Determination of the xylan backbone distribution of arabinoxylan-oligosaccharides

Bioactive Carbohydrates and Dietary Fibre 2 (2013) 84 –91

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Determination of the xylan backbone distribution of arabinoxylan-oligosaccharides Jeroen Sneldersa,b, Emmie Dorneza,b, Willem F. Broekaertc, Jan A. Delcoura,b, Christophe M. Courtina,b,n a

Laboratory of Food Chemistry and Biochemistry, KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium c Fugeia NV, Science Park Arenberg, Gaston Geenslaan 1, B-3001 Leuven, Belgium b

ar t ic l e in f o

abs tra ct

Article history:

Arabinoxylan-oligosaccharide samples (AXOS) present themselves as mixtures of different

Received 10 June 2013

molecular entities with xylan backbones of different length and with different levels of

Received in revised form

arabinose substitution. Their prebiotic properties depend on their degree of polymerisation

28 August 2013

(DP) and degree of arabinose substitution (DAS). Therefore, structural characterisation of

Accepted 28 August 2013

AXOS samples is important. Gas chromatography (GC) is most frequently used for quantification of AXOS levels and for determination of the average DP (avDP) and average

Keywords:

DAS (avDAS), yet it does not provide information on the molecular mass distribution of the

AXOS

xylan backbones of the different AXOS entities present in the mixture. This manuscript

Structural characterisation

evaluates a method based on high performance anion exchange chromatography (HPAEC)

HPAEC

involving quantification of xylo-oligosaccharides (XOS) after either acidic or enzymic

GC

removal of arabinose substituents for its ability to determine such distribution. Results

Prebiotic

show that despite the fact that a small fraction of the arabinoses could not be removed, representative DP distributions of xylan backbones in complex AXOS samples were obtained. The similarity of the avDP determined with GC or determined with the new HPAEC method using enzymic removal of arabinose substituents confirmed this. It can be concluded that the HPAEC method involving enzymic removal of arabinoses provides useful insight in the DP distribution of the xylan backbones in complex AXOS samples. & 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

The demand for functional foods with health benefits is increasing. Arabinoxylan-oligosaccharides (AXOS), hydrolytic degradation products of arabinoxylan (AX), are a good example of a functional food constituent. They not only possess prebiotic properties (Broekaert et al., 2011; Cloetens et al., 2010; Grootaert et al., 2009; Van Craeyveld et al., 2008), but are

also potential antioxidants, due to the presence of ferulic acid (FA), the major phenolic acid in wheat, as AXOS substituent (Smith & Hartley, 1983). In the EU (European Commission directive 2008/100/EC) and a lot of countries worldwide, some oligosaccharides (DP 3–9), including AXOS, classify as dietary fibre (de Menezes, Giuntini, Dan, Sardá, & Lajolo, 2013). The positive impact of dietary fibre on human health is already known for decades and the study of its effects is of great

n Corresponding author at: Laboratory of Food Chemistry and Biochemistry, KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium. Tel.: þ32 16 32 19 17; fax: þ32 16 32 19 97. E-mail address: [email protected] (C.M. Courtin).

2212-6198/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcdf.2013.08.005

Bioactive Carbohydrates and Dietary Fibre 2 (2013) 84 –91

importance (Champ, Langkilde, Brouns, Kettlitz, & Collet, 2003). AXOS consist of a linear backbone of β-1,4 linked Dxylopyranosyl residues which can be un-, mono- or disubstituted with α-L-arabinofuranosyl units on their C-(O)-2 and/ or C-(O)-3 position (Cleemput, Roels, Van Oort, Grobet, & Delcour, 1993). Some arabinose residues are esterified with FA (Izydorczyk & Biliaderis, 1995). Other minor constituents on the xylan backbone include glucuronic acid, D-glucose, Dgalactose or small oligomeric side-chains consisting of two or more arabinofuranosyl residues (Izydorczyk & Biliaderis, 1995; Schooneveld-Bergmans, Beldman, & Voragen, 1999). While individual AXOS molecules are structurally characterised by a degree of polymerisation (DP) and degree of arabinose substitution (DAS), AXOS products, comprising a mixture of different molecules, are typically characterised by an average DP (avDP) and an average DAS (avDAS). The prebiotic properties of AXOS products depend on their structural characteristics. Especially, the (av)DP has a substantial influence on the prebiotic properties of AXOS (Pollet et al., 2012; Van Craeyveld et al., 2008). AXOS with the same avDP but different avDAS have either been reported to be equally fermentable (Kabel, Kortenoeven, Schols, & Voragen, 2002; Van Craeyveld et al., 2008) or to be more fermentable when less arabinose substituents are present (Damen et al., 2011; Pollet et al., 2012). So, next to chain length, the level of the arabinose substituents presents a second structural characteristic that can be used to fine-tune AXOS functionality. The main techniques for quantitative carbohydrate analysis are gas chromatography (GC) (Englyst & Cummings, 1984), highperformance liquid chromatography (HPLC) [including normal phase HPLC, high-performance size-exclusion chromatography, and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC–PAD)] (Lebet, Arrigoni, & Amadò, 1997; Pollet, Beliën, Fierens, Delcour, & Courtin, 2009; Swennen, Courtin, Van der Bruggen, Vandecasteele, & Delcour, 2005), colorimetric methods (Douglas, 1981) and to a lesser extent proton nuclear magnetic resonance (1H-NMR) (Kiemle, Stipanovic, & Mayo, 2004). Most of the methods quantifying oligo- and polysaccharides are based on analysing the monosaccharide composition after hydrolysis. Acid hydrolysis is typically used, although this can lead to further degradation of released monosaccharides. Enzymic hydrolysis, in contrast, converts oligo- or polysaccharides to monomeric sugars without further degradation (Virkki, Maina, Johansson, & Tenkanen, 2008). A major disadvantage of these methods is that, due to the hydrolysis step, information on the structure (DP and DAS) is lost. A GC method developed by Courtin, Van den Broeck, and Delcour (2000), which determines both total, reducing end and free sugar content through shuffling of the hydrolysis, reduction and acetylating steps, allows to determine the avDP and avDAS of oligo- and polysaccharides, and hence also of AXOS. Yet this method does not provide information on the DP distribution of individual entities in complex oligo- and polysaccharide mixtures. For analysis of both monomeric (Lv et al., 2009) and oligomeric (Berthod, Chang, Kullman, & Armstrong, 1998) carbohydrates, some HPLC techniques have been developed. Of these, HPAEC–PAD is most widely used, because of its excellent resolution and the highly sensitive detection based

85

on pulsed amperometry. HPAEC quantification methods for fructo-oligosaccharides (Borromei et al., 2010; Verspreet et al., 2012), cello-oligosaccharides (Konno, Sakamoto, & Kamaya, 1996; Yu & Wu, 2009) and xylo-oligosaccharides (XOS) (Pollet et al., 2009) exist. Such methods not only allow quantifying oligosaccharides, but also allow evaluation of the DP distribution of oligosaccharides. To our knowledge, a procedure for the quantification of AXOS with HPAEC up to a DP of 9 has not yet been described in literature. HPAEC for AXOS quantification is faced with two major difficulties. Firstly, the elution pattern of AXOS is highly complex due to the impact of both xylan backbone length and arabinose substituents on the retention time and detector response. When dealing with a complex mixture of AXOS, structurally different AXOS often co-elute (Pastell, Tuomainen, Virkki, & Tenkanen, 2008). Secondly, AXOS standards and high DP XOS standards are not commercially available, thus hampering quantitative analysis. Nevertheless, HPAEC analyses in combination with enzymic treatment of AX with endoxylanase and arabinofuranosidases with different substrate specificity have been used to identify AXOS, whereas the structures were confirmed using other techniques (such as mass spectrometry and 1H-NMR) (Pastell et al., 2008; Pitkänen, Tuomainen, Virkki, Aseyev, & Tenkanen, 2008; Rantanen et al., 2007; Rivière et al., 2013). In view of the impact of AXOS structure on its functional properties and the need for proper characterisation of materials for which. health claims are proposed, structural characterisation of AXOS is required. In this manuscript, we set out to obtain quantitative information on the xylan backbone distribution of AXOS through HPAEC, to complement the information that can be obtained by GC analysis (Courtin et al., 2000). To this end, two variants for selectively stripping the xylan backbone of the AXOS prior to HPAEC analysis were tested. A first variant consists of trimming off the arabinose substituents through mild acidic hydrolysis. In the second variant, enzymes were used to selectively strip the xylan backbone. GC analysis was used for monitoring the degree of arabinose (and xylose) hydrolysis during the mild acidic or enzymic treatment.

2.

Materials and methods

2.1.

Materials

All chemicals, solvents, and reagents were purchased from Sigma-Aldrich (Bornem, Belgium) and were of at least analytical grade unless specified otherwise. Xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6) and FA esterase from Clostridium thermocellum (E-FAEZCT) were purchased from Megazyme (Bray, Ireland). Grindamyl H640, containing a Bacillus subtilis endoxylanase, was from Danisco (Copenhagen, Denmark). Birchwood xylan was from Sigma-Aldrich. XOS, produced by enzymic hydrolysis with endoxylanases after mild pre-treatment of corncobs, were obtained from Shandong Longlive Bio-technology (Qingdao, China). AXOS-1 and -2 were produced by enzymic treatment of wheat bran AX using the procedure described by Swennen, Courtin, Lindemans, and Delcour (2006) with adaptations and

86

Bioactive Carbohydrates and Dietary Fibre 2 (2013) 84 –91

was obtained from Fugeia (Leuven, Belgium). AXOS-3 were produced starting from AXOS-1 using an Amberlite XAD-4 resin column and elution with increasing ethanol concentration (Saulnier et al., 1999). The fractions eluting at an ethanol concentration exceeding 50% were pooled. They had a higher avDP and were more enriched in FA. Following rotary evaporation of ethanol and freeze-drying, they were ready for further use.

2.2. Preparation of purified xyloheptaose (X7), xylooctaose (X8), xylononaose (X9) Pure xyloheptaose (X7), xylooctaose (X8), xylononaose (X9) were prepared by xylanolytic hydrolysis of birchwood xylan with Grindamyl H640, followed by gel filtration chromatography of the hydrolysis products using Bio-Gel P4 and Bio-Gel P2 columns from Bio-Rad (Hercules, CA, USA) as described by Pollet et al. (2010).

2.3. Recombinant expression and purification of arabinofuranosidases Recombinant expression in Escherichia coli of Bifidobacterium adolescentis AXH-d3 (UniProtKB: Q5JB56), AbfA (UniProtKB: A1A0H1) and AbfB (UniProtKB: A1A3M2), coding sequences and purification of the obtained arabinofuranosidases was executed as described by Lagaert et al. (2011). Enzyme purity was estimated with the 2100 Bioanalyzer system using the Protein 80 Kit (Agilent Technologies, Diegem, Belgium). Combined enzyme fractions were dialysed against sodium citrate buffer (25 mM, pH 6.0). Protein concentrations were determined on the basis of the extinction at 280 nm measured with a NanoDrop-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and using molar extinction coefficients based on the amino acid sequence (96,720, 115,850 and 158,140 M  1 cm  1 for AXH-d3, AbfA and AbfB, respectively).

2.4. Mild acidic and enzymic hydrolysis of AXOS to trim off arabinose prior to analysis 2.4.1.

Mild acidic hydrolysis

Approximately 150 mg of XOS or AXOS sample was accurately weighed and dissolved in 140 mL Milli-Q water. The pH of the solution was adjusted to pH 7.0 by adding either 0.10 M NaOH or 0.10 M HCl before the addition of Milli-Q water to make a total volume of 150 mL. 30 mL of the sample solution was transferred to three glass test tubes with screw cap to execute hydrolysis in triplicate. Several hydrolysis conditions were tested by adapting either pH (2.6–3.0) of the samples, using 1.0 M HCl, or temperature (80–100 1C). After heating the samples for 24 h in an oven, they were cooled to room temperature and neutralised by adding of 1.0 M NaOH. Rhamnose was added as internal standard (IS) before further analysis. As XOS needed to be diluted more than AXOS before analysis due to its more homogeneous nature, more rhamnose was added to XOS (32 mg per 30 mg sample) than to AXOS (4 mg per 30 mg sample), so that the peak of the IS was always in the

same range as the peaks in the sample. All further treatments were performed at pH 2.8 and 90 1C, as these proved optimal for hydrolysis of the bonds between the xylan backbone and arabinose substituents based on the current results, which were in agreement with Swennen et al. (2006).

2.4.2.

Enzymic hydrolysis

For the enzymic release of arabinose from AXOS, three different arabinofuranosidases were used in combination with a FA esterase, as this enzyme works synergistically with the arabinofuranosidases (Panagiotou, Olavarria, & Olsson, 2007; Yu, McKinnon, & Christensen, 2005). AbfA removes arabinose from the positions C-(O)-2 and C-(O)-3 of monosubstituted xylose residues, whereas AbfB and AXH-d3 cleave arabinose on the C(O)-3 position of disubstituted xyloses (Lagaert et al., 2010). FA esterase was used for removal of FA of the C-(O)-5 position of arabinose. XOS or AXOS samples (75 mg) were dissolved in 75 mL sodium citrate buffer (25 mM, pH 6.0). Enzymic hydrolysis was executed in triplicate on 15.0 mL aliquots from this solution. The samples were incubated for 24 h at 35 1C with 200 mg of purified AXH-d3, AbfA and AbfB together with 10 mL of FA esterase. The incubation conditions were based on pH and temperature optima of the arabinofuranosidases (Lagaert et al., 2010). After enzyme inactivation for 15 min at 100 1C, IS was added (2 and 16 mg rhamnose for 15 mg samples of AXOS and XOS, respectively) and the samples were ready for further analysis. The hydrolysis conditions were evaluated using sample AXOS-1. Adding more enzyme or extending the hydrolysis time did not increase the arabinose yield.

2.5. Analysis of total, reducing end and monomeric carbohydrate content and composition using GC Total, reducing end and monomeric carbohydrate contents and compositions, and derived parameters, of native AXOS and AXOS treated as in Section 2.4 were determined by GC as in Courtin et al. (2000). For determination of total carbohydrate content, samples were hydrolysed in 2.0 M trifluoroacetic acid (TFA) at 110 1C for 60 min, followed by reduction of the released monosaccharides by NaBH4 and acetylation with acetic acid anhydride to form alditol acetates. Alditol acetates (1.0 mL) were analysed on a Supelco SP-2380 column (30 m  0.32 mm i.d., 0.2 mm film thickness; Supelco, Bellefonte, PA, USA) with helium as the carrier gas in a Agilent 6890 series chromatograph (Agilent, Wilmington, DE, USA) equipped with an autosampler, splitter injection port (split ratio 1:5), and flame ionisation detector. Separation was at 225 1C with injection and detection temperatures at 270 1C. βD-Allose was used as IS and calibration samples, containing known concentrations of the expected monosaccharides, were included with each set of the samples. For determination of the reducing end carbohydrate content and composition, the hydrolysis step was executed after reduction whereas for determination of the free monosaccharide content no hydrolysis was performed. The AXOS level (%), avDP of the xylan backbone (avDPxyl), avDAS, oligomeric glucose level (%) and avDP of the glucose

Bioactive Carbohydrates and Dietary Fibre 2 (2013) 84 –91

oligomers (avDPglu) were calculated according to formulas (1)– (5), respectively. AXOS level ð%Þ ¼ ð%total  %free AÞ 

132 150

þð%total X %free XÞ 

avDPxyl ¼

avDAS ¼

ððavDPxyl 1Þ  132 þ 150Þ 150  avDPxyl

%total X%free X %reducing end X %free X

%total A %free A %total X %free X

ð1Þ

ð2Þ

ð3Þ

Oligomeric glucose level ð%Þ ¼ ð%total G %free GÞ 

ððavDPglu 1Þ  162 þ 180Þ 180  avDPglu

avDPglu ¼

%total G %free G %reducing end G  %free G

ð4Þ

ð5Þ

In the above formulas, arabinose is abbreviated as A, xylose as X and glucose as G. For total and reducing end xylose, arabinose, and glucose, percentages are weight percentages after hydrolysis. The factors 132 and 150 in formula (1) correspond to the molecular masses of anhydroarabinose (or anhydroxylose) and arabinose (or xylose), respectively. Likewise, the factors 162 and 180 in formula (4) correspond to the molecular masses of anhydroglucose and glucose, respectively.

2.6. Analysis of XOS and AXOS carbohydrate composition by HPAEC–PAD The samples prepared in Section 2.4, both treated as well as untreated, were diluted appropriately and filtered (0.22 mm) prior to HPAEC–PAD analysis on a CarboPac PA-100 anion exchange column (250 mm  4 mm) using a Dionex ICS3000 chromatography system (Sunnyvale, CA, USA). The injection volume was 25 mL and elution (1.0 mL min  1) was with 100 mM sodium hydroxide for 5 min followed by a linear gradient of 0–125 mM sodium acetate in 100 mM sodium hydroxide for 30 min. Standard solutions containing arabinose, xylose and XOS from DP 2 up to DP 9 and IS were used to identify and quantify the AXOS samples upon mild acidic or enzymic hydrolysis (Pollet et al., 2009). HPAEC analytical data allow determining the avDPxyl using formula (6) in which concentrations are expressed on molar base. The abbreviations for arabinose and xylose are those used in Section 2.5. In addition, Xn stands for XOS with n xylose residues. Total X and free X stand for the amount of monomeric xylose present in the sample with and without acidic or enzymic hydrolysis, respectively. Total Xn corresponds to the amount of Xn present in the sample after acidic or enzymic hydrolysis. avDPxyl ¼

%total X %free X þ ∑9n ¼ 2 %total Xn  n %total X%free X þ ∑9n ¼ 2 %total Xn

ð6Þ

87

2.7. Analysis of the ferulic acid content by use of reversed phase high performance liquid chromatography (RP-HPLC) Phenolic acids were saponified according to Antoine et al. (2003) with some modifications. NaOH (2.5 mL, 4.0 M) was added to 2.5 mL of the prepared XOS and AXOS samples (1.0 mg/mL). Samples were saponified under slow stirring for 120 min at 35 1C under nitrogen atmosphere and protected from light. Samples were acidified to pH 2.0 with HCl (4.0 M) followed by addition of caffeic acid as IS. Phenolic acids were extracted twice with 2.0 mL diethyl ether. Ether fractions were collected and evaporated under a nitrogen flow. Phenolic acids were dissolved in MeOH and filtered (0.45 mm). Monomeric phenolic acids were analysed on a Luna Phenyl-Hexyl column (250 mm  4.6 mm i.d., 5 μm particle size, plus 3 mm  4.6 mm i.d. guard column; Phenomenex, Utrecht, The Netherlands) in a LC-20AT modular HPLC system (Shimadzu, Kyoto, Japan) using a ternary gradient system of 1.0 mM aqueous TFA, acetonitrile/1.0 mM aqueous TFA [90/ 10 (v/v)] and methanol/1.0 mM aqueous TFA [90/10 (v/v)] according to Dobberstein and& Bunzel (2010). The injection volume was 20 mL and the separation was performed at 45 1C. UV detection (Shimadzu, UV-10A detector) was carried out at 280 nm.

3.

Results and discussion

3.1.

Production of XOS standards

Most often XOS quantification with HPAEC is limited to XOS with a DP up to 6, due to a lack of commercially available XOS standards with higher DP. Because AXOS with DP from 3 to 9 classify as dietary fibre (at least) in Europe (de Menezes et al., 2013), quantification of XOS with a DP higher than 6 is desirable. XOS samples with DP 7 to 9 were produced by hydrolysis of birchwood xylan with an endoxylanase followed by purification using gel filtration chromatography. Purity as assessed by HPAEC was very high (495%). Fig. 1 shows that rhamnose, arabinose, xylose and XOS from DP 2 up to DP 9 standards are nicely separated in the HPAEC profile.

3.2. Evaluation of the efficiency of mild acidic and enzymic treatment to remove arabinose from AXOS Since the presence of arabinose on the xylan backbone of AXOS typically confers a blurred HPAEC–PAD profile, two

Fig. 1 – HPAEC profile of rhamnose (Rh), arabinose (A), xylose (X) and XOS (Xn) standards.

88

Bioactive Carbohydrates and Dietary Fibre 2 (2013) 84 –91

different methods were assessed to trim off these arabinose residues. Hereby, a mixture of unsubstituted XOS can be obtained that yields unambiguous well-resolved HPAEC– PAD profiles. Both acidic and enzymic treatments were evaluated for their capacity to remove arabinose and other minor substituents from the xylan backbone of AXOS mixtures, with minimal internal xylose backbone degradation. This was done on sample AXOS-1. Efficiency of the treatments was assessed using GC analysis of reducing end sugars as formation of reducing end sugars is a measure for the degree of hydrolysis of oligosaccharides. Therefore, the degree of hydrolysis is expressed based on the formation of reducing end xylose or arabinose relative to the initial non-reducing end xylose and arabinose concentration, respectively. For the acidic hydrolysis, several combinations of pH (2.6–3.0) and temperature (80–100 1C) were tested to determine optimal conditions for arabinose release, while at the same time avoiding hydrolysis of the xylan backbone. Table 1 lists the results. Hydrolysis at pH 2.8 and 90 1C for 24 h provided the best results, in agreement with Swennen et al. (2006). Up to 96% of the initial non-reducing end arabinoses were released under this hydrolysis condition, while only 15% of the initial non-reducing end xyloses were hydrolysed. Although increasing the pH from 2.8 to 2.9 and 3.0 led to less xylose backbone hydrolysis, arabinose hydrolysis was far more incomplete at these conditions, which would leave more AXOS entities unidentified and unresolved upon HPAEC–PAD analysis. Lowering the pH to 2.7 and 2.6 gave similar results as pH 2.8, but less harsh conditions are preferred. Temperature also had a big impact on hydrolytic release of arabinose and xylose. Increasing the hydrolysis temperature to 100 1C (pH 2.8, 24 h) led to hydrolysis of more than 40% of the initial non-reducing end xyloses. At 80 1C

(pH 2.8, 24 h) approximately 60% of the initial non-reducing end arabinose residues only were released. Between 81% and 96% of the initial non-reducing end arabinose of AXOS samples were hydrolysed under optimal acidic hydrolysis conditions (90 1C, pH 2.8, 24 h), indicating that arabinose removal occurred to near completion (Table 2). However, 7% to 15% of xylose of AXOS and XOS were also removed under this condition. For the enzymic hydrolysis, three arabinofuranosidases with complementary action were used in combination with a FA esterase. Conditions (pH, temperature, time and amount of enzyme) were first tested for maximal hydrolysis (data not shown). The arabinofuranosidases, in combination with FA esterase, were able to release 79% to 89% of the bound arabinose. A big advantage of the enzymic treatment was that it did not impact the xylan backbone (Table 2). Therefore, enzymic hydrolysis was preferred over acidic treatment, for our purpose.

3.3. Structural characterisation of AXOS based on GC, RP-HPLC and HPAEC analyses For quantification and structural characterisation of the AXOS carbohydrate structures, mass percentages, avDAS, avDPxyl and FA contents are important characteristics as they can influence functional properties. Carbohydrate characteristics were determined with GC (Table 3A) and HPAEC (Table 3B). FA content was quantified by RP-HPLC (Table 3A). The AXOS levels determined by GC ranged between 73% and 77% for the AXOS samples and amounted to 80% for the XOS sample. XOS had a low avDPxyl (3.1) and avDAS (0.04), whereas the avDPxyl of the AXOS samples ranged between 3.9 and 5.5 and the avDAS between 0.22 and 0.37. Due to their low avDAS, XOS contained only 0.07% FA. AXOS-1 and

Table 1 – Hydrolysed arabinose and xylose (%), determined with GC analysis, during mild acidic treatment for 24 h of AXOS-1. pH T (1C)

2.6 90

2.7 90

2.8 90

2.9 90

3.0 90

2.8 80

2.8 100

Reducing end arabinose formed (% of initial non-reducing end arabinose) Reducing end xylose formed (% of initial non-reducing end xylose)

90 (71)

92 (74)

96 (73)

79 (72)

76 (74)

57 (79)

97 (77)

14 (72)

12 (72)

15 (72)

7 (72)

10 (77)

6 (78)

42 (73)

Table 2 – Reducing end arabinose and xylose formation during mild acidic treatment and enzymic treatment as determined using the GC analysis method. Sample

XOS

Hydrolysis treatment

Acid

Enzyme

Acid

Enzyme

Acid

Enzyme

Acid

Enzym

Reducing end arabinose formed (% of the initial non-reducing end arabinose) Reducing end xylose formed (% of the initial non-reducing end xylose)

n.d.

n.d.

96 (73)

84 (77)

89 (75)

79 (75)

81 (75)

89 (74)

12 (72)

0

15 (72)

0

10 (73)

0

n.d.: not determined.

AXOS-1

AXOS-2

AXOS-3

7 (74)

0

89

Bioactive Carbohydrates and Dietary Fibre 2 (2013) 84 –91

Table 3 – Structural characteristics of a set of XOS and AXOS samples according to different methods [GC and RP-HPLC (A) and HPAEC with acidic or enzymic hydrolysis (B)]. Xnh stands for the level of XOS with n xylose residues after hydrolysis. Levels are expressed on dry matter base (dm). Analytical method

Characteristic

XOS

AXOS-1

AXOS-2

AXOS-3

GC

AXOS level (% dm) avDPxyl avDAS Oligomeric glucose (% dm)

80 (71) 3.1 (70.1) 0.04 (70.01) 16 (71)

73 3.9 0.22 12

77 5.0 0.24 8

74 5.5 0.37 12

RP-HPLC

Total FA (% dm)

0.07 (70.01)

1.68 (70.06)

HPAEC with enzymic pre-treatment

avDPxyl X2h (% dm) X3h (% dm) X4h (% dm) X5h (% dm) X6h (% dm) X7h (% dm) X8h (% dm) X9h (% dm)

2.8 (70.1) 26.0 (72.0) 27.1 (72.1) 12.7 (70.9) 4.1 (70.3) 1.5 (70.1) 1.4 (70.1) o0.1 o0.1

3.4 16.7 9.6 8.6 6.3 4.6 4.6 3.5 3.3

A (71) (70.2) (70.01) (71)

(72) (70.6) (70.01) (71)

1.59 (70.04)

(72) (70.3) (70.03) (71)

7.72 (70.24)

AXOS-2 had a similar FA content (1.7% and 1.6%, respectively), while AXOS-3 had the highest FA content (7.7%). By use of GC, other sugars such as galactose, mannose and glucose were also determined. Glucose was also present in relatively high amounts in the XOS and AXOS samples (from 8% to 16%) and mainly in an oligomeric form. The avDPxyl determined with HPAEC analysis after enzymic hydrolysis ranged from 2.8 to 5.3 for the tested samples. These values were comparable with the avDPxyl determined with GC and only gave small underestimations, ranging from 0.2 to 0.5, which are in the order of magnitude of the standard deviation on the GC measurements. Therefore, it can reasonably be assumed that, although some arabinose residues had not been hydrolytically removed, the relative DP distribution of the xylose backbone based on molar percentages of total XOS (up to DP 9) after hydrolysis approximates the actual DP distribution of the xylan backbone of the different AXOS entities in the sample. Furthermore, expressing the DP distribution in relative concentrations (molar base) as done in Fig. 2 facilitates the comparison of different samples. Table 3B summarises the concentrations of the XOS of different DP after enzymic hydrolysis. XOS and AXOS-1 had a rather similar avDPxyl determined with HPAEC after enzymic hydrolysis (2.8 and 3.4, respectively). The xylan backbones of both samples consist of almost 50% xylobiose. Xylotriose backbone entities were also very abundant in the XOS sample (34% relative to total XOS after enzymic hydrolysis) but less so in AXOS-1 (19%). Xylotetraose backbone entities were (based on relative concentrations) present in similar levels in the XOS and AXOS-1 sample, while XOS and AXOS with a DP of the xylan backbone ranging from 5 to 9 were more abundant in AXOS-1. The enzymically hydrolysed XOS sample did only contain entities with a xylan backbone up to DP 7. Although AXOS-2 and AXOS-3 had comparable avDPxyl (4.5 and 5.3, respectively, determined with HPAEC after enzymic hydrolysis), differences in the DP distribution of

Relative concentration (mole%)

B (70.1) (71.3) (70.8) (70.8) (70.6) (70.5) (70.5) (70.4) (70.3)

4.5 5.8 8.0 7.6 7.0 6.6 7.9 6.5 6.4

(70.1) (70.2) (70.4) (70.4) (70.4) (70.5) (70.6) (70.5) (70.6)

5.3 1.5 1.4 5.6 6.4 5.6 6.5 4.8 4.9

(70.1) (70.2) (70.1) (70.6) (70.6) (70.6) (70.6) (70.4) (70.5)

50 40 30 20 10 0 XOS AXOS-1 AXOS-2 AXOS-3 DP distribution of xylan backbone

Fig. 2 – Relative DP distribution of the xylan backbone (molar percentage of total XOS composition after hydrolysis) of XOS and AXOS after enzymic hydrolysis of XOS, AXOS1, AXOS2, and AXOS3, respectively. The colour of the bars representing XOS with DP 2 to 9 are depicted in decreasing intensity, with the highest intensity corresponding to X2 and the lowest to X9.

the xylan backbone were clear. Xylobiose and xylotriose backbone entities were predominant in AXOS-2 (about 22% and 21% of total, respectively), while they were clearly less abundant in AXOS-3 (11% and 7%, respectively). AXOS with a DP of the xylan backbone ranging from DP 4 to 9 were present at higher relative concentrations in AXOS-3 than in AXOS-2. Besides the avDPxyl, the DP distribution of the xylan backbone helped distinguishing different XOS and AXOS samples and contributed to a more profound characterisation of their composition.

4.

Conclusion

AXOS-containing samples typically consist of a mixture of very diverse structurally related molecules. As functional

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properties are related to structural characteristics, thorough structural characterisation of AXOS is important. As AXOS are so diverse, it is impossible to quantify every single molecule of complex AXOS mixtures. GC analysis only allows determining structural characteristics on an average basis for the population of different molecular entities. Here, an HPAEC method was tested to characterise the DP distribution of xylan backbone of AXOS, based on quantification of XOS after the removal of arabinose substituents. Enzymic release of arabinose proved specific, removing 80% to 90% of the arabinoses, while leaving the xylan backbones uncleaved. HPAEC analysis of AXOS pretreated by enzymic hydrolysis provides insight in the DP distribution of the xylan backbones of the different AXOS entities. The fact that avDPxyl values of the new HPAEC method corresponded well with those of the standard GC method, provides validation of the new method (within the tested range). Furthermore, the HPAEC method requires relatively little manual handling. HPAEC analysis therefore is a useful and complementary addition to the standard GC and RP-HPLC methods that allow accurate determination of the AXOS level, avDP, avDAS and FA content of XOS and AXOS.

Acknowledgements Financial support from the ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO, Brussels, Belgium) for the postdoctoral fellowship of E. Dornez and from the European Commission in the Communities 7th Framework Programme (FP7/2007– 2013) for the Biocore Project (Grant agreement no. FP7-241566) is gratefully appreciated. This publication reflects only the author's views and the Community is not liable for any use that may be made of the information contained in this publication. Mira Beke is gratefully thanked for the technical assistance. This research is also part of the Methusalem Programme Food for the Future (2007–2014). Jan A. Delcour is W.K. Kellogg Chair of Cereal Science and Nutrition at KU Leuven.

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