Arabinan and Galactan Oligosaccharide Profiling by High

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Arabinan and Galactan Oligosaccharide Profiling by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) Daniel Wefers, and Mirko Bunzel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01121 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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Journal of Agricultural and Food Chemistry

Arabinan and Galactan Oligosaccharide Profiling by High-Performance AnionExchange Chromatography with Pulsed Amperometric Detection (HPAECPAD)

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]) 1 ACS Paragon Plus Environment


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ABSTRACT

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Arabinans and galactans are complex pectic polysaccharides, which greatly influence the

3

physicochemical and physiological properties of plants and plant based foods. Conventional

4

methods to characterize these challenging polymers are based on derivatization and/or

5

unselective chemical cleavage of the glycosidic bonds of the polysaccharides resulting in

6

partial loss of essential information such as anomeric configuration. Here, endo-arabinanase

7

and endo-galactanase were used to selectively cleave pectic arabinans and galactans. The

8

liberated oligosaccharides were purified and characterized by LC-MS and one- and two-

9

dimensional NMR spectroscopy resulting in known but also several previously unknown

10

pectic structural elements. For the routine analysis of pectin hydrolyzates by HPAEC-PAD,

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incubation conditions, chromatographic parameters, and relative response factors of the

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isolated pectic oligosaccharides against an internal standard were determined. The

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applicability of the method was demonstrated by analyzing different well-characterized plant

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cell wall materials. It was demonstrated that the developed method yields additional

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information about pectic arabinan and galactan structures that is not obtained from

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conventional methods such as methylation analysis.

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KEYWORDS

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Pectic arabinans and galactans, pectins, screening, oligosaccharide profiling, dietary fiber,

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plant cell wall constituents, selective enzymatic hydrolysis, HPAEC-PAD.

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2 ACS Paragon Plus Environment

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INTRODUCTION

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Pectins are a group of complex, ubiquitous plant cell wall polysaccharides. Especially in

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dicotyledons, pectic polysaccharides are often major plant cell wall constituents,1 having a

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large impact on quality parameters such as the texture of fruits and vegetables.2 Also, pectins

26

are dietary fiber constituents with potential health benefits. Depending on their chemical

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structure, pectins are divided into certain subgroups with homogalacturonan and

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rhamnogalacturonan I being the quantitatively dominating polymers. Homogalacturonan is

29

made up of α-(1→4)-linked

30

composed of repeating units of α-(1→4)-linked D-galacturonic acid and α-(1→2)-linked L-

31

rhamnose. Complexity is added to rhamnogalacturonan I polymers through neutral side chains

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of arabinans and galactans attached to the O-4 position of the rhamnose residues. Arabinans

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have a backbone of α-(1→5)-linked

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substituted with different side chains at multiple positions.3 The O-3 position was reported as

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the main arabinan branching point for various plant species such as sugar beet, amaranth,

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quinoa, and buckwheat.4-7 In other plant materials, however, the arabinans were mainly

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branched at position O-2.8, 9 Another common structural element is the substitution of both,

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the O-2 and O-3 arabinose position.4, 5 Substitution of adjacent and nearby arabinose units at

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the same or different positions has also been described.4,

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demonstrated that dimeric side chains composed of a β-arabinose substituted arabinose are

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linked to the O-3 position of quinoa arabinans.10 Structural elements such as β-arabinose

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containing side chains are, however, difficult to analyze by commonly applied methods of

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polysaccharide analysis because information about the anomeric configuration is usually lost.

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Galactans were also suggested to have several branching patterns with substitution of the β-

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(1→4)-linked backbone at positions O-3 or O-6 by arabinofuranoses and galactopyranoses

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being commonly reported.3 However, the existence of some of these structural elements has

D-galacturonic

acid residues, while rhamnogalacturonan I is

L-arabinofuranose

residues, which are partially


5, 7

Most recently, it was


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not yet been unambiguously proven by the isolation and characterization of diagnostic

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oligosaccharides. Recently, it was demonstrated that arabinopyranoses can be linked to

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galactans, both as internal and terminal units.5,

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units are not detected by methylation analysis, because they result in the same partially

51

methylated alditol acetate (PMAA) as (1→5)-linked arabinofuranoses.

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Promising approaches to structurally characterize cell wall polysaccharides in more detail are

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based on enzymatic liberation of oligosaccharides followed by their analysis by HPAEC-

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PAD. For example, Ordaz-Ortiz et al.12 used an HPAEC-PAD profiling method to get detailed

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information about arabinoxylan structures in different wheat varieties. Comparable

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approaches for pectin analysis were, however, based on the qualitative analysis of a limited

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number of oligosaccharides only.5,

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comprehensive, semiquantitative HPAEC-PAD profiling method for the analysis of endo-

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arabinanase and endo-galactanase liberated pectic oligosaccharides. By using this approach,

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data from conventional methods can be complemented by detailed information about

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structural elements of neutral pectic side chains.

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MATERIALS AND METHODS

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General

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endo-Arabinanase (EC 3.2.1.99, from A. niger, 9 U/mg), endo-galactanase (EC 3.2.1.89, from

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A. niger, 408 U/mg), arabinan from sugar beet, polygalacturonic acid from citrus pectin, and

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galactan from potato were purchased from Megazyme (Bray, Ireland). Thermostable α-

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amylase (Termamyl 120 L, EC 3.2.1.1, from B. licheniformis, 120 KNU/g), protease

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(Alcalase 2.4L, EC 3.4.21.62, from B. licheniformis, 2.4 AU/g), and amyloglucosidase (AMG

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300L, EC 3.2.1.3, from A. niger, 300 AGU/g) were kindly donated by Novozymes

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(Bagsvaerd, Denmark). Bio-Gel P-2 was from Bio-Rad Laboratories (Hercules, CA).

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10, 11

Internal (1→4)-linked arabinopyranose

Therefore, the aim of this study was to develop a


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Driselase enzyme preparation from Basidiomycetes sp., malto-oligosaccharides, and raffinose

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were purchased from Sigma Aldrich (Schnelldorf, Germany).

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Materials

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Quinoa seeds (Chenopodium quinoa Willd., grown and harvested 2012 in Bolivia) and apples

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(Malus domestica cv. Braeburn, grown and harvested 2013 in Germany) were purchased from

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local suppliers. Soybean extraction meal was kindly provided by ADM Rothensee (Hamburg,

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Germany). Apples were peeled and the pulp without seeds and apple-core was freeze-dried

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prior to milling. The materials were milled to a particle size < 0.5 mm using an MF10 basic

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mill (IKA-Werke, Staufen, Germany). Quinoa meal was defatted with acetone before further

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analysis as described previously.6

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Dietary fiber isolation

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To isolate preparative amounts of dietary fiber, 20 g of apple, quinoa, or soybean extraction

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meal was suspended in 200 mL of 0.08 M phosphate buffer (pH 6.2), and the suspension was

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incubated with 1.5 mL of thermostable α-amylase at 92 °C for 20 min. The pH was adjusted

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to 7.5, and 700 µL of protease were added. The mixture was incubated for 30 min at 60 °C.

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The pH was adjusted to 4.5 with hydrochloric acid, 700 µL of amyloglucosidase was added,

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and the suspension was incubated for 30 min at 60 °C. Insoluble fiber was obtained by

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centrifugation and washed with water, ethanol (99%, v/v), and acetone.

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Isolation and purification of standard compounds

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To isolate arabino-oligosaccharides (Figure 1), arabinans from sugar beet, amaranth insoluble

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fiber, and quinoa insoluble fiber were incubated with endo-arabinanase as described

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previously.5, 10 In addition, arabinans from sugar beet (1 g) were dissolved in 100 mL of water

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and incubated with 100 mg of Driselase enzyme preparation for 24 h at 40 °C. G-2b, G-2c, 5 ACS Paragon Plus Environment


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and G-3c (Figure 2) were liberated by endo-galactanase (20 U/g insoluble fiber) from

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soybean extraction meal insoluble fiber after 40 min of autoclave extraction (8 g fiber were

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suspended in 160 mL water, autoclaved for 40 min at 121 °C, and filled up to 400 mL).

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Galacto-oligosaccharides G-2a, G-3a, G-3b, and G-4 were liberated from potato galactan and

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polygalacturonic acid as described previously.5, 10

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The hydrolyzates were fractionated by Bio-Gel P-2 chromatography (bed volume: 85 cm x

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2.6 cm) at 45 °C with water as eluent at 1 mL/min. Oligosaccharide fractions were detected

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using a Smartline RI detector (Knauer, Berlin, Germany). Fractions were collected every 2.5

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min, analyzed by HPAEC-PAD, and freeze dried. Further purification was achieved by

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additional fractionation on an HPLC system (L-7100 pump, L-7490 RI detector)

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(Merck/Hitachi, Darmstadt, Germany) equipped with a 250 mm x 8 mm i.d., 5 µm

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semipreparative Eurosphere 100 C18 column (Knauer) or a 250 mm × 4.6 mm i.d., 5 µm

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Luna C18 column (Phenomenex, Torrance, CA). Water at 0.5 - 1 mL/min was used as eluent.

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Impure compounds were further fractionated on an HPLC-ELSD system (AZURA P 2.1L

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pumps, Knauer; Sedex 85 ELSD detector) (Sedere, Alfortville, France) equipped with an

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adjustable flow splitter (Analytical Scientific Instruments, Richmond, CA). A 100 mm x 4.6

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mm i.d., 5 µm porous graphitized carbon (PGC) Hypercarb column (Thermo Fisher Scientific,

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Waltham, MA) was used at a flow rate of 3 mL/min and 70 °C. A gradient composed of water

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(A) and acetonitrile (B) was applied: 0-1 min, isocratic 100% A; 1-20 min, linear to 80% A

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and 20% B; 20-24 min, isocratic 20% A and 80% B; 24-28 min, isocratic 80% A and 20% B.

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HPAEC-PAD analysis

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Arabino- and galacto-oligosaccharides were analyzed on an ICS-5000 System (Thermo

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Scientific Dionex, Sunnyvale, CA) equipped with a 250 mm x 3 mm i.d., 5.5 µm CarboPac

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PA200 column (Thermo Scientific Dionex). The injection volume was 25 µL. Flow rates of

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0.45 mL/min (arabino-oligosaccharides) and 0.4 mL/min (galacto-oligosaccharides) and 6 ACS Paragon Plus Environment

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gradients composed of the following eluents were used at 25 °C: (A) bidistilled water, (B) 0.1

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M sodium hydroxide, (C) 0.1 M sodium hydroxide + 0.5 M sodium acetate. Before every run,

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the column was washed with 100% C for 10 min and equilibrated with 90% A and 10% B for

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20 min. The following gradient was used for the arabino-oligosaccharides: 0-10 min, from

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90% A and 10% B linear to 50% A and 50% B; 10-60 min, linear to 50% A, 30% B, and 20%

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C; 60-75 min, linear to 40% B, and 60% C; 75-90 min, isocratic 100% C. The following

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gradient was used for the galacto-oligosaccharides: 0-10 min, isocratic 90% A and 10% B;

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10-20 min, linear to 50% A and 50% B; 20-60 min, linear to 50% A, 40% B, and 10% C; 60-

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75 min, linear to 50% B and 50% C; 75-80 min, linear to 100% C; 80-90 min, isocratic 100%

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C.

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Characterization of standard compounds

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An aliquot of the oligosaccharides was hydrolyzed with 2 M TFA at 121 °C for 30 min. After

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evaporation, the samples were redissolved in water and analyzed for their monosaccharide

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composition by HPAEC-PAD on an ICS-5000 System (Thermo Scientific Dionex) using a

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150 mm x 3 mm i.d., 6.5 µm CarboPac PA20 column (Thermo Scientific Dionex) at 25 °C. A

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flow rate of 0.4 mL/min and a gradient composed of (A) bidistilled water, (B) 0.1 M sodium

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hydroxide, (C) 0.1 M sodium hydroxide + 0.2 M sodium acetate were used: Before every run,

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the column was rinsed with 100% B for 10 min and equilibrated for 20 min with 90% A and

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10% B. After injection, the following gradient was applied: 0-1.5 min, from 90% A and 10%

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B to 96% A and 4% B; 1.5-22 min, isocratic, 96% A and 4% B; 22-32 min from 96% A and

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4% B to 100% B; 32-42 min, isocratic, 100% C.

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To determine the monosaccharide

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heated overnight at 130 °C with 150 µL of (R)-octanol and 5 µL of TFA as described by

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Leontein et al.13 The solvent was removed and samples were silylated by using 80 µL of N,O-

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bis(trimethylsilyl)trifluoroacetamide and 20 µL of pyridine. Silylated sugar derivatives were 7

D/L-configuration,

evaporated TFA hydrolyzates were

ACS Paragon Plus Environment


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analyzed by GC-MS with GC-2010 Plus and GCMS-QP2010 Ultra instruments (Shimadzu,

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Kyoto, Japan) on a 30 m x 0.25 mm i.d., 0.25 µm Rxi-5Sil MS column (Restek, Bad

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Homburg, Germany) with the following conditions: Initial column temperature, 150 °C;

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ramped at 1 °C/min to 200 °C; then ramped at 15 °C/min to 300 °C. Split injection was

148

performed at a split ratio of 10:1, and the injection temperature was 275 °C. Helium was used

149

as carrier gas at 40 cm/sec. The transfer line was held at 275 °C and electron impact

150

ionization was performed at 70 eV.

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LC-PGC-MS analysis was carried out on a Surveyor HPLC System, coupled to an LXQ linear

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ion trap MSn system (Thermo Fisher Scientific). A 100 x 2.1 mm, 3 µm analytical PGC

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Hypercarb column (Thermo Fisher Scientific) at a flow rate of 0.2 mL/min was used at 70 °C.

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The following gradient composed of 25 µM aqueous LiCl (A) and acetonitrile (B) was used:

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0-1 min, 100% A; 1-20 min, linear to 85% A and 15% B; 20-28 min, linear to 30% A and

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70% B; 28-31 min, linear to 20% A and 80% B; 31-35 min, isocratic 20% A and 80% B; 35-

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36 min, linear to 100% A; 36-41 min, isocratic 100% A.

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Structural elucidation and determination of concentrations were carried out by NMR

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spectroscopy. The oligosaccharides were hydrogen-deuterium exchanged and dissolved in

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D2O. Spectra were acquired on an Ascend 500 MHz NMR spectrometer (Bruker,

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Rheinstetten, Germany) equipped with a Prodigy cryoprobe. Acetanilide (0.5 mg/mL) was

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used as an internal standard and for spectrum calibration (2.17 ppm (1H) / 23.36 ppm (13C),

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determined relative to acetone at 2.22 ppm (1H) / 30.89 ppm (13C)), according to Gottlieb et

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al.14 To identify the structures of the oligosaccharides, 1H, H,H-Correlated Spectroscopy

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(COSY), Heteronuclear Single Quantum Coherence (HSQC), and Heteronuclear Multiple

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Bond Correlation (HMBC) experiments were performed. 1H spectra (pulse sequence: zg, 16

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Scans) with a relaxation delay of 60 s were acquired to determine the oligosaccharide

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concentrations. Oligosaccharide signals representing a known number of protons were chosen 8 ACS Paragon Plus Environment

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for integration, and the peak area relative to the peak area of the signal of the acetanilide

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methyl group was determined.

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Method development

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Linearity of the detector response was tested for wide concentration ranges of oligosaccharide

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standard solutions. Concentrations within the linear ranges (Table 1) were used to evaluate

174

relative response factors, which were determined by three-fold injection of at least five

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concentrations of the oligosaccharide solutions containing the internal standard raffinose (10

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µM). HPAEC-PAD analyses were very reproducible, and relative standard deviations of the

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signal areas were constantly < 5%. Values of concentration(oligosaccharide) x area(raffinose)

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were plotted against values of concentration(raffinose) x area(oligosaccharide), and the slope

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of the regression line was used as relative response factor. Limit of quantitation (signal to

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noise ratio 9:1) and limit of detection (signal to noise ratio 3:1) were also determined. In

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endo-arabinanase and endo-galactanase hydrolyzates of cell walls or dietary fiber

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preparations, the main matrix compounds are oligosaccharides (other than the individual

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oligosaccharide detected) and soluble polysaccharides. To test whether matrix carbohydrates

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influence the determined response factors, the stock solution of arabinobiose, A-2a, was

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exemplarily diluted with different oligosaccharides (arabino- and malto-oligosaccharides) and

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polysaccharides (arabinan from sugar beet), and relative response factors were determined. To

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study a potential impact of buffer salts on the chromatographic behavior and the response

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factors of the oligosaccharide analytes, 0.05 M sodium acetate buffer (pH 5) was used to

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dilute different oligosaccharide standard solutions.

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To determine suitable enzyme activities and incubation times, apple insoluble fiber (5 mg)

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was suspended in 500 µL of water and incubated with different enzyme activities (endo-

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arabinanase: 1, 2, and 5 U/100 mg insoluble fiber, endo-galactanase: 2, 5, 10, and 20 U/100

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mg insoluble fiber) for 24 h at 40 °C. After incubation, enzymes were inactivated by heating 9 ACS Paragon Plus Environment


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to 95 °C for 5 min. After centrifugation (20000 rcf, at least 3 min) an aliquot of the

195

hydrolyzate was appropriately diluted, and raffinose was added as an internal standard to

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yield a concentration of 10 µM.

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Method application

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Autoclave extraction was carried out by suspending 5 mg of fiber in 500 µL of water and

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incubation at 121 °C for 40 min. Insoluble fiber samples and cooled autoclave extracts were

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hydrolyzed as described above using 2 U endo-arabinanase / 100 mg insoluble fiber or 10 U

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endo-galactanase / 100 mg insoluble fiber. All analyses were performed in duplicate, and an

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incubation mixture without enzyme was analyzed as a control.

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To compare the results of the developed HPAEC-PAD profiling method with results of the

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commonly used methylation analysis, this standard method of polysaccharide analysis was

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performed as described by Nunes et al.15 with minor modifications. After dissolving the

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sample (5 mg) in dimethyl sulfoxide (2 mL), 100 mg of freshly ground sodium hydroxide

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pellets were added, and the mixture was incubated for 90 min in an ultrasonic bath and 90 min

208

at room temperature. Methyl iodide (1 mL) was added, followed by sonication for 30 min and

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incubation for 30 min at room temperature. The solution was extracted with dichloromethane,

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and the organic phase was washed once with 3 mL of 0.1 M sodium thiosulfate and twice

211

with 3 mL of water. After evaporation of the organic phase, the sample was dried overnight in

212

a vacuum oven at 40 °C. Following the addition of 2 M TFA (2 mL), the residue was

213

incubated at 121 °C for 90 min. The TFA was removed by evaporation, and NaBD4 (20 mg)

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in aqueous NH3 solution (2 M) was added. Reduction was carried out at room temperature for

215

1 h and terminated by addition of glacial acetic acid. For acetylation, 450 µL of 1-

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methylimidazole and 3 mL of acetic anhydride were added under ice cooling, and the solution

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was incubated for 30 min at room temperature. After the addition of water (3 mL), the

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solution was extracted with 5 mL of dichloromethane. The organic phase was washed with 10 ACS Paragon Plus Environment

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water (three times, 5 mL), and residual water was removed by freezing overnight at -18 °C.

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The PMAAs in dichloromethane were analyzed by GC-MS (GC-2010 Plus and GCMS-

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QP2010 SE) (Shimadzu) on a 30 m x 0.25 mm i.d., 0.25 µm DB-225MS column (Agilent

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Technologies, Santa Clara, CA) using the following conditions: Initial column temperature,

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140 °C, held for 1 min; ramped at 20 °C/min to 220 °C, held for 25 min. Helium was used as

224

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

225

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

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

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FID (GC-2010 Plus) (Shimadzu) on a 30 m x 0.25 mm i.d., 0.25 µm DB-225 column (Agilent

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Technologies) with the same conditions as described above, but using a 10:1 split ratio. FID

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temperature was 240 °C, and nitrogen was used as makeup gas. Molar response factors

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according to Sweet et al.16 were used for semiquantitative analyses.

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RESULTS AND DISCUSSION

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Standard compounds

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Arabino-oligosaccharide standard compounds were prepared from sugar beet arabinans (A-

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2a/3a/3b/4a/5a/5d/6a/6b/7a/7b), red clover sprouts (A-4b), amaranth (A-2b), and quinoa (A-

235

5b/5c) insoluble fiber after endo-arabinanase (main products) or Driselase (side products)

236

(Figure 1) treatment. Following Bio-Gel P-2 and C18-HPLC fractionation, the

237

oligosaccharides were further purified by PGC-chromatography. The purity of the individual

238

compounds was generally > 90%, an acceptable purity for their use as standard compounds.

239

The structures of the oligosaccharides A-2a/2b/3a/4a/4b/5a/5b/6a/7b were described earlier.4,

240

5, 10

241

and/or O-2 positions. Compounds A-6a and A-7b, with adjacent and nearby backbone

242

arabinose units being branched, represent highly ramified areas of the arabinan backbone.

243

Compound A-7a, isolated from sugar beet arabinan, also represents a highly branched area of 11

They represent different side chains of the arabinan backbone, with branches at O-3



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the arabinan backbone with one O-3- and one double-substituted backbone arabinose unit. A

245

similar oligosaccharide was already described by Westphal et al.4 but with an additional

246

arabinose unit at the reducing end. NMR data of this compound were in good agreement with

247

previously published data. Compound A-2b is composed of an arabinofuranose unit and a

248

terminal xylopyranose unit (which was originally interpreted as an arabinopyranose) and was

249

previously isolated from an endo-arabinanase digest of amaranth insoluble fiber.5 Just like the

250

previously described compound A-5b, compound A-5c was liberated from quinoa insoluble

251

fiber, but in lower amounts. Also, some impurities were difficult to remove from this fraction

252

by C18-HPLC. Thus, the digestion procedure was repeated and the pooled A-5c fractions

253

were purified by PGC-chromatography. By using this technique, a sufficient amount of the

254

oligosaccharide was purified for its characterization by NMR spectroscopy. The

255

oligosaccharide represents the same complex dimeric side chain as A-5b, where a β-arabinose

256

is attached to O-3 of another arabinose. However, the oligosaccharides differ in their

257

substitution position of the arabinan backbone (A-5b: O-3, A-5c: O-2). The

258

shifts of the dimeric side chains of these two oligosaccharides were in good agreement,10

259

whereas the chemical shifts of the backbone arabinoses of A-5c were in good agreement with

260

an 1,2,5-substituted arabinose.5

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Compounds A-3b/5d/6b, which were isolated from a Driselase digest of sugar beet arabinans,

262

had similar substitution patterns as compound A-4a (A-3b), compound A-6a (A-5d), and

263

compound A-7b (A-6b), but are devoid of terminal arabinose units attached to position O-5.

264

Because the multienzyme preparation Driselase contains substantial arabinofuranosidase

265

activity, the oligosaccharides A-3b/5d/6b likely result from continued degradation of the

266

oligosaccharides A-4a/6a/7b. Compounds A-3b and A-5d were already characterized by

267

Westphal et al.4 and the NMR data were in good agreement. The oligosaccharides A-

268

3b/5d/6b were included in the method, because they may also be part of the arabinans of 12 ACS Paragon Plus Environment

13

C chemical

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some plants. However, in the fiber hydrolyzates studied here, they were only of low

270

abundance and probably resulted from an arabinofuranosidase side activity of the endo-

271

arabinanase preparation; therefore, they were claimed as side products.

272

The oligosaccharides representing linear galactans (G-2a and G-3a) were isolated from citrus

273

pectin and potato galactan and were already extensively characterized.5, 17, 18 Usually, G-2a is

274

the main product of the endo-galactanase hydrolysis and G-3a is only detected after

275

incomplete hydrolysis of linear galactans. Compound G-3b and compound G-4, which were

276

isolated from potato galactan, represent oligosaccharides with terminal, O-4-attached

277

arabinopyranose units and internal, α-(1→4)-linked arabinopyranose units and were

278

characterized previously.5,

279

conventional methods of polysaccharide analysis, because an internal (1→4)-linked

280

arabinopyranose yields the same PMAA as (1→5)-linked arabinofuranose. G-2b, G-2c, and

281

G-3c were isolated from an endo-galactanase hydrolyzate of autoclave extracted soy

282

extraction meal. Their structures were evaluated by HPAEC, LC-MS, and NMR

283

spectroscopy. In the course of the method development it was evaluated that G-2b and G-2c

284

are end products of endo-galactanase digestion as demonstrated by endo-galactanase

285

incubation of G-3b and G-4. Galactose and G-2b were the only digestion products of G-3b,

286

while G-4 was mainly digested to G-2a and G-2c. In addition, G-3c and galactose were

287

detected in the G-4 hydrolyzate, while only trace amounts of G-2b were present. Arabinose

288

was not detected in either hydrolyzate. Thus, G-2b and G-2c are the main products of the

289

enzymatic digestion representing terminal (G-2b) and internal (G-2c) arabinopyranoses. The

290

side products G-3a/3b/3c/4 were also included into the method to detect incomplete

291

hydrolysis.

292

Stock solutions of the standard compounds were prepared after determination of their

293

concentrations by quantitative 1H NMR. Acetanilide was used as an internal standard as

10, 11

These structural elements are difficult to assess by



294

previously suggested.19 This compound was very suitable, because it has sufficient water

295

solubility and is not detected by HPAEC-PAD avoiding its removal after NMR analysis.

296

Method development

297

Chromatography, validation parameters. All isolated oligosaccharides were sufficiently well

298

separated by HPAEC on a CarboPac PA200 column. To determine the amounts of arabinan

299

and galacto-oligosaccharides by HPAEC-PAD, response factors relative to an internal

300

standard were used. Various compounds, which do not naturally occur in plants, were tested

301

for their use as internal standard compounds, but all of them co-eluted with either a standard

302

compound or matrix peaks. Raffinose, however, suited all requirements, despite its natural

303

occurrence in some vegetables. Thus, it is necessary to carefully remove oligosaccharides

304

from the analyzed cell wall or dietary fiber material by suitable washing procedures. Relative

305

response factors, retention times, limits of quantitation, and limits of detection of the arabino-

306

and galacto-oligosaccharides are listed in Table 1. If trace amounts of some oligosaccharides

307

are present only or if the correct assignment of the oligosaccharides is difficult for various

308

reasons, LC-PGC-MS2 proved to be a useful tool for unambiguous identification, a technique

309

used more recently for the analysis of oligosaccharides.5, 20, 21 Thus, PGC retention times of

310

the oligosaccharides are also listed in Table 1. To be analyzed by PGC, galacto-

311

oligosaccharides were reduced as described previously to avoid anomeric peak broadening.5,

312

22

313

The PAD response of single compounds depends on the exact concentration and purity of the

314

eluents used, but relative response factors remain largely constant. This was demonstrated by

315

determining the response factors of arabinobiose (compound A-2a) relative to raffinose with

316

eluents prepared on different days. Thus, a recalibration is not necessary. The response factors

317

were determined at a raffinose concentration of 10 µM. Other concentrations were also tested,

318

but yielded slightly different relative response factors. Thus, it is mandatory to use a constant 14 ACS Paragon Plus Environment

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319

raffinose concentration for every measurement. To achieve this, raffinose is added to the

320

hydrolyzates after enzymatic digestion. By using this approach, different dilutions of the

321

hydrolyzates can be analyzed without repeating the digestion procedure.

322

To study potential matrix effects, the relative response factor of arabinobiose, A-2a, was

323

evaluated in different matrices. Due to incomplete enzymatic degradation, arabinan and

324

galactan polysaccharides but also higher amounts of other oligosaccharides may be present in

325

the hydrolyzate. Arabinobiose, A-2a, to raffinose response factors were measured with and

326

without the addition of arabinan from sugar beet (polysaccharides), other arabino-

327

oligosaccharides, and malto-oligosaccharides. The relative response factors were not

328

influenced by the presence of these compounds, demonstrating sufficient robustness.

329

Sodium acetate buffer was reported as a suitable buffer system for endo-arabinanase and

330

endo-galactanase incubations.23,

331

solubilized in sodium acetate buffer, a major impact of the buffer salts on both

332

chromatography and response factors was demonstrated. Retention times were significantly

333

reduced, double peaks were observed for most of the oligosaccharides, and the values for the

334

relative response factors also differed from those determined in water. Therefore it was tested

335

whether the enzymatic digestion can be performed in water instead of buffer. The enzymatic

336

digestion in water yielded a very similar pattern if compared to an enzymatic digestion in the

337

buffer system, yet with decreased intensities. Thus, water was chosen as the incubation

338

medium.

339

Quantitative aspects, sample preparation. Because oligosaccharide liberation may not be

340

quantitative, it has to be estimated whether an absolute quantitation is deemed reasonable. It

341

needs to be considered that enzymatic liberation of oligosaccharides is dependent on the cell

342

wall architecture potentially impeding enzymatic accessibility. In addition, endo-arabinanase

343

and endo-galactanase preparations are only able to cleave linear (α-(1→5)-linked 15

24

However, by injecting different standard compounds



344

arabinofuranoses and β-(1→4)-linked galactopyranoses) areas of the arabinan and galactan

345

polysaccharides. The resulting branched oligosaccharides can only be analyzed to a degree of

346

polymerization of 7 by using the method described here. This results in an

347

underrepresentation of highly branched high molecular-weight compounds. However,

348

absolute quantitation appears reasonable to compare similar samples or to follow individual

349

samples over a certain period of time, for example during storage or ripening of plant based

350

products.

351

Because both amounts and types of liberated oligosaccharides are also dependent on the

352

amount of enzyme used, suitable incubation conditions were evaluated. Apple insoluble fiber

353

(10 mg/mL incubation medium) was chosen as a substrate because it contains both arabinans

354

and galactans incorporated into a comparably simple cell wall architecture (mostly primary

355

parenchymatous cell walls). The optimum amounts of endo-arabinanase and endo-galactanase

356

were established by incubating batches with various enzyme activities for 24 h. Compounds

357

A-3a and A-3b were chosen as marker oligosaccharides to evaluate endo-arabinanase

358

mediated degradation. Large concentrations of A-3a indicate insufficient enzyme activity,

359

while large concentrations of A-3b demonstrate unsuitably high arabinofuranosidase side

360

activities in the incubation batch. Using 2 U endo-arabinanase/100 mg insoluble fiber was

361

determined to be a suitable enzyme activity. The relative proportions of the main fermentation

362

products (G-2a/2b/2c) and the appearance of potential side products were used to evaluate the

363

endo-galactanase concentration. Using an activity of 10 U/100 mg insoluble fiber, no further

364

modification of the relative proportions among G-2a/2b/2c was observed. These studies also

365

revealed that the endo-galactanase preparation used here contained side activities.

366

Considerable amounts of glucose and oligosaccharides other than galacto-oligosaccharides,

367

eluting after most of the galacto-oligosaccharides (Figure 3), were observed in the endo-

368

galactanase hydrolyzates, which were absent in the endo-arabinanase hydrolyzates and 16 ACS Paragon Plus Environment

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Page 17 of 34


369

incubation batches without enzyme. Isolation and monosaccharide analysis of some of the

370

later eluting peaks indicated xylogluco-oligosaccharides, suggesting a β-(1→4)-glucanase

371

side activity.

372

Method application

373

To demonstrate the applicability of the method, various plant materials were studied. For

374

some materials such as sugar beet pulp only very low amounts of arabino-oligosaccharides

375

and no galacto-oligosaccharides were liberated. This could be due to the incorporation of

376

these polysaccharides into a complex cell wall architecture. A low liberation yield of galacto-

377

oligosaccharides was also observed for amaranth fiber. However, yields were increased by

378

enzymatic degradation of polysaccharides extracted with calcium hydroxide.5 Here, however,

379

calcium hydroxide extraction was not used, because it involves various time consuming steps

380

and the use of strong salt solutions, which have to be removed due to their potential effect on

381

HPAEC analysis and enzyme activities. Oosterveld et al.25 used an autoclave assisted

382

extraction of sugar beet polysaccharides. Because this extraction can be performed in water

383

and there is no need for further treatment of the extract, autoclave assisted extraction was

384

tested for this profiling approach by applying it to different plant materials (apple insoluble

385

fiber, quinoa insoluble fiber, and soybean extraction meal insoluble fiber) (Table 2).

386

The precision of the method was good (half range uncertainty < 5%) for the individual

387

oligosaccharides, with and without autoclave extraction. A significantly higher amount of

388

oligosaccharides was liberated for all materials by applying the autoclave assisted extraction

389

procedure, but the arabino-oligosaccharide composition was roughly comparable between

390

both procedures. Although the relative proportion is somewhat different for some

391

oligosaccharides, no structural elements are specifically degraded or only liberated by either

392

of the treatments. The numeric changes in the composition are likely due to the autoclave

393

assisted liberation of structurally different polysaccharides that are strongly incorporated into 17 ACS Paragon Plus Environment


394

the cell wall. Thus, materials with hard to degrade cell wall architectures hindering enzymatic

395

cleavage can effectively be analyzed following the autoclave assisted extraction approach.

396

Methylation analysis was performed to compare the information gathered from the approach

397

described here to the information obtained from a standard procedure of polysaccharide

398

analysis. The composition of the PMAAs is given in Table 3. As stated above, the

399

oligosaccharides analyzed here are not fully representative for the whole cell wall

400

composition because higher oligosaccharides are not detected. On the other hand, structural

401

elements such as β-arabinose units and (1→4)-linked arabinopyranose units cannot be

402

detected by methylation analysis. Thus, the data of both methods are not directly comparable,

403

but give the same trends for certain structural elements. As demonstrated by the

404

corresponding PMAAs, apple insoluble fiber consists of rather complex arabinans, which are

405

mainly branched at position O-3 and contain lower amounts of O-2 and double substituted

406

backbone arabinose units. A comparable PMAA composition was reported for quinoa

407

insoluble fiber.6 The endo-arabinanase liberated oligosaccharides indicate a similar

408

composition, as the main branching position of the detected oligosaccharides is also O-3. The

409

other branching patterns were also unambiguously identified by the analyzed oligosaccharides

410

(A-4b and A-5a). Different from the oligosaccharide profiling approach, unambiguous

411

identification of double substituted arabinose units by methylation analysis is difficult

412

because the corresponding PMAA may also arise from undermethylation. Additionally, the

413

enzymatic approach points out that both materials analyzed by methylation analysis also

414

contain highly branched areas as demonstrated by the detection of the corresponding

415

oligosaccharides (A-6a and A-7b); an information that cannot be obtained from methylation

416

analysis. As demonstrated by methylation analysis data, the arabinans from soybean

417

extraction meal have a higher portion of linear structures than apple insoluble fiber, which are

418

probably short, with large amounts of PMAAs representing terminal arabinose units. In 18 ACS Paragon Plus Environment

Page 18 of 34

Page 19 of 34


419

addition, arabinan O-2 branches are more abundant than in apple insoluble fiber. The same

420

trend is demonstrated by the liberated oligosaccharides, with O-2-branched oligosaccharides

421

being detected in slightly higher amounts than O-3-branched oligosaccharides. In addition, a

422

high amount of A-2a is detected, suggesting larger amounts of linear arabinans. The

423

oligosaccharides representing more heavily branched areas are also of low abundance,

424

suggesting a rather simple arabinan structure for soybean extraction meal.

425

A comparison of the galactan data of both methods used is difficult because information about

426

terminal galactose units is not obtained by the oligosaccharide profiling, whereas methylation

427

analysis does not give any information about internal arabinopyranose units. In addition,

428

comparison of monosaccharide analysis and methylation analysis data from numerous

429

samples analyzed in our laboratory suggests that galactans are underestimated by methylation

430

analysis under the conditions used. For example, methylation analysis of the soluble galactan

431

from potato polysaccharides yielded a lower amount of galactan derived PMAAs ((1→4)-

432

linked Galp in particular) than expected from its monosaccharide composition. However, both

433

galactans from soybean extraction meal and from apple seem to contain considerable amounts

434

of terminal arabinopyranoses, which is demonstrated by both methylation analysis and

435

oligosaccharide profiling. The advantage of the latter is that the arabinopyranoses can be

436

clearly assigned to be attached to galactans and not to other structural elements. In addition,

437

the portion of internal arabinopyranoses can be assessed by the developed profiling approach.

438

These results demonstrate that the developed method can be readily applied to various plant

439

materials. It provides additional information about the structural elements of different

440

polysaccharides. In particular β-arabinose units and internal arabinopyranose units can be

441

analyzed by this approach, which is not possible by conventional approaches such as

442

methylation analysis. The fate of these structural elements during, for example, ripening or

443

post-harvest storage and processing can now be elucidated by using this profiling approach. 19 ACS Paragon Plus Environment


444

ABBREVIATIONS USED

445

COSY, Correlated Spectroscopy; HSQC, Heteronuclear Single Quantum Coherence; HMBC,

446

Heteronuclear Multiple Bond Correlation; PGC, porous graphitized carbon; PMAA, partially

447

methylated alditol acetate.

448

ACKNOWLEDGMENT

449

SUPPORTING INFORMATION DESCRIPTION

450

Supporting Information Available: NMR data for the standard compounds A-5c, A-7a, A-3b,

451

A-5d, A-6b, G-2b, G-2c, and G-3c. This material is available free of charge via the Internet

452

at http://pubs.acs.org.


Page 20 of 34

Page 21 of 34

453


REFERENCES

454 455

1.

Harris, P. J.; Smith, B. G., Plant cell walls and cell-wall polysaccharides: Structures,

456

properties and uses in food products. Int. J. Food Sci. Technol. 2006, 41, 129-143.

457 458

2.

Pena, M. J.; Carpita, N. C., Loss of highly branched arabinans and debranching of

459

rhamnogalacturonan I accompany loss of firm texture and cell separation during prolonged

460

storage of apple. Plant Physiol. 2004, 135, 1305-1313.

461 462

3.

Voragen, A. G. J.; Coenen, G. J.; Verhoef, R. P.; Schols, H. A., Pectin, a versatile

463

polysaccharide present in plant cell walls. Struct. Chem. 2009, 20, 263-275.

464 465

4.

Westphal, Y.; Kühnel, S.; de Waard, P.; Hinz, S. W. A.; Schols, H. A.; Voragen, A. G.

466

J.; Gruppen, H., Branched arabino-oligosaccharides isolated from sugar beet arabinan.

467

Carbohydr. Res. 2010, 345, 1180-1189.

468 469

5.

Wefers, D.; Tyl, C. E.; Bunzel, M., Neutral pectin side chains of amaranth

470

(Amaranthus hypochondriacus) contain long, partially branched arabinans and short

471

galactans, both with terminal arabinopyranoses. J. Agric. Food Chem. 2015, 63, 707-715.

472 473

6.

Wefers, D.; Gmeiner, B. M.; Tyl, C. E.; Bunzel, M., Characterization of diferuloylated

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pectic polysaccharides from quinoa (Chenopodium quinoa WILLD.). Phytochemistry 2015,

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116, 320-328. 21 ACS Paragon Plus Environment


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7.

Wefers, D.; Bunzel, M., Characterization of dietary fiber polysaccharides from

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dehulled, common buckwheat (Fagopyrum esculentum) seeds. Cereal Chem. 2015, 92, 598-

479

603.

480 481

8.

Pustjens, A. M.; Schols, H. A.; Kabel, M. A.; Gruppen, H., Characterisation of cell

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wall polysaccharides from rapeseed (Brassica napus) meal. Carbohydr. Polym. 2013, 98,

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1650-1656.

484 485

9.

Navarro, D. A.; Cerezo, A. S.; Stortz, C. A., NMR spectroscopy and chemical studies

486

of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos.

487

Carbohydr. Res. 2002, 337, 255-263.

488 489

10.

Wefers, D.; Tyl, C. E.; Bunzel, M., Novel arabinan and galactan oligosaccharides from

490

dicotyledonous plants. Front. Chem. 2014, 2, 100.

491 492

11.

Huisman, M. M. H.; Brull, L. P.; Thomas-Oates, J. E.; Haverkamp, J.; Schols, H. A.;

493

Voragen, A. G. J., The occurrence of internal (1→5)-linked arabinofuranose and

494

arabinopyranose residues in arabinogalactan side chains from soybean pectic substances.

495

Carbohydr. Res. 2001, 330, 103-114.

496 497

12.

Ordaz-Ortiz, J. J.; Devaux, M. F.; Saulnier, L., Classification of wheat varieties based

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on structural features of arabinoxylans as revealed by endoxylanase treatment of flour and

499

grain. J. Agric. Food Chem. 2005, 53, 8349-8356. 22 ACS Paragon Plus Environment

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13.

Leontein, K.; Lindberg, B.; Lonngren, J., Assignment of absolute configuration of

502

sugars by GLC of their acetylated glycosides formed from chiral alcohols. Carbohydr. Res.

503

1978, 62, 359-362.

504 505

14.

Gottlieb, H. E.; Kotlyar, V.; Nudelman, A., NMR chemical shifts of common

506

laboratory solvents as trace impurities. J. Org. Chem. 1997, 62, 7512-7515.

507 508

15.

Nunes, F. M.; Reis, A.; Silva, A. M. S.; Domingues, M. R. M.; Coimbra, M. A.,

509

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee

510

arabinogalactans. Phytochemistry 2008, 69, 1573-1585.

511 512

16.

Sweet, D. P.; Shapiro, R. H.; Albersheim, P., Quantitative-analysis by various GLC

513

response-factor theories for partially methylated and partially ethylated alditol acetates.

514

Carbohydr. Res. 1975, 40, 217-225.

515 516

17.

Lichtenthaler, F. W.; Oberthur, M.; Peters, S., Directed and efficient syntheses of

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β(1→4)-linked galacto-oligosaccharides. Eur. J. Org. Chem. 2001, 2001, 3849-3869.

518 519

18.

Fransen, C. T. M.; Van Laere, K. M. J.; van Wijk, A. A. C.; Brull, L. P.; Dignum, M.;

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Thomas-Oates, J. E.; Haverkamp, J.; Schols, H. A.; Voragen, A. G. J.; Kamerling, J. P.;

521

Vliegenthart, J. F. G., α-D-Glcp-(1 ↔ 1)-β-D-Galp-containing oligosaccharides, novel

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products from lactose by the action of β-galactosidase. Carbohydr. Res. 1998, 314, 101-114.



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19.

Rundlöf, T.; McEwen, I.; Johansson, M.; Arvidsson, T., Use and qualification of

525

primary and secondary standards employed in quantitative

526

pharmaceuticals. J. Pharm. Biomed. Anal. 2014, 93, 111-117.

1

H NMR spectroscopy of

527 528

20.

Westphal, Y.; Schols, H. A.; Voragen, A. G. J.; Gruppen, H., Introducing porous

529

graphitized carbon liquid chromatography with evaporative light scattering and mass

530

spectrometry detection into cell wall oligosaccharide analysis. J. Chromatogr. A 2010, 1217,

531

689-695.

532 533

21.

Westphal, Y.; Kühnel, S.; Schols, H. A.; Voragen, A. G. J.; Gruppen, H., LC/CE-MS

534

tools for the analysis of complex arabino-oligosaccharides. Carbohydr. Res. 2010, 345, 2239-

535

2251.

536 537

22.

Pitson, S. M.; Voragen, A. G. J.; Vincken, J. P.; Beldman, G., Action patterns and

538

mapping of the substrate-binding regions of endo-(1→5)-α-L-arabinanases from Aspergillus

539

niger and Aspergillus aculeatus. Carbohydr. Res. 1997, 303, 207-218.

540 541

23.

Dunkel, M. P. H.; Amado, R., Purification and physicochemical properties of an endo-

542

1,5-α-L-arabinanase (EC 3.2.1.99) isolated from an Aspergillus niger pectinase preparation.

543

Carbohydr. Polym. 1994, 24, 247-263.

544 545

24.

Van de Vis, J. W.; Searle-van Leeuwen, M. J. F.; Siliha, H. A.; Kormelink, F. J. M.;

546

Voragen, A. G. J., Purification and characterization of endo-1,4-β-D-galactanases from 24 ACS Paragon Plus Environment

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547

Aspergillus niger and Aspergillus aculeatus: Use in combination with arabinanases from

548

Aspergillus niger in enzymatic conversion of potato arabinogalactan. Carbohydr. Polym.

549

1991, 16, 167-187.

550 551

25.

Oosterveld, A.; Beldman, G.; Schols, H. A., Arabinose and ferulic acid rich pectic

552

polysaccharides extracted from sugar beet pulp. Carbohydr. Res. 1996, 288, 143-153.

553 554 555 556



557

Page 26 of 34

FIGURE CAPTIONS

558 559

Figure 1: Structures of arabino-oligosaccharide standard compounds. Letters R, A, B, C, T, a,

560

and b are used to describe the sugar units for the NMR spectroscopic assignment, R and T are

561

additionally used to abbreviate the sugar units.

562 563

Figure 2: Structures of galacto-oligosaccharide standard compounds. Letters R, A, and T are

564

used to describe the sugar units for the NMR spectroscopic assignment.

565 566

Figure 3: HPAEC chromatograms of the endo-arabinanase hydrolyzate of quinoa insoluble

567

fiber and of the endo-galactanase hydrolyzate of apple insoluble fiber. The structures of the

568

marked

peaks

are

shown

in

Figure


1

and

Figure

2.

Page 27 of 34


TABLES

Table 1: Relative response factors (RRF), the linear concentration range (CR) of the RRF, limit of quantitation (LOQ), limit of detection (LOD), retention time on a CarboPac PA200 column (Rt HPAEC), and retention time on a PGC column (Rt PGC) of the arabino- and galactooligosaccharides. Compound

RRF

CR [µM]

LOQ

LOD

Rt

[µM]

[µM]

[min]

HPAEC

Rt

PGC

[min]

Raffinose

1.000

-

-

-

13.7

14.1

A-2a

0.880

0.1 - 30

0.088

0.036

28.0

4.6

A-2b

0.827

0.5 - 10

0.083

0.032

21.1

5.9

A-4a

0.597

0.1 - 24

0.066

0.032

49.5

15.5

A-4b

0.719

0.1 - 20

0.073

0.038

49.8

15.8

A-5a

0.571

0.1 - 18

0.056

0.023

51.5

18.0

A-5b

0.567

0.1 - 18

0.060

0.028

54.3

19.5

A-5c

0.697

0.5 - 10

0.065

0.023

55.4

18.6

A-6a

0.561

0.1 - 18

0.057

0.025

67.5

22.7

A-7a

0.611

0.1 - 15

0.050

0.014

68.6

23.7

A-7b

0.504

0.1 - 18

0.055

0.027

71.2

24.2

A-3a

0.616

0.1 - 24

0.073

0.036

38.6

10.8

A-3b

0.659

0.1 - 24

0.062

0.024

40.2

9.9

A-5d

0.593

0.1 - 20

0.077

0.042

62.1

19.0

A-6c

0.655

0.1 - 15

0.082

0.047

68.8

22.7



Page 28 of 34

Raffinose

1.000

-

-

-

17.2

14.1

G-2a

0.874

0.5 – 24

0.116

0.049

19.3

3.1

G-2b

1.134

0.5 – 24

0.108

0.040

15.4

3.1

G-2c

0.780

0.25 – 12

0.067

0.013

12.2

-

G-3a

0.578

0.1 – 24

0.061

0.023

35.2

8.7

G-3b

0.596

0.1 – 24

0.075

0.040

33.1

8.1

G-3c

0.598

0.1 – 24

0.071

0.028

30.8

8.6

G-4

0.686

0.1 – 24

0.060

0.019

40.2

12.3


Page 29 of 34


Table 2: Relative composition of oligosaccharides liberated with and without autoclave extraction from apple insoluble fiber (apple-IF), quinoa insoluble fiber (quinoa-IF), and soybean extraction meal insoluble fiber by endo-arabinanase and endo-galactanase treatment. Compound

apple-IF

apple-IF

quinoa-IF

extracted

quinoa-IF

SEM-IF

extracted

SEM-IF extracted

A-2a

80.7%

82.2%

76.5%

74.6%

87.0%

92.0%

A-4a

13.0%

12.0%

13.4%

14.9%

5.4%

3.0%

A-4b

1.0%

0.8%

1.3%

1.7%

4.9%

3.6%

A-5a

0.6%

0.6%

0.5%

0.6%

0.6%

0.2%

A-5b

-

-

3.0%

2.0%

-

-

A-5c

-

-

0.8%

0.5%

-

-

A-6a

2.1%

2.2%

1.6%

2.1%

tr

tr

A-7a

-

-

-

-

-

-

A-7b

2.7%

2.2%

3.0%

3.5%

2.1%

1.2%

G-2a

93.4%

89.8%

-

tr

85.6%

87.8%

G-2b

3.8%

6.6%

-

-

7.9%

6.8%

G-2c

2.8%

3.6%

-

-

6.5%

5.3%

tr = traces



Table 3: Percentages of the partially methylated alditol acetates from apple insoluble fiber and soybean extraction meal insoluble fiber. Glycosidic linkage

apple insoluble fiber

soybean extraction meal insoluble fiber

t-Araf

9.5%

14.9%

t-Arap

0.5%

0.9%

1,5-Araf/1,4 Arap

7.6%

14.5%

1,3,5-Araf

5.7%

4.1%

1,2,5-Araf

0.4%

2.0%

1,2,3,5-Araf

2.4%

3.3%

t-Galp

3.1%

3.9%

1,4-Galp

1.1%

8.2%

1,6-Galp

0.3%

0.6%

1,2-Rhap

0.3%

1.1%

1,2,4-Rhap

0.3%

0.7%

t-Glcp

1.3%

0.8%

1,4-Glcp

42.2%

23.4%

1,4,6-Glcp

7.6%

2.5%

t-Xylp

10.5%

7.9%

1,4-Xylp

1.3%

3.4%

1,2-Xylp

3.8%

4.4%

t-Manp

0.2%

0.6%

1,4-Manp

2.1%

2.8%

Ara = arabinose, Gal = galactose, Rha = rhamnose, Glc = glucose, Xyl = xylose, Man = mannose, f = furanose, p = pyranose.


Page 30 of 34

Page 31 of 34


FIGURES Figure 1:



Page 32 of 34

Figure 2: Main products G-2a

OH OH

G-2b

OH O

O O OH

HO

HO

O

T

O OH

HO

T

OH

OH

G-2c

OH OH

O OH

O

O OH

HO

HO

R

O OH

HO

R

OH

OH

OH OH

Side products

G-3a

OH OH

G-3b

OH

O

O O OH

HO

O OH

HO

OH

OH O

O O OH

HO

O OH

HO

OH

OH O

O OH

HO OH

G-3c

OH

HO OH

G-4

OH OH O

OH OH

HO

O OH

O HO

O

T

O

O OH

HO

OH

OH O

O HO

A

O OH

O OH

HO

OH

OH O

HO R

O OH

HO

OH

OH OH


Page 33 of 34


Figure 3:



TABLE OF CONTENTS GRAPHICS For Table of Contents Only


Page 34 of 34

Arabinan and Galactan Oligosaccharide Profiling by High

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