GC-2010 Plus) (Shimadzu) using the same conditions as described above, but

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applying a 10:1 split ratio. Nitrogen was used as makeup gas, and the FID temperature was

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240°C. Molar response factors according to Sweet et al.16 were used for semiquantitative

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analyses. All analyses were performed in duplicate.

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RESULTS AND DISCUSSION

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Evaluation of marker signals

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The two-dimensional HSQC experiment, which provides information about short-range (1J)

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couplings between protons and

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galactan structural elements. Application of the HSQC experiment improves virtual resolution

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by using a second dimension: overlapping proton signals may become distinguishable by

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carbon shift differences, and overlapping carbon shifts are pulled apart by proton shift

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differences. Being an inverse experiment, the application of an HSQC experiment is less

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time-consuming than a one-dimensional carbon experiment, resulting in acceptable spectra

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acquisition times. Different from one-dimensional experiments, volume integration of the

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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

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needed. Ideally, these marker signals are specific for a single structural element and do not

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overlap with signals derived from the same or other structural elements. To evaluate the

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suitability of specific signals as marker signals, the HSQC spectra of various arabinan and

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galactan oligosaccharide standard compounds were compared. These oligosaccharides were

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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

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Figures 1 and 2. Different from the ideal situation, some marker signals represent more than

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one structural element due to the very similar chemical environment of the corresponding

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protons/carbons. In these cases the intensities of other marker signals had to be subtracted to

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get information about a single structural element (Table 1; 5-/1,5-Araf, t-O4-Galp, t-O4-

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Arap).

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For example, this approach was used to calculate the intensity of an 1,5-disubstituted

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backbone α-arabinofuranose unit (1,5-arabinofuranose) from signal 1, which represents the

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C5/H5 correlation peaks of all arabinofuranose units that are substituted at position 5 (signal 1

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in Figure 1). In addition, this signal represents two protons and, therefore, needs to be divided

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by two. It was not possible to use the signal of the anomeric proton/carbon because it showed

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the same chemical shift as a terminal α-arabinofuranose unit attached to another

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arabinofuranose unit through a (1→5)-linkage (t-O5-α-arabinofuranose). However, due to the

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different substitution patterns, all branched backbone arabinose units (additional substitution

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sites next to position 1 and 5) showed characteristic marker signals. Therefore, it was possible

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to subtract their intensities from signal 1 to obtain the intensity of 1,5-disubstituted α-

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arabinose units. However, reducing arabinose units that are only substituted in position 5 (5-

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arabinofuranose) cannot be distinguished from 1,5-disubstituted α-arabinofuranose units by

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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

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(signal 6 and signal 7, respectively, in Figure 1). The C1/H1 HSQC signal of the 1,2,5-

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substituted α-arabinofuranose also showed a characteristic position, but it was very close to

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signal 6. This makes the detection of small amounts of 1,2,5-substituted α-arabinofuranose

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units besides large amounts of 1,3,5-substituted α-arabinofuranose units, a common

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composition for many pectic materials, difficult. Therefore, the well-separated C2/H2

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correlation peak was chosen as marker signal for a 1,2,5-substituted α-arabinofuranose unit

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(signal 5 in Figure 1). The correlation peaks of the anomeric protons/carbons were suitable to

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identify terminal α-arabinofuranose units that are linked through (1→3)-linkages (t-O3-α-

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arabinofuranose) or (1→2)-linkages (t-O2-α-arabinofuranose) to the arabinan backbone,

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respectively (signal 3 and signal 4 in Figure 1). The t-O5-α-arabinofuranose did not show any

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specific signal, except for the C4/H4 correlation peak (signal 2 in Figure 1). As shown in

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Figure 3, this peak showed a partial overlap with the C4/H4 signals of t-O2-α-arabinofuranose

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units. Thus, some inaccuracies may be observed when high amounts of t-O2-α-

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arabinofuranose and t-O5-α-arabinofuranose units (Figure 3, C) or high amounts of t-O2-α-

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arabinofuranose, t-O3-α-arabinofuranose, and t-O5-α-arabinofuranose (Figure 3, D) are

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present (both of which being possible but rather unlikely scenarios). Dimeric, β-(1→3)-linked

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arabinobiose side chains were recently established as structural elements of quinoa

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arabinans.10, 13 The analysis of arabinose units in their β-configuration is of particular interest

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because they may have an impact on the enzymatic degradability of arabinans but cannot be

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detected by conventional methods such as methylation analysis. By using the NMR

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spectroscopic approach, terminal β-arabinose units can easily be distinguished from all other

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structural elements, because the C1/H1 correlation peak shows a unique

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(signal 8 in Figure 1). Finally, 3-O-(β-arabinofuranosyl)-α-arabinofuranose residues, which

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were identified as side chains in quinoa arabinans can be identified by their C2/H2 correlation

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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-

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galactopyranose units (1,4-galactopyranose) and terminal β-galactopyranose units forming the

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non-reducing end of the backbone by being attached through a (1→4)-linkage (t-O4-β-

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galactopyranose). The analysis of internal 1,4-disubstituted α-arabinopyranoses (1,4-

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arabinopyranose) and terminal α-arabinopyranose units attached to the backbone through a

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(1→4)-linkage (t-O4-β-arabinopyranose), however, might be more important because the role

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of these recently established structural elements is yet unknown. In addition, these structural

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elements, which constitute relatively low portions of galactans, are difficult to analyze by

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using conventional methods. For example, internal 1,4-disubstituted α-arabinopyranose units

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cannot be detected by methylation analysis because they yield the same PMAA as arabinan

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derived 1,5-disubstituted α-arabinofuranose units. The anomeric HSQC correlation peaks of

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β-galactopyranoses and α-arabinopyranose units show minor differences in their 1H chemical

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shifts and about the same 13C chemical shifts. Although 1,4-disubstituted β-galactopyranose,

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t-O4-β-galactopyranose, 1,4-disubstituted α-arabinopyranose, and t-O4-α-arabinopyranose

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can be differentiated through their anomeric signals if standard compounds are measured, this

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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.

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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

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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

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arabinopyranose units substituted at position O4 (4-α-arabinopyranose, formed through 11

proton

of

1,4-disubstituted

α-arabinopyranose

ACS Paragon Plus Environment

units

and

reducing

α-


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galactan hydrolysis or being the reducing end of the polymer) can be used to unambiguously

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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

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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

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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

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containing disaccharides.13 Therefore, the signal in the selected part of the anomeric region

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(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).

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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.

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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

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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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Page 15 of 33


322

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


Page 16 of 33

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

17

To evaluate the

Page 17 of 33


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


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.


Page 18 of 33

Page 19 of 33


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


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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Page 21 of 33


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.



490

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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535 536 537 538 539 540 541 542 543


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



559

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


Page 24 of 33

Page 25 of 33


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



Page 26 of 33

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.


Page 27 of 33


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.



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


HO

OH

Page 29 of 33


Figure 2:



Figure 3:


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Page 31 of 33


Figure 4:



Figure 5:


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NMR Spectroscopic Profiling of Arabinan and Galactan Structural

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