Journal of the Science of Food and Agriculture J Sci Food Agric 88:1905–1910 (2008)

A simplified method for extractingwater-extractable arabinoxylansfrom wheat flourNishath K Ganguli1,2 and Matthew A Turner1,2∗1The University of Sydney, Plant Breeding Institute, Cobbitty, PMB 11, Camden, NSW 2570, Australia2Value Added Wheat Cooperative Research Centre Ltd., P.O. Box 7, North Ryde, NSW 1670, Australia

Abstract

BACKGROUND: Water-extractable arabinoxylan (WEAX) is a minor constituent of wheat grain which influencesthe properties of wheat dough and its end products while conferring numerous health benefits. Consequently,various applications have been proposed for it in foodstuffs. Fortification of food products with pure WEAXextracts represents a simple means to evaluate its effect in these systems. However, rapid methods to isolaterelatively pure WEAX are not available. This study aimed to develop a rapid, technically simple means to extractrelatively pure WEAX on a small scale.

RESULTS: A simplified process was developed to extract WEAX from wheat flour. After heating, flour WEAXwas extracted with water and starch was eliminated by digestion with amyloglucosidase. Most of the proteinwas removed by adsorption to bentonite and precipitation with 65% ethanol. The final product generated by thedeveloped method consisted of 93 ± 4% arabinoxylan. The WEAX yield was 0.43 ± 0.02% (0.43 ± 0.02 g WEAX/100 gof starting flour) which is higher than that of other methods that generate WEAX of similar purity.

CONCLUSION: This method provides a technically simple means to perform small-scale isolation of WEAX thatcontains no detectable contaminating protein. This attribute renders it a preferred input for studies assessing theimpact of WEAX fortification on food product quality. 2008 Society of Chemical Industry

Keywords: water-extractable arabinoxylans; arabinogalactan peptides; pentosans; wheat flour

INTRODUCTIONWater-extractable arabinoxylan (WEAX) and arabino-galactan peptide (AGP) are water-extractable ara-binopolymers (WEAPs) in wheat,1 which togetheraccount for approximately 0.5–0.8% of wheat flourby mass.1,2 A number of methods to isolate WEAPs,which were historically referred to as pentosans,from wheat flour are reported.3–9 The extracts thatare produced by these approaches typically containapproximately 60% WEAX, 30% AGP and contami-nants which include glucose polymers (α-D-glucans orβ-glucans) and protein.3–11

WEAP extracts that contain both WEAX and AGPare not appropriate for studies that aim to investigatethe influence of either of the WEAP components onthe properties of food products because they possessdifferent chemical properties12 and behave differentlyin bread making.13 WEAX positively influences theproperties of wheat dough and its end products1,2,14

and confers health benefits when included in humandiets.15,16 Applications have been proposed for it as afood additive15 and as an input in the production ofindustrial products.17

Some methods to isolate WEAX either includeavoidable steps or employ complex procedures,12,18–21

while other approaches generate WEAX that containsa substantial amount of contaminating protein,18,22

some β-glucans,22 or are low yielding.23 In somereports extracts have been referred to as WEAX, butthese must contain AGP because no process to removeit was used in the methods of extraction.24,25

Supplementation studies to elucidate the influenceof WEAX on food product quality are reliant on theincorporation of pure extracts into food products.Small-scale food quality studies require gram amountsof pure WEAX. Therefore, the aim of the present workwas to develop a streamlined procedure that may beapplied to isolate relatively pure WEAX from wheatflour for studies of this type.

MATERIALS AND METHODSMaterialsCultivar Sunlin (Triticum aestivum, L.) was grown atThe University of Sydney, Plant Breeding Institute,Narrabri, NSW in 2002 and flour samples were

∗ Correspondence to: Matthew A Turner, The University of Sydney, Plant Breeding Institute, Cobbitty, PMB 11, Camden, NSW 2570, AustraliaE-mail: mturner@camden.usyd.edu.au(Received 5 November 2007; revised version received 27 February 2008; accepted 4 March 2008)Published online 25 June 2008; DOI: 10.1002/jsfa.3273

2008 Society of Chemical Industry. J Sci Food Agric 0022–5142/2008/$30.00

N K Ganguli, M A Turner

generated by milling on a Buhler ML202 mill (BuhlerAG, Uzwil, Switzerland) according to AACC Method26-21A.26

Amyloglucosidase (EC 3.2.1.3), bentonite,phloroglucinol and xylose were purchased fromSigma–Aldrich, St Louis, MO, USA. All other chem-icals used were of analytical grade.

Assessment of preliminary extract compositionWEAP content was quantified throughout the purifi-cation process by measuring total pentose sugars.Duplicate analysis was performed on two samplesthat were derived from separate extractions and meanvalues are presented. The total WEAP content ofaqueous flour extracts was determined using a mod-ification of the procedure developed by Rouau andSurget27 where samples were prepared differently. Inthe current study aqueous flour extracts were gener-ated by mixing flour (1% w/v) with distilled waterby vortexing for 1 min. The samples were then cen-trifuged (3220 × g for 10 min) and an aliquot of theextract was used to measure the concentration ofpentose units using the phloroglucinol reagent.28

The protein content of extracts and aqueoussolutions generated during the WEAX extractionprocedure was determined in the same samples asdescribed for WEAP determination using the TotalProtein Kit (Micro Lowry, Onishi & Barr modification,product codes TP0200 and B3934; Sigma–Aldrich).

The presence of starch in the extract was evaluatedusing iodine–potassium iodide.29

Assessment of the composition and yield of theWEAX extractAnalysis of WEAX content and yield was determinedby analysing three WEAX extracts that were eachisolated from 500 g of flour. Data are presented as themean ± 1 SD.

The yield of the procedure was determined byweighing extracts in duplicate. The purity of isolatedWEAX was determined by quantifying in duplicatethe concentration of pentose units in a 0.01% (w/v)aqueous solution of the extracts. The concentrationof pentose units was measured as described forquantification of WEAPs.

Refined WEAX (25 mg) was hydrolysed for separa-tion of monomeric sugars using paper chromatographyand determination of glucose content. Samples werehydrolysed by addition of 1 mol L−1 sulphuric acid,and incubation at 80 ◦C for 6 h. After addition ofbarium carbonate to neutralise pH, the samples werecentrifuged (3220 × g for 10 min) and the supernatantwas retained for subsequent analysis.

Paper chromatography was performed to verifypurity of the extracts and to identify the presence of anycontaminant sugars. An isopropanol–water (80:20)solvent system was used to separate sugars. Acidhydrolysed WEAX and reference markers (arabinose,galactose, glucose and xylose) were spotted onWhatman No. 1 chromatography paper and placed

in a chamber for descending chromatography. After16 h the paper was dried and sugars were detectedusing aniline–phosphate reagent (100 ◦C for 5 min).

Glucose in the extract was detected by paperchromatography. The concentration of glucose wasdetermined in acid hydrolysates of the WEAX extractsusing the Glucose Assay Kit (product code GAGO-20;Sigma–Aldrich).

The Lowry method grossly overestimates the proteincontent of samples that contain various phenolic acidsin concentrations greater than 1 µmol L−1.30 Becausephenolic acids are associated with WEAX the proteincontent of the final extract sample (N × 5.7) wasdetermined by nitrogen combustion (AACC, Method46-30).26 Duplicate measurements were taken andEDTA was used as a standard.

Extraction of WEAX from milled wheat flourMilled wheat flour (100 g) containing 0.5% WEAPwas heated at 130 ◦C for 90 min to inactivate endoge-nous enzymes. After cooling to room temperature theflour was added to 1 L of distilled water while themixture was stirred to obtain homogeneous slurry.After stirring for 90 min, the slurry was centrifuged(3220 × g for 10 min) and the supernatant was filteredusing Whatman No.1 filter paper. The filtrate wasadjusted to pH 4.5 with 0.1 mol L−1 HCl and 2 mL ofdistilled water containing 31.5 U mL−1 of amyloglu-cosidase from Aspergillus niger was added to degradestarch. The solution was then incubated at 55 ◦C for16 h until no starch was detectable in the extract.After starch hydrolysis, the solution was heated in aboiling water bath for 30 min to inactivate the amy-loglucosidase and to precipitate soluble proteins. Theextract was then centrifuged (3220 × g for 10 min)and filtered using Whatman No. 1 filter paper.

The filtrate (100 mL) was adjusted to pH 5.0 imme-diately with 0.1 mol L−1 HCl, added to 10 mL of a0.2% bentonite aqueous slurry to adsorb contaminat-ing protein and stirred for 30 min. The suspension wascentrifuged as previously described to separate solubleprotein that was adsorbed to bentonite from WEAPs,and the filtrate was adjusted to pH 7.0 by addition of0.1 mol L−1 NaOH. To selectively precipitate WEAX,ethanol was added whilst stirring to a final concen-tration of 65% (v/v). The solution was stirred for anadditional 30 min and left to stand overnight at 25 ◦C.Precipitated WEAX was recovered by decanting mostof the supernatant and subsequent centrifugation.Samples were then washed once with 65% ethanol,twice with acetone, and air dried. A 1% aqueoussolution of the precipitate was centrifuged to removeinsoluble protein. The supernatant was lyophilisedand powdered in a coffee grinder (Breville Coffee andSpice Grinder, Model CG-2; Breville Pty, Botany,Australia). A schematic representation of the arabi-noxylan extraction procedure is presented in Fig. 1.

A series of concentrations of bentonite wereevaluated in the slurry (0.1, 0.15. 0.2, 0.25, 0. 3,0.4, 0.5 and 1.0% w/v) to identify the concentration

1906 J Sci Food Agric 88:1905–1910 (2008)DOI: 10.1002/jsfa

Extraction of arabinoxylans from wheat flour

Refined WEAX

Crude WEAP

Heat at 130°C for 90 minutesInactivation of endogenous enzyme

Add ethanol until 65% (v/v) whilststirring, rest overnight, centrifuge,wash with 65% alcohol, acetone andair dry

Removal of arabinogalactanpeptides and protein

Add 0.2% bentonite slurry, adjust topH 5.0, stir for 30 min, centrifuge,filter and adjust to pH 7.0

Removal of protein

Removal of protein

Heat in boiling water bath for 30min, cool to room temperature,centrifuge and filter

Inactivation of amylolytic enzymeand removal of protein

Degradation of starch

Centrifuge and filter

Refined WEAP

Crude WEAX

Milled Wheat Flour

Heat Treated Flour

Wheat Flour Slurry

Aqueous Flour Extract

Starch Free Extract

Dissolve in distilled water, centrifuge,lyophilize supernatant and grind

Adjust pH to 4.5, add amyloglucosidaseand incubate for 16h at 55°C

Cool, mix with distilled water whilststirring and stir for 90 min

Figure 1. A schematic representation of the WEAX isolationprocedure.

that maximises protein removal whilst minimisingadsorption of WEAX. To identify the pH optimum forextraction, the adsorption procedure was performedwith a 0.2% bentonite slurry at 0.5 pH unit intervalsranging from pH 3.0 to 9.5.

RESULTS AND DISCUSSIONThe procedure used to prepare WEAX is shownin Fig. 1. The first step involved heating flourto inactivate endogenous arabinoxylan degradingenzymes1,18,31 that are active in aqueous cereal flourextracts.32,33 Hot aqueous ethanol (80%) treatmentsare reported to be an effective alternative primary stepin the extraction process as they denature proteinsincluding endogenous enzymes,34 but they reduceWEAX yield by up to 60%.12,23 Most methods ofWEAP extraction do not apply a heat treatment beforepreparing aqueous flour extracts,3–14,18,20,35 leavingpotential for degradation by native arabinoxylandegrading enzymes which may reduce yield and alterWEAX structure.

The iodine test revealed that starch was presentin the aqueous flour extract. Therefore, amyloglu-cosidase was used to catalyse the hydrolysis of solu-ble starch. Preliminary time course studies showedstarch could not be detected after a 16 h incu-bation (data not shown). Other investigators haveused amyloglucosidase3,5,23,36 or a combination ofα-amylase and amyloglucosidase8,9,13,24,37 to degrade

Table 1. Removal of protein contaminant at different steps of the

WEAX extraction procedure

Sample

Extractprotein

contenta

Proteinremoval (%of total)b

Stepwiseprotein

removal (%)c

Aqueous flour extract 5.73 – –Crude WEAP 5.22 8.9 8.9Refined WEAP 0.74 78.3 85.9Crude WEAX 0.09 11.3 88.3Refined WEAX 0.02 1.2 81.4Total 99.7

a Protein present in extracts derived from 100 g flour.b Percentage of protein removed in the step relative to that containedin the aqueous flour extract.c Percentage of protein removed from the previous sample.

starch in WEAP extracts. Results of this study showthat amyloglucosidase alone is sufficient to degradestarch that is present in the aqueous flour extract.

Proteins in the extract were eliminated at four dif-ferent stages during the isolation of WEAX (Table 1,Fig. 1). The first of these steps was a heat treatmentwhich was applied directly after enzyme-catalysedstarch degradation. Heat treatment is commonly usedin the extraction of WEAPs to remove some of thecontaminating protein.5,20,32,36,38 In this study, whenthe starch-free extract was heated to inactivate amy-loglucosidase and centrifuged, only 8.9% of the solubleprotein in the extract was removed (Table 1). Previousstudies have claimed to remove 20–50% of contam-inating protein by using a heating step.5,32,38 Differ-ences in results may be accounted for by differences inthe extraction procedure, properties and compositionof flour19 and methods of protein estimation.

Bentonite has been successfully used to adsorb pro-tein in procedures to extract arabinopolymers fromcereals.13,24,35,39 When applied in extracts of rye itadsorbed most of the protein that was present.39

The incorporation of different concentrations of ben-tonite was trialled to determine the concentration thatmaximised adsorption of protein from the aqueousextract whilst minimising adsorption of WEAP. With-out incorporation of the bentonite step approximately5 g of protein that was present in the crude WEAPextract (Table 1) remained as a contaminant. Substan-tial increases in protein adsorption were observed asslurry bentonite concentrations increased up to 0.2%,where approximately 86% was adsorbed (Fig. 2).Small increases in protein adsorption occurred asbentonite concentration was further increased. Themaximum protein adsorption of approximately 91%occurred when a slurry bentonite concentration of 1%was employed.

The effect of bentonite concentration on the adsorp-tion of WEAP was also investigated. When a slurrycontaining 0.15% bentonite was used, no detectableWEAP adsorption was observed (Fig. 3). As the con-centration of bentonite in the slurry was increased,WEAP adsorption increased in a near linear fashion

J Sci Food Agric 88:1905–1910 (2008) 1907DOI: 10.1002/jsfa

N K Ganguli, M A Turner

0

10

20

30

40

50

60

70

80

90

100

0 0.2 0.4 0.6 0.8

Slurry bentonite concentration (%)

Pro

tein

ads

orpt

ion

(%)

1

Figure 2. Adsorption of protein (%) from a crude arabinopolymerextract at different bentonite slurry concentrations.

0

10

20

30

40

50

60

70

80

90

100

0 0.2 0.4 0.6 0.8

Slurry bentonite concentration (%)

WE

AP

ads

orpt

ion

(%)

1

Figure 3. Adsorption of WEAPs (%) from a crude arabinopolymerextract at different bentonite slurry concentrations.

up to approximately 88% when using a 1.0% ben-tonite slurry. The bentonite concentration for WEAXisolation that was chosen for incorporation was 0.2%because it adsorbed approximately 86% of the contam-inating protein that was present in the crude WEAPextract. This represented 78.3% of the protein thatwas present in the aqueous flour extract (Table 1). Atthe same bentonite concentration approximately 9%of WEAP in the crude WEAP extract was adsorbed.

The interaction between protein and bentonite isionic in nature. Therefore the influence of pH onlevels of free protein and WEAP was investigated. AtpH 3.0 free protein was at its lowest level (5.4 µg mg−1

flour; Fig. 4) because adsorption to bentonite wasat its maximum. As the pH of the slurry increasedup to pH 6.0, the amount of free protein increasedsteadily to 6.7 µg mg−1 of flour as a result of decreasedadsorption. When the pH of the slurry increased abovepH 6.0 rapid increases in free protein were observedup to 13.8 µg mg−1 of starting flour at pH 9.5.

Free WEAP increased in an almost linear fashionfrom pH 3.0 (4.7 µg mg−1 of starting flour) to pH 7.5(5.84 µg mg−1 of starting flour), reflecting decreasingadsorption to bentonite. Free WEAP declined slightlyfrom pH 7.5 to pH 9.5 but the free WEAP contentat pH 9.5 (5.4 µg mg−1 flour) was greater than thatobserved at all pH values below pH 7.0.

Filtrate pH

WE

AP

and

sol

uble

pro

tein

con

tent

(µg/

mg

of f

lour

)

0

2

4

6

8

10

12

14

16

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

WEAP Protein

Figure 4. Content of WEAPs and soluble protein in a crudearabinopolymer extract treated with bentonite (0.2%) at pH 3.0–9.5.

Protein adsorption to bentonite represents a simplepractical approach to remove protein in WEAXextraction. Furthermore, bentonite is widely usedin wineries to remove protein and represents a safeprocedure for the preparation of food additives.Bentonite can adsorb WEAX and reduce the yieldof such extracts, so a protein adsorption stepincorporating it must be carefully optimised.

This study identified a 0.2% bentonite slurry (pH 5)as the best for generating WEAP extracts (5.3 µg mg−1

flour) that contained a low level of protein (6.4 µg mg−1

of flour) which was removed later in the WEAXextraction process. The WEAP yield was 5% lowerthan for the 0.2% (pH 7.0) bentonite treatment whichwould be appropriate in circumstances where thepresence of protein in the final extract was acceptable.

Ethanol precipitation of refined WEAP extractremoved 88.3% of the protein that was present inthe filtrate generated by the bentonite step, which isequivalent to 11.3% of the protein that was present inthe aqueous flour extract (Table 1, Fig. 1). This stepselectively precipitates WEAX, leaving the majority ofcontaminating residual protein and AGP in solution.22

After ethanol precipitation the precipitate wasdissolved in distilled water to generate a crude WEAXextract (Fig. 1). Centrifugation was employed toremove residual insoluble protein instead of dialysis21

because it is simpler to perform and quicker. Thesupernatant was lyophilised and the extract wasground with a coffee grinder to produce a whitepowder.

The yield of refined WEAX obtained was 0.43 ±0.02% of starting flour (0.43 ± 0.02 g 100 g−1 flour).This is in the range of reported WEAX yields of0.06%18 to 0.71%23 (w/w of starting flour). TheLowry method determined that a total of 99.7% ofthe protein that was present in the aqueous flourextract was removed in this procedure (Table 1),and the combustion method did not identify anydetectable protein in the final extract. Furthermore,the developed method generates WEAX that containssubstantially lower levels of contaminating proteinthan approaches that have similar yields, and is

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Extraction of arabinoxylans from wheat flour

substantially higher yielding than other methods thatgenerate WEAX that is contaminated with low levelsof protein (up to 0.32%).12,18 Acid hydrolysates ofthe WEAX extract contained 93 ± 4% pentose sugars,and approximately 1% glucose. Glucose may representan integral part of the arabinoxylan structure10 orcould have originated from contaminating glucosepolymers.10,22

Incorporation of arabinoxylan-rich wheat fibre,which is composed of approximately 50% WEAX, infood products revealed that it is more palatable thanguar gum.15,16 Furthermore, its inclusion in humandiets has positive effects on glycaemic control.15,16

There is a need to determine the applicability offortification of WEAX in various food products.The developed process could be applied to generateWEAX extracts from wheat flour that have low levelsof contaminants, enabling studies to elucidate itsinfluence on food quality.

CONCLUSIONA streamlined procedure to isolate WEAX was devel-oped in this study. To ensure that its structure was notaltered by native arabinoxylan-degrading enzymes aheat treatment was applied to flour. Treatment of theaqueous flour extract with amyloglucosidase was suf-ficient to reduce the starch content below the level ofdetection. Protein was removed in four different stepsin the isolation process. However, most of the protein(78% of protein that was present in the initial aqueousflour slurry) was removed by adsorption to bentonite.Lengthy steps like dialysis were not employed.

The final product contained 93 ± 4% arabinoxylan.No protein was detected in the extract by thecombustion method. The extract generated representsan ideal input in food fortification studies.

ACKNOWLEDGEMENTSWe wish to thank the Value Added Wheat CRC forproviding funds for the research. We would also liketo thank Dr Mike Sissons for valuable comments onthe manuscript.

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