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doi:10.1016/j.jc

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Journal of Cereal Science 47 (2008) 357–364

www.elsevier.com/locate/jcs

A systematic micro-dissection of brewers’ spent grain

Andrew J. Jay, Mary L. Parker, Richard Faulks, Fiona Husband, Peter Wilde,Andrew C. Smith, Craig B. Faulds, Keith W. Waldron�

Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK1

Received 28 February 2006; received in revised form 8 May 2007; accepted 16 May 2007

Abstract

Brewers’ spent grain (BSG), one of the co-products of the brewing industry, has been mainly used as cattle feed. Spent grain was shown

to contain a number of potentially high-value components such as feruloylated arabinoxylan and protein, as confirmed by microscopy

and chemical analysis. A significant quantity of starch was also identified, a polysaccharide generally considered to be removed through

the malting and mashing steps of brewing. As part of a study to increase the exploitation of spent grain, five separate fractions were

prepared through combined milling and vibratory sieving and characterised in terms of chemical composition (polysaccharide

composition and linkage; phenolic composition) and by fluorescence microscopy. Material retained on sieve mesh plates of 500, 250 and

150 mm consisted mainly of arabinoxylan-rich palea and lemma, while material passing through 106 and 55mm sieves was fine, crumb-like

material enriched in protein and starch. Lignin was present in all fractions, and originated from the fragmented palea and lemma.

The results are discussed in relation to the potential for whole BSG exploitation.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Arabinoxylan; Barley; Brewers’ spent grain; Carbohydrate; Cereal processing co-products; Fractionation; Phenolic acids; Ferulic acid

1. Introduction

Brewers’ spent grain (BSG), the main low-value solidresidue which results from the use of barley (Hordeum

vulgare L.) in brewing, represents more than 25% (w/w) ofthe starting material (malted barley). The EnvironmentAgency estimates that UK brewers produce over halfmillion tonnes of waste annually, and across Europe thisfigure has been estimated as 3.5 million tonnes. Until now,BSG has been sold as cattle feed, composted or disposed aslandfill. Due to legislative drivers (e.g. EC Council LandfillDirective 199/31/EC, 1999), the cost for the disposal of

e front matter r 2007 Elsevier Ltd. All rights reserved.

s.2007.05.006

: AIR, Alcohol insoluble residue; BSG, Brewers’ spent

iferulic acid; FA, Ferulic acid; NSP, Non-starch

ing author. Sustainability of the Food Chain Exploitation

ute of Food Research, Colney, Norwich NR4 7UA, UK.

255385; fax: +44 1603 507723.

ess: keith.waldron@bbsrc.ac.uk (K.W. Waldron).

p://www.ifr.ac.uk/, http://www8.ifr.ac.uk/sustainability/,

ro-food.net/index.htm

BSG has increased and with the decline in traditionaldisposal routes for the solid material (such as animal feed),alternative commercial uses for BSG are being sought.These have included using the residual cell wall material asbaking flour supplements in extruded bread products(Rasco et al., 1990; Wampler and Gould, 1984), andincorporating it into snack products (Demiranda et al.,1994a, b; Ozturk et al., 2002). This has met with limitedsuccess, as the BSG can impart unwanted flavours andaromas (Townsley, 1979) which may limit its usefulness,although the use of fresh BSG in breadmaking has beencommercialised on a small scale by a Seattle bakery(Nathan, 1994). However, these constitute relatively lowvalue uses of BSG. Non-food applications have includeduse as a fermentation substrate due to its high nitrogenconcentration (Bogar et al., 2002; Okita et al., 1985) andas a fuel source (Kepplinger and Zanker, 2003; Okamotoet al., 1999, 2002). The potential for using BSG in humanfood products is of considerable interest due to its highfibre and mineral content (Ranhotra et al., 1982) togetherwith recent studies, which have demonstrated the beneficial

ARTICLE IN PRESSA.J. Jay et al. / Journal of Cereal Science 47 (2008) 357–364358

effects of BSG in treating conditions such as constipation(Odes et al., 1985) and ulcerative colitis (Bamba et al.,2002; Kanauchi et al., 2001).

Brewers’ spent grain is approximately 80% cell wallmaterial, which is partly lignified and rich in (feruloylated)arabinoxylan polysaccharides (Hernanz et al., 2001;Valverde, 1994). The remaining 20% is mainly protein.Cost-effective separation of BSG into its individualcomponents, through mechanical and/or (bio)chemicalmeans, combined with a reduction in biomass, couldprovide commercially valuable streams for exploitation in anumber of different applications, both food and non-food.

Presently, only a few empirical separation methods havebeen tested, while some employing a simple wet separationof BSG have been patented (e.g. Zucher and Gruss, 1990).In this patent, a protein fraction was separated from a‘‘roughage’’ fraction, the latter being recovered for use as ahigh-fibre food ingredient. BSG has also been used asa source of carbohydrate for bioethanol production,and higher-protein animal feed with an acid pretreatment(Tucker et al., 2004). Dried spent grain can be roll-milledand sieved into three fractions to produce materials whichhave been tested empirically for a variety of applications:medicinal, agricultural, recycled materials manufacture(Ishiwaki et al., 2000) and microbial growth medium(Szponar et al., 2003). Pentose-rich growth medium canalso be made by simple acid hydrolysis (Carvalheiro et al.,2004; Duarte et al., 2004). Other work has focussed on thespecific release of individual types of component, i.e.arabinoxylans and ferulic acids using enzymes (Bartolomeet al., 2002; Faulds et al., 2004) or oligosaccharides usinghydrothermal treatment (Carvalheiro et al., 2004; Kabelet al., 2002).

In this paper, we describe an in-depth evaluation of thecomposition of a range of fractions that can be obtainedfrom BSG under controlled conditions. This forms partof a strategy to optimise the exploitation of the wholematerial. The compositions of BSG fractions were char-acterised in relation to the botanical structure of barleyand its key chemical components, taking account of post-harvest processing history.

2. Experimental

2.1. Materials

Brewers’ spent grain was provided by Scottish CourageLtd. (Edinburgh, Scotland), and frozen to �20 1C within6 h of collection from the mash tun in order to avoidmicrobial spoilage. It was then lyophilised and stored atambient temperature and humidity in sealed plasticcontainers. All chemicals were of analytical grade.

2.2. Milling and sieving

Using a vibrating feeder, dried BSG was fed into anultracentrifugal mill (Retsch, Germany). Three batches

were milled using screens of mesh size 1.0, 0.5 and 0.25mm,respectively. A portion of each milled batch (64 g) waspassed through a series of sieves with mesh sizes 500, 250,150, 106 and 53 mm, respectively. The sieves were shaken ona vibratory sieve shaker (Fritsch Analysette 03, Germany),set to amplitude 4 for 30min at 10-s intervals.

2.3. Light microscopy

Samples of whole and sieved BSG were viewed at lowmagnification using a dissecting microscope (Wild M8).Samples of sieved BSG were also suspended in 5%ammonia and were viewed at a higher magnification usingan Olympus BX60 (Olympus, Japan) microscope withAcquis software (Syncroscopy, Cambridge, UK). Theautofluorescence in unstained sections was recorded usingthe UV filter cube (U-MWU, exciter filter BP330-385,barrier filter BA420) of the microscope.

2.4. Moisture content

Weighed samples of the sieved fractions were dried in avacuum oven at 55 1C, 0.099Torr for 42 h over calciumoxide. The oven was cooled to 20 1C, re-pressurised withdry air, and the samples reweighed immediately.

2.5. Neutral sugar composition

Sugars were released from the fractions by hydrolysiswith 72% H2SO4 for 3 h, followed by dilution to 1M(Saeman hydrolysis). An internal standard of 2-deoxyglu-cose was added prior to neutralisation with ammonia. Themonosaccharides were analysed as their alditol acetates byGLC (Blakeney et al., 1983), on a cross-bonded 50%cyanopropyl methyl-, 50% phenyl methyl-polysiloxanecolumn (Thames Chromatography, Maidenhead, UK)using a flame ionisation detector. The oven temperatureprogramme used was: 140 1C (0min), +2.5 1Cmin�1

(5min), 210 1C (45min). A second set of samples washydrolysed with 1M H2SO4 acid only (100 1C, 2.5 h), butotherwise treated the same as the first set, so as to quantifynon-cellulosic glucose.

2.6. Starch analysis

Starch content was determined using a starch assay kitsupplied by Megazyme (Bray, Republic of Ireland),according to the manufacturer’s instructions.

2.7. Methylation analysis

Linkage positions for the milled material and sievefractions were determined by methylation analysis. Freeze-dried samples (5–6mg) were freeze-milled in a SPEX 6700freeze mill, and then dispersed in dry dimethyl-sulfoxide(DMSO) at 20 1C for 16 h after flushing with argon. Theywere methylated by sequential addition of freshly powdered

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sodium hydroxide (0.5 g) and iodomethane (4ml) (Ciucanuand Kerek, 1984; Needs and Selvendran, 1993). Afterelution–extraction on a C18-bonded cartridge (Sep-Pak,Waters, Watford, UK), the methylated carbohydrates weredried, extracted into CHCl3/CH3OH (50/50, v/v), andevaporated to dryness. The samples were hydrolysed using2M trifluoroacetic acid (Blakeney et al., 1983), andconverted to partially methylated alditol acetates (PMAAs)by NaBD4 reduction and acetylation with acetic anhydrideand N-methylimidazole (Albersheim et al., 1967).

The PMAAs were analysed using the same GC asabove, with a temperature programme, 55 1C (2min),+45 1Cmin�1 (1.9min), 140 1C (2min), +2 1Cmin�1

(35min), 210 1C (40min). The PMAAs were identified bymeasuring their retention times relative to myo-inositolhexaacetate, and comparing the relative retention timeswith those of external standards. A mixture of standards foreach sugar was prepared by deliberate under-methylationof methyl glycosides (Doares et al., 1991). Peak areas wererepresented as relative molar quantities using effectivecarbon response factors (Sweet et al., 1975).

Identities of PMAAs were confirmed by their electron-ionisation mass spectra (Carpita and Shea, 1989). GC–MSanalysis was performed on an identical GC in series with anAnalytical Trio 1S Mass Spectrometer (Fisons Instruments,San Jose, CA, USA), using a source temperature of 200 1Cand an ionisation potential of 70 eV.

2.8. Uronic acid content

Total uronic acid content was determined by the methodof Blumenkranz and Asboe-Hansen (1973) using glucuronicacid as a standard.

2.9. Phenolic acid analysis

The total alkali-extractable hydroxycinnamate contentof BSG fractions was determined by hydrolysis with 4Msodium hydroxide (deoxygenated), under nitrogen for 24 hin the dark at room temperature. Samples were centrifuged(1000 rpm, 5min) and 800 ml of supernatant was taken foranalysis. trans-Cinnamic acid (10 mg/50 ml) was added andthe solution adjusted to pH 2 with 6M hydrochloric acid.Phenolic acids were extracted with ethyl acetate (3� 3ml)which was evaporated to dryness. The dry extract wasredissolved in aqueous methanol (MeOH/H2O, 50/50, v/v,500 ml), filtered (0.22 mm fluoropore membrane) andinjected onto a LUNA C18 reverse-phase HPLC column(Phenonomex, Macclesfield, UK). Ferulic and p-coumaricacid levels were quantified against standard curves. Ferulicacid dehydrodimers were quantified according to themethod of Waldron et al. (1996).

2.10. Protein content

Protein was determined using the Kjeldahl method.

2.11. Lignin analysis

Lignin was determined using a method adapted fromTheander and Westerlund (1986). Samples (50mg) weretreated with 72% (w/v) sulphuric acid (0.75ml) for 3 h at20 1C. Water (9ml) was added, mixed and incubationcontinued for 2.5 h at 100 1C. Residues were recovered byfiltration through pre-weighed sintered glass funnels undervacuum. The solid was washed three times with warmwater until the residue was free of acid. The glass filterswere dried at 50 1C in an oven until a constant weight wasobtained.

2.12. Lipid analysis

Total lipid content and fatty acids were determined usingthe method described by Mondello et al. (2000).

3. Results and discussion

3.1. Fractionation profiles

BSG contains flowering glume material, rachilla andlodicule remains, and parts of the embryonic axis, inaddition to bran layers, and partially degraded fragmentsof the endosperm. In comparison to wheat bran, this makesthe composition of BSG physically more heterogeneousand chemically more complex. Initial milling involved theevaluation of three mill mesh sizes and their impact on thesubsequent distribution of particles after sieving for 1 h(Fig. 1). A 1mm mill mesh resulted in a sieved profile inwhich the first major fraction (30%) was retained by the250-mm sieve. A further 40% was retained by the 150-mmsieve and 19% by the 106-mm sieve. Less than 10% wasretained by the 53-mm sieve and less than 0.3% passedthrough (results not shown). In contrast, a 0.5mm millmesh resulted in a significant shift in this profile towardsthe smaller sieve size, with a decrease in material retainedby the 250 and 150 mm sieves, and an increase to 40% ofmaterial retained by the 106-mm sieve. However, millingthrough a 0.25mm mill mesh did not result in a furthershift in the profile, but resulted in an increase in materialretained by the 150-mm sieve. Low-power microscopyrevealed that this was due to blockage of that sieve withclumps of particles which individually would be expected topass through (blinding). Prolonged sieving of this material(2 and 16 h) failed to improve on this. As a result, the sievefractions derived from material milled through the 0.5-mmmesh were chosen for further study. The production of fiveseparated fractions using this method improves on thethree fraction separation described previously by Ishiwakiet al. (2000).

3.2. Light microscopy

Examination of whole, thawed BSG under low magni-fication revealed large, flat fragments consisting essentially

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an

tity

Re

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ed

(%

w/w

)

70.0

30.0

20.0

0.0

10.0

40.0

50.0

60.0

500µm 250µm

Sieve Fraction

150µm 106µm 53µm

Fig. 1. Distribution of particle sizes after milling and sieving BSG. After milling lyophilised BSG through 1mm (E), 0.5mm (’) and 0.25mm (m) mesh

screens, samples were separated by a vibratory sieve shaker for 1 h. Samples of the 0.25mm milled material were also separated for 2 h (J) and 16 h (&).

The percentage retention corresponds to the weight of material retained by the sieve.

A.J. Jay et al. / Journal of Cereal Science 47 (2008) 357–364360

of grain enveloped in flowering glumes (lemma and palea)with fragments of endosperm and embryo (Fig. 2a).The endosperm stained with iodine (KI3 solution)(Fig. 2b), indicating the presence of undigested starch.Under higher magnification, the dried, milled BSG sampleshowed that, even after milling, fragments of many sizesand tissue types are present (Fig. 2c). The use ofautofluorescence enhanced differentiation of the tissuetypes (Fig. 2d).

Low magnification imaging of the sieve fractions showedthat the first three fractions (retained by 500, 250 and150 mm sieves) comprised predominantly different sizedfragments of lemma and palea, and attached material(Figs. 2e–g). The last two fractions (retained by 106 and53 mm sieves) comprised much finer, crumb-like material(Figs. 2h–i). Using autofluorescence microscopy, materialderived from the lignin-rich glume appears blue, consistentwith the presence of p-coumaric acid (see below). Materialof aleurone origin autofluoresces green, probably due tothe presence of ferulic acid (see below). Both types ofmaterial were found to be present in the first three fractions(Figs. 2j–m). A closer examination of the third fraction(150 mm) showed significant amounts of outer epidermis ofthe glume (Fig. 2n); bran layers, i.e. pericarp (green) andinner layers of glume (blue) (Fig. 2o); aleurone (Fig. 2p);tissue containing lipid (orange), derived from the embryo(Fig. 2q).

The last two fractions comprised a greater mixture oftissue types (Figs. 2r–u). In addition to small fragments ofthose types already identified, a greater number of lipidcontaining fragments were seen (e.g. Figs. 2v), and alsogranular fragments of starch (Fig. 2w–x). These stainedreddish-blue with iodine and appeared slightly gelatinised

(Fig. 2). Other elements were visible in small numbers,e.g. fibrovascular bundles from the glume ribs (green) andthin walled fibres (blue) (not shown).

3.3. Total chemical composition

The initial BSG and each sieved fraction was analysedfor protein, starch, cell-wall carbohydrate, lignin, phenolicacids and moisture content (there was insufficient materialfrom the 500mm fraction, preventing moisture determination).The initial material was also analysed for lipid content,but insufficient material was available to do this for thesieved fractions. The percentage total recoveries of thesecomponents are shown in Fig. 3. The results show thatthere was a segregation of components between the sievedfractions, resulting in a considerable enrichment in theproportion of protein and starch, and a concomitantdecrease in cell-wall polymers, in the small particle sizefractions (retained by the 106 and 53 mm sieves) ascompared with the original milled BSG. Klason ligninremained relatively constant within all fractions. Thepresence of residual starch in the BSG was surprising asit is generally thought that most of the starch in the rawmaterial had been removed and converted to fermentablesugars during the mashing process (Tang et al., 2005). Themoisture level (�8%) does not vary greatly between thesieved fractions and is probably similar in the 500 mmfraction. However, the amount of material not accountedfor by these analyses is clearly greater in the firstthree fractions. This may be due to unquantified lipidicand cuticular components, and possibly due to the diffi-culty in quantifying polysaccharide components that arecross-linked with lignin.

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Fig. 2. Light, bright field and autofluorescence micrographs of total and sieved fractions of BSG. Bars: (a–b) bar ¼ 1mm; (e–i) bar ¼ 500mm; (c–d, j–m, r–u)

bar ¼ 200mm; (n–q, v–w) bar ¼ 100mm; (x) bar ¼ 50mm. (a) Unstained light micrograph of whole BSG; (b) as for (a) but stained in iodine solution;

(c) unstained light micrograph of milled BSG; (d) as for (c) but under autofluorescence, pH 10; (e–i) light micrographs of milled BSG retained by 500, 250,

150, 106 and 53mm sieves, respectively; (j–m) autofluorescence micrographs (pH 10) of mixtures of aleurone and glumes in 500, 250 and 150mm fractions;

(n) autofluorescence micrograph (pH 10) of the outer epidermis of the glume; (o) autofluorescence micrograph (pH 10) of the pericarp and inner layers of

the glume; (p) autofluorescence micrograph (pH 10) of the outer aleurone layer; (q) autofluorescence micrograph (pH 10) of the lipidic material from the

embryo; (r–u) light and fluorescence micrographs of mixtures of cell types from 106 and 53mm fractions; (v–x) light and autofluorescence micrographs of

lipidic components and granular starch (w–x).

A.J. Jay et al. / Journal of Cereal Science 47 (2008) 357–364 361

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0%

20%

40%

60%

80%

100%

BG1 500µm 250µm 150µm 106µm

Sieve fraction

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l w

eig

ht com

positio

nNot determined

Water

Lipid

Phenolics

Lignin

Protein

Carbohydr. (non-starch)

Starch

53µm

Fig. 3. Chemical composition of unsieved and sieved BSG fractions. Values are expressed as weight percentages of the material retained on the sieve.

Table 1

Sugar composition of BSG and sieved fractions

Sieve fraction Mole fraction (%)

Rha Fuc Ara Xyl Man Gal Glc (non-c.) Glc (cel.) UA Total (m/mga) Ara/Xyl ratio

BSG 0.20 0.11 15.36 29.84 1.24 2.00 25.71 12.60 12.95 444 0.515

500mm 0.24 0.14 9.18 47.03 0.34 1.04 8.96 19.57 13.50 488 0.195

250mm 0.29 0.08 14.33 41.06 0.72 1.51 12.97 17.53 11.51 450 0.349

150mm 0.21 0.09 17.06 32.87 0.99 1.83 16.79 14.69 15.47 472 0.519

106mm 0.30 0.13 15.54 20.42 1.61 2.44 44.56 5.33 9.67 419 0.76

53mm 0.22 0.13 12.76 14.44 1.72 2.25 32.57 20.52 15.40 516 0.883

Ara, arabinose; Fuc, fucose; Gal, galactose; Glc, glucose; Man, mannose; Rha, rhamnose; Xyl, xylose; non-c., non-cellulose; cel., cellulose.aAnhydrous.

A.J. Jay et al. / Journal of Cereal Science 47 (2008) 357–364362

3.4. Polysaccharide composition

The sugar composition of the milled BSG and sievedfractions is shown in Table 1. Information on the linkagesof the component sugars in all samples except for the500 mm fraction are shown in Table 2. The non-starchglucose (i.e. hydrolysed by 1M sulphuric acid) is accountedfor principally by the starch component which was assayeddiagnostically (Fig. 3; see Section 2.1). The small quantitiesof (1–3)-linked Glcp are probably derived from mixed-linkage glucans which are present in BSG in only minorquantities (Robertson, J.A., et al., unpublished results).The ratio of the (starch-derived) non-cellulose glucans tocellulose exhibits a considerable increase across the 500 to106 mm fractions, and then decreases in the 53 mm fraction,reflecting the partitioning of starch shown in Figs. 2 and 3.The high proportion of cellulosic glucose in the 500, 250and 150 mm fractions, as inferred from the release of Glcby acid hydrolysis conditions (Table 1) is in keepingwith the dominant palea and lemma fragments (Figs. 2e–g).The majority of the glucose was (1–4)-linked with smallquantities of 1,4-6-linked glucose consistent with the

presence of starch-derived amylopectin (Table 2). Interest-ingly, significant quantities of terminal glucose was alsopresent.The bulk of the xylose component comprised (1–4)-

linked xylose, indicative of xylan hemicellulose. Only tracequantities of (1,3-4)- and (1,2-4)-linked Xylp could bedetected. However, the presence of terminal Araf, and theratio of ara:xyl shown in Table 2 is indicative ofarabinoxylan hemicelluloses from which one would expectto see larger quantities of branched xylose residues. It isprobable that the arabinoxylans of the palea and lemmaare difficult to evaluate by methylation analysis. This mayreflect their insolubility in the highly-lignified palea andlemma fragments, resulting in their removal from theanalytical process during filtration. Such insolubility maywell be enhanced through the cross-linking of ester-linkedphenolics present on terminal arabinose branches. Thismay explain discrepancies between the relative levels ofsugars in Table 1 compared with levels in Table 2, and thepresence of small amounts of under-methylated xylose andglucose. The significant levels of uronic acid probablyderive from glucuronic acid as found in other cereal cell

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Table 3

Phenolic acid composition of BSG and sieved fractions

Phenolic compound Mole fraction (%)

BG1 500 mm 250 mm 150 mm 106 mm 53 mm

p-Hydroxybenzaldehyde n.d. 7.58 0.19 n.d. n.d. n.d.

Vanillin n.d. 1.16 0.83 n.d. n.d. n.d.

trans-p-Coumaric acid 27.72 35.41 40.17 22.22 15.98 14.58

8,80:6,70-Diferulic acid (AT) n.d. 28.11 n.d. n.d. n.d. n.d.

trans-Ferulic acid 51.00 15.70 38.91 52.19 55.63 56.72

cis-p-Coumaric acid 4.52 3.17 6.44 5.50 4.26 3.83

8,50-Diferulic acid 2.21 0.44 1.81 2.54 3.49 4.36

cis-Ferulic acid 6.16 2.03 4.90 7.72 8.98 9.44

5,50-Diferulic acid 2.03 1.44 1.49 1.89 3.16 3.43

8-0-40-Diferulic acid 6.36 2.63 5.26 7.94 8.50 7.64

8,50:7-O-40-Diferulic acid (BF) n.d. 2.32 n.d. n.d. n.d. n.d.

n.d. denotes that the free acid was not detected in the sample.

Table 2

Methylation analysis of milled and sieved BSG

Sugar linkage residue Fraction

BSG 250mm 150mm 106mm 53 mm

t-Araf 1.34 1.64 1.47 2.08 2.62

1,4-Arap/1,5-Araf 1.1 1.15 1.31 0.69 0.79

t-Xylp 0.64 0.9 0.84 0.69 0.79

1,4-Xylp (+ t-Galp) 6.33 9.75 9.97 4.94 5.21

1,3,4-Xylp (+ 1,2,4-Xylp) T 3.42 2.39 t t

1,2,3,4-Xylp 1.5 1.83 1.56 1.27 1.18

t-Glcp 9.47 6.02 7.3 8.61 9.25

1,3-Glcp 1.48 0.82 1.13 1.33 0.94

1,4-Glcp 66.69 64.64 66.2 74.24 73.46

1,4,6-Glcp 5.39 3.53 4.29 4.15 3.64

2,3,4,6-Glcp 2.35 3.83 1.53 0.27 0.29

1,4-Manp 1.46 0.69 0.88 1.13 1.18

1,6-Galp 2.23 1.78 1.12 0.6 0.65

Values are expressed as mol%.

n.d. signifies linkage not detected.

Ara, arabinose; Gal, galactose; Glc, glucose; Man, mannose; Xyl, xylose.

A.J. Jay et al. / Journal of Cereal Science 47 (2008) 357–364 363

walls (Brett and Waldron, 1996; Waldron and Faulds,2007). The amount of pectin-derived galacturonic acidis likely to be low in the lignified tissues, but may besignificant in the growing embryonic tissues, componentsof which are present in the small particle-size fractions.

Significant differences were observed in the phenoliccompositions of the fractions (Table 3). The proportion oftrans-p-coumaric acid decreased with particle size, and isprobably associated with lignified palea and lemma tissueswhich are enriched in the larger particle-sized fractions.Interestingly, the trans-ferulic acid component shows anopposite trend to that of trans-p-coumaric acid, and ishighest in the smaller particle-sized fractions. This indicatesthat it is associated with poorly lignified cell walls includingthe remnants of endosperm and growing embryonic tissueswhere, as indicated by the similar trends of the 8-0-40-, 5,50-and 8,50-diferulic acids, it is involved in interpolymericcross-linking, probably of arabinoxylan hemicelluloses.

However, p-hydroxybenzaldehyde, vanillin and the twopolycyclic diferulic acids, 8-80:6,70 (aryltetralin) diferulateand 8,50:8-O-40 (benzofuran) diferulate, are seen only in thefirst and second fractions; these have been associated withthe outermost, highly lignified layers of BSG, and may bebreakdown products of lignin.Recent papers have described the fractionation of BSG

using chemical (alkali) or hydrothermal (autoclaving)treatments (Kabel et al., 2002; Mandalari et al., 2005).Both systems started with whole BSG and resulted in theextraction of feruloylated arabinoxylan-rich extracts. Theuse of alkali caused the removal of most of the ferulate anddiferulate moieties from this arabinoxylan and no otherpolymer was isolated. The hydrothermal treatment of BSGresulted in the degradation of certain sugars, mainlyarabinose, with a conversion of this sugar to furfural,formic and levulinic acid (Kabel et al., 2002). By thecombined milling and vibratory sieving method describedin this paper, together with economic drying regimes, suchas thin layer drying in superheated steam (Tang et al.,2005), enriched fractions of phenolic acid-containingarabinoxylan or protein/starch can be produced. Thiswould allow a cleaner and much wider utilisation of allthe spent grain components for a number of potential foodand non-food industrial applications, such as prebiotics(Fooks et al., 1999), plant-based protein source for animalfeed, emulsifying and thickening agents, pulp papersubstitutes (Ishiwaki et al., 2000), or direct acid hydrolysisto produce pentose sugars for microbial fermentations(Carvalheiro et al., 2004).In summary, this investigation demonstrates that the

components of BSG can be fractionated effectively bysimple milling and sieving. The fractionation involvesseparation of palea and lemma lignified glumes from themore protein- and starch-rich embryonic tissues. It ispossible that with further physical treatments, starch-richendosperm that is attached to the lignified tissues couldalso be separated and recovered. Further characterisationof the starch and non-carbohydrate polymers will lead tofurther information on the properties and exploitation ofvalue-added components in BSG.

Acknowledgements

This work was supported by the Biotechnology andBiological Sciences Research Council (BBSRC), UK, andthe Department of the Environment, Food and RuralAffairs (DEFRA), UK, through a BridgeLINK grant(AFM201). We would like to express our gratitude toScottish Courage Limited, Edinburgh, UK, for supplyingthe brewers’ spent grain.

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