Characterization of (Glucurono) arabinoxylans from Oats Using

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Characterization of (glucurono)arabinoxylans from oats using enzymatic fingerprinting Lingmin Tian, H. Gruppen, and Henk A. Schols J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04419 • Publication Date (Web): 04 Dec 2015 Downloaded from http://pubs.acs.org on December 5, 2015

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Journal of Agricultural and Food Chemistry

Characterization of (glucurono)arabinoxylans from oats using enzymatic fingerprinting

Lingmin Tian, Harry Gruppen, Henk A. Schols*

Laboratory of Food Chemistry, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands

* Corresponding author Tel: +31 317482239. E-mail: [email protected].

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Abstract

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Cell wall material from whole oat grains was sequentially extracted to study the

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structural characteristics of individual arabinoxylan populations. Araf was singly

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substituted at both O-3 (mainly) and O-2 positions of Xylp, and no di-substitution of

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Xylp with Araf residues was found in oat AXs. Both highly substituted and sparsely

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substituted segments were found in AXs in Ba(OH)2 extracts, while AXs in 1 M and 6

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M NaOH extracts were rarely branched and easily aggregated. Both O-2 linked GlcA

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and 4-O-MeGlcA residues were present in oat AXs. A series of AX oligomers with

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galactose as a substituent was detected for the first time in oats. The present study

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suggested that the distribution of Araf was contiguous in oat AXs, different from the

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homogeneous distribution of Araf in wheat and barley AXs, which might result in

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different fermentation patterns in human and animals.

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Keywords: oats, Avena sativa, cell wall polysaccharides, arabinoxylan, enzymatic

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fingerprinting, distribution

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1. Introduction

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In recent years, human and animal experiments have clearly shown that consumption

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of cereals rich in non-starch polysaccharides (NSP) is associated with reduced risk of

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obesity, type 2 diabetes, cardiovascular diseases, and some forms of cancer.1,2 The

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health benefits are partly ascribed to fermentation of NSP by the microbiota in the

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gastro-intestinal tract. Microbial degradation of NSP, however, is subject to restrictions

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due to their complexity and lack of accessibility by bacterial enzymes.3 In addition, NSP

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limits accessibility of starch. Due to the limited degradation of the complex structure of

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NSP, the starch can be poorly accessed by enzymes in the gastrointestinal tract.4

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Whole grain oat (Avena sativa), widely used as human food and animal feed, contains

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up to 23% (w/w) of NSP,1,5 mainly composed of mixed linkage (1-3),(1-4)-β-D-glucans

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(β-glucan) and arabinoxylans (AXs) besides cellulose.5 To understand the mechanism of

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their biological functions and to improve the fermentability, detailed studies on the

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chemical structure, physicochemical properties and enzymatic degradation of NSP are

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needed.6,7

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The mixed-linked glucans are mainly composed of two major building blocks:

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cellotriose (DP3) and cellotetraose (DP4) units linked by β-(1-3) linkages to form the

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polymer.8 The ratio of DP3 to DP4 (DP3/DP4) is used to characterize different types of

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cereal β-glucans.9 Oat β-glucan, constituted of 70% 4-O-linked and 30% 3-O-linked β-

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D-Glup units, has been extensively characterized.8-10 In contrast, relatively little is

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known on the oat AXs.

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Oat AXs are composed of a main chain of β-(1-4)-Xylp units, which can be mono-

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substituted by α-L-Araf residues at the O-2 or O-3 position, and/or di-substituted at O-2

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and O-3.11 Cell walls of oats have been sequentially extracted to study details of AXs

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present.12 AXs with different ratios of arabinose to xylose (A/X) were found in different

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extracts. However, the A/X ratio is not fully indicative for the precise structure of AXs, -3-Environment ACS Paragon Plus


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especially in relation to the distribution of substituents over the main chain.13 Specific

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enzymes could be used to reveal such a distribution of substituents, as previously done

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for barley and wheat.14,15 The distribution of the substituents over the xylan backbone

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may differ for individual AXs populations, but also depends on the source of AXs.16

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Besides α-L-Araf substitution, also the presence of uronic acids attached through O-2

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Xylp have been reported for oats.5,17 However, glucuronic acid (GlcA) and/or its 4-O-

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methylated derivative (4-O-MeGlcA) containing oligosaccharides are scarcely

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identified in oat (glucurono)arabinoxylan enzyme hydrolysates.18

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In the present study, whole-grain oats were used to obtain NSP, which were

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fractionated using sequential extraction. The NSP fractions were characterized by their

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constituent

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Oligosaccharides obtained after enzymatic degradation of oat AXs were identified and

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quantified, in order to understand the substitution patterns of the AXs.

monosaccharide

composition

and

molecular

weight

distribution.

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2. Materials and methods

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2.1. Materials

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Oat (Avena sativa) grains were harvested in Poland (2011) and supplied by Agrifirm

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(Apeldoorn, The Netherlands). Wheat arabinoxylan (medium viscosity), α-L-

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arabinofuranohydrolase-D3

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adolescentis19 and endo-1,3(4)-β-D-glucanase (lichenase, GH 16) from Bacillus subtilis

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were purchased from Megazyme (Bray, Ireland). Endo-(1,4)-β-D-xylanase I (Endo I,

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GH 10) was purified from Aspergillus awamori CMI 142717.20 Endo-(1,4)-β-D-

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xylanase III (Endo III, GH 11) was purified from Aspergillus niger A16 DSM 5082M1.

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α-Glucuronidase was purified from Trichoderma viride, and contained traces of β-

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xylosidase activity.21 Xylose was purchased from Sigma Aldrich (Steinheim, Germany).

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Xylobiose, xylotriose and xylotetraose were purchased from Megazyme (Bray, Ireland).

(AXH-d3,

GH

family

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from

Bifidobacterium

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2.2. Preparation of alcohol insoluble solids and sequential extraction

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The whole oat grains with hulls were milled using a centrifugal mill (ZM 200,

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Retsch, Haan, Germany) to a particle size of BE1>1 MASS>6

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MASS. The complexities of AXs, nevertheless, are not only affected by the degree of

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substitution but also by the distribution of substituents.13

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In addition to arabinose and xylose, both the BE2 and 6 MASS contained substantial

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amount of glucose that could arise from mixed-linked β-glucan, representing 19% and

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10% of the monosaccharides (mol%) in the two fractions. Enzymatic degradation using

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the (1-3),(1-4)-β-D-glucan specific enzyme lichenase showed that (1-4)-linked

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cellooligosaccharides with one (1-3)-linked glucose residue as reducing end were

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released from BE2 (DP3/DP4=2.1), but not from 6 MASS (data not shown). The

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inaccessibility of 6 MASS for lichenase may be explained by the low solubility of oat β-

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glucan in 6 MASS as a result of the presence of short cellulose-like sequences.9

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The protein contents in BE1 (19% w/w) and 1 MASS (19% w/w) were found to be

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much higher than that in BE2 (4% w/w) and 6 MASS (3% w/w). The sum of

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carbohydrate and protein contents in the fractions ranged from only 58% (w/w, 6 MASS)

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to 90% (w/w, BE2) (Table 2). This could be partly explained by the insufficient

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removal of buffer salts by dialysis, and the presence of lignin.34

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3.4. Molecular weight distribution of oat NSP fractions

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The molecular weight (Mw) distribution of the different fractions from oat AIS were

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analyzed by HPSEC (Figure 1). The patterns indicated that different polysaccharide

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populations were present. BE1 contained multiple AXs populations with Mw ranging

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from 6 kDa to 2000 kDa, calculated using pullulan standards. Since Ba(OH)2 was used

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as first extractant of oat AIS, water soluble AXs present in AIS will be solubilized in

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saturated Ba(OH)2 adding up to the multiple populations present in this fraction. Given

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the water extract would be composed of a mixture of AXs and β-glucans, water

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extraction was not included to undermined the use of Ba(OH)2. A large proportion of

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mixed-linked β-glucans insoluble in saturated Ba(OH)2 was recovered in BE2. BE2 and

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1 MASS fractions from oats consisted of AXs with an average Mw of approximately

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100 kDa. AXs with same Mw range were also found in fraction 6 MASS. However, at

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similar carbohydrate contents for the three fractions, 6 MASS showed a much lower RI-

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response than BE2 and 1 MASS, indicating the lower solubility of 6 MASS. The

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relatively poor solubility of AXs in 6 MASS was ascribed to self-aggregation of AXs

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with low degree of substitution (A/X=0.11, Table 2) in buffer and eluent.35

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3.5. Enzymatic fingerprinting of oat NSP fractions

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3.5.1. Changes of molecular weight distribution after endo-xylanase incubation

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The backbone of AXs in all fractions was degraded by Endo I (GH-family 10) and

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Endo III (GH-family 11). The different modes of action of the two enzymes on AXs

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have been described by Kormelink et al.36 Endo I is able to split the glycosidic linkage

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at the non-reducing side of a single or double substituted Xylp residue. Endo III requires

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at least one unsubstituted Xylp residue at the non-reducing side of an Araf substituted

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Xylp residue. To be able to split the glycosidic linkage at the reducing side of a Xylp

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residue substituted with on Araf unit, Endo I needs at least one, and Endo III at least two -11ACS Paragon Plus Environment


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unsubstituted Xylp units towards the reducing end. To display the effects of the

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hydrolysis, Mw distributions of the digests are shown in Figure 1 together with Mw

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distributions of parental fractions as discussed above.

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For BE1 and 1 MASS, at the endpoint approximately 50% (w/w) of the AXs were

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degraded by both Endo I and Endo III as calculated based on the area under the curve

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(retention time, Rt: 7-12 min) in HPSEC patterns (Figures 1A and 1C). The populations

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were mostly degraded into fragments with a Mw between 6 kDa and 200 kDa by Endo I,

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and were degraded less by Endo III. Given the mode of action of the endo-xylanases

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used, at least three contiguous unsubstituted Xylp residues must be present in AXs for

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Endo III to be able to act.36 The observed difference in degradation indicates that the

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AXs backbones in BE1 and 1 MASS contain considerable amounts of unsubstituted

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Xylp residues present within regions with only one or two contiguous units being

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inaccessible for Endo III.36 For BE2 (Figure 1B), the AXs were much less degraded by

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both Endo I and Endo III, resulting in fragments of Mw between 6 kDa and 500 kDa.

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The high proportion (≈60%) of undegradable material in BE2, as shown by HPSEC,

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might be due to the presence of β-glucan and relatively highly substituted xylans

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(A/X=0.61) that are not degradable by the endo-xylanases used. For 6 MASS (Figure

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1D), the polysaccharides present were mostly degradable by both Endo I and Endo III,

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indicating the presence of contiguous unsubstituted Xylp units (n≥3) that are enzyme

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accessible.

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3.5.2. Oligosaccharide populations released from fractions treated with endo-xylanases

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Oligosaccharides released from oat AXs by endo-xylanases were monitored using HPAEC and MALDI-TOF MS. HPAEC elution patterns are presented in Figure 2.

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The digestion of oat AXs with Endo I mainly generated xylose (X), xylobiose (X2),

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xylotriose (X3) and a series of AX oligosaccharides (A2XX, XA3X, A3X, XA3A3X and

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A3A3X, Figure 2A) identified using known reference compounds.36 The AX -12ACS Paragon Plus Environment

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oligosaccharides were annotated using a series of single letters, superscripted number

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and superscripted lowercase letter to describe an oligosaccharide, linkage position and

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non-glycosidic group, respectively.37 Since Endo III is hindered more by Araf than

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Endo I, the larger oligomers released by Endo III provided additional information on the

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intact structure oat AXs. The generated AX oligosaccharides include A2XX, XA3XX,

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XXA3XX, XA3A3XX, XXA3A3XX, and XXA3XA3XX, in addition to unsubstituted

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xylan oligomers (Figure 2B). The total AXs hydrolyzing capacity by Endo I and Endo

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III after 24 h for oat fractions is displayed in Table 3, and is defined as the sum of

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arabinose and xylose present as monomer and oligomers relative to that present in the

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parental AXs. It shows that BE1 was most degraded (53%) by Endo I, followed by 1

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MASS (51%), BE2 (35%) and 6 MASS (23%). Compared to Endo I, Endo III was less

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efficient towards fractions BE1 (49%) and BE2 (28%), but slightly more efficient

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towards fractions 1 MASS and 6 MASS mainly ascribed to the release of unsubstituted

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xylan oligomers (Table 3). This might be explained by the degradation of unsubstituted

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polymer segments in the insoluble parts of 1 MASS and 6 MASS by Endo III as has

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been revealed for GH 11 xylanases from Myceliophthora thermophila C1.38

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3.5.3. Substitution patterns of AXs

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Structural information of the parental polymer can be obtained from the structures

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and relative amounts of those released fragments. The proportion of released xylose

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present in the monomer and oligomers from BE1, BE2, 1 MASS and 6 MASS after

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Endo I and III digestion are displayed in Figure 3. The proportion of total xylose

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released present in X, X2 and X3 increased (black rectangle, Figure 3) with the decrease

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of A/X ratio in the digests of both enzymes (Table 3). The similar mole percentages of

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released monomer and oligomers by Endo I for BE1 and BE2 revealed that the relative

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abundance of substituted units in the degradable AXs were similar (Figure 3A). For

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Endo III, the mole percentage of released monomer and oligomers for BE1 and BE2 -13ACS Paragon Plus Environment


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were different (Figure 3B). This reflects a different distribution of substituents in AXs

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in the BE1 and BE2. The percentage of xylose present as unsubstituted oligosaccharide

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X3 was much higher in BE2 digests (17%) than in BE1 digests (0%) (Figure 3B). Given

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the mode of action of Endo III, unsubstituted regions (n≥6) of xylan backbone are

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absence in BE1, but are frequently present in BE2. The presence of three contiguous

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unsubstituted Xylp residues is less frequent in BE2 than BE1, as indicated by the lower

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percentage of released xylose present in oligosaccharide XA3XX (Figure 3B) for Endo

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III. Following the same reasoning, three contiguous unsubstituted Xylp residues are less

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frequently present in 6 MASS than 1 MASS.

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To compare the substitution pattern of oat AXs with that of other cereal AXs,

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previous results of AXs in wheat flour14 and barley15 were used. The variety of main

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AX oligosaccharides from endo-xylanase digested oat fractions was much lower than

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that from wheat and barley (Figure 3C). The proportion of total released xylose present

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in X, X2 and X3 from wheat BE1-U30 (22%)14 and barley BE-20 (45%)15 after Endo I

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digestion are much lower than that for oat BE1 (60%) (Figure 3C), although the A/X

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ratio for AXs in wheat BE1-U30 (0.46)14 and barley BE-20 (0.43)13 is comparable to

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that for oat BE1 (0.43). The different proportions indicated that the distribution of Araf

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was random in wheat AXs (Figure 4A), but was slightly clustered in barley AXs (Figure

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4B). In oat AXs, Araf substituents were present as clusters on the xylan backbone

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forming contiguously substituted regions (Figure 4C).

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3.5.4. A/X ratio for undegraded residues reflects the solubility of AXs

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The degradable AXs regions of AXs in the oat fractions were lowly substituted,

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which was reflected by the low overall A/X ratio for the oligosaccharides released

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(≤0.15, Table 3). In BE1 and BE2, the actions for both Endo I and Endo III were

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presumably hindered by the high degree of substitution as indicated by the calculated

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high A/X ratio for resistant segments (Rres, Table 3). The Rres values were close to 1. -14ACS Paragon Plus Environment

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Since di-substituted Xylp residues were absent (see discussion below), this indicates

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that the substitutions were contiguous in the xylan backbone of these resistant segments.

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The highly substituted fragments might be oligosaccharides, which were too large to be

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analyzed by HPAEC. These segments are released from the AXs with unsubstituted

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Xylp residues frequently present (Figure 4C). The segments might also be high Mw

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fragments released from AXs with unsubstituted Xylp residues occasionally present in

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the middle of the molecules, but frequently present at the ends of the molecules (Figure

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4D). Because of the high substitution, the (high Mw and low Mw) fragments in the

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digests were presumably well solubilized as shown by the high RI response of resistant

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residue after enzyme incubation (Figures 1A and 1B). In contrast to BE1 and BE2, 1

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MASS and 6 MASS showed much lower Rres for the undegraded residues.

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Consequently, this indicates the presence of rather unbranched but enzyme resistant

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AXs. The resistant AXs might be partly oligomers and polymers with contiguous

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substitution, since soluble fragments were detected after incubation (Figure 1C and 1D).

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Given the low Rres for the undegraded residues, the resistant AXs in 1 MASS and 6

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MASS partly are supposed to be aggregated.35 Consequently, in 1 MASS and 6 MASS,

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the presence of unsubstituted, lowly substituted, and contiguously substituted regions

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might be combined in a single parental AXs molecule (Figure 4E).

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3.6. Diversity of substituents in released oligosaccharides

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3.6.1 Araf mono-substituted Xylp unit

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Araf O-3 mono-substituted Xylp (A3) was the main unit present in the

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oligosaccharides released. Most of the A3 units (>90%) in Endo I degradable AXs were

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separated from each other by at least one unsubstituted Xylp residue (Figure 3).

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Correspondingly, a low abundance (

Characterization of (Glucurono) arabinoxylans from Oats Using

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