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Journal of Cereal Science 47 (2008) 357364 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 conrmed by microscopy and chemical analysis. A signicant quantity of starch was also identied, 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, ve 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 uorescence 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 55 mm sieves was ne, 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 solid residue which results from the use of barley (Hordeum vulgare L.) in brewing, represents more than 25% (w/w) of the starting material (malted barley). The Environment Agency estimates that UK brewers produce over half million tonnes of waste annually, and across Europe this gure has been estimated as 3.5 million tonnes. Until now, BSG has been sold as cattle feed, composted or disposed as landll. Due to legislative drivers (e.g. EC Council Landll Directive 199/31/EC, 1999), the cost for the disposal of
Abbreviations: AIR, Alcohol insoluble residue; BSG, Brewers spent grain; DIFA, Diferulic acid; FA, Ferulic acid; NSP, Non-starch polysaccharide Corresponding author. Sustainability of the Food Chain Exploitation Platform, Institute of Food Research, Colney, Norwich NR4 7UA, UK. Tel.: +44 1603 255385; fax: +44 1603 507723. E-mail address: keith.waldron@bbsrc.ac.uk (K.W. Waldron). 1 URLs: http://www.ifr.ac.uk/, http://www8.ifr.ac.uk/sustainability/, http://www.repro-food.net/index.htm 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.05.006

BSG has increased and with the decline in traditional disposal 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 as baking our supplements in extruded bread products (Rasco et al., 1990; Wampler and Gould, 1984), and incorporating it into snack products (Demiranda et al., 1994a, b; Ozturk et al., 2002). This has met with limited success, as the BSG can impart unwanted avours and aromas (Townsley, 1979) which may limit its usefulness, although the use of fresh BSG in breadmaking has been commercialised on a small scale by a Seattle bakery (Nathan, 1994). However, these constitute relatively low value uses of BSG. Non-food applications have included use as a fermentation substrate due to its high nitrogen concentration (Bogar et al., 2002; Okita et al., 1985) and as a fuel source (Kepplinger and Zanker, 2003; Okamoto et al., 1999, 2002). The potential for using BSG in human food products is of considerable interest due to its high bre and mineral content (Ranhotra et al., 1982) together with recent studies, which have demonstrated the benecial

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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 wall material, which is partly lignied 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 individual components, through mechanical and/or (bio)chemical means, combined with a reduction in biomass, could provide commercially valuable streams for exploitation in a number of different applications, both food and non-food. Presently, only a few empirical separation methods have been tested, while some employing a simple wet separation of 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 a high-bre food ingredient. BSG has also been used as a 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-milled and sieved into three fractions to produce materials which have 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 can also be made by simple acid hydrolysis (Carvalheiro et al., 2004; Duarte et al., 2004). Other work has focussed on the specic release of individual types of component, i.e. arabinoxylans and ferulic acids using enzymes (Bartolome et al., 2002; Faulds et al., 2004) or oligosaccharides using hydrothermal treatment (Carvalheiro et al., 2004; Kabel et al., 2002). In this paper, we describe an in-depth evaluation of the composition of a range of fractions that can be obtained from BSG under controlled conditions. This forms part of a strategy to optimise the exploitation of the whole material. The compositions of BSG fractions were characterised in relation to the botanical structure of barley and its key chemical components, taking account of postharvest processing history. 2. Experimental

were milled using screens of mesh size 1.0, 0.5 and 0.25 mm, respectively. A portion of each milled batch (64 g) was passed through a series of sieves with mesh sizes 500, 250, 150, 106 and 53 mm, respectively. The sieves were shaken on a vibratory sieve shaker (Fritsch Analysette 03, Germany), set to amplitude 4 for 30 min at 10-s intervals. 2.3. Light microscopy Samples of whole and sieved BSG were viewed at low magnication using a dissecting microscope (Wild M8). Samples of sieved BSG were also suspended in 5% ammonia and were viewed at a higher magnication using an Olympus BX60 (Olympus, Japan) microscope with Acquis software (Syncroscopy, Cambridge, UK). The autouorescence in unstained sections was recorded using the UV lter cube (U-MWU, exciter lter BP330-385, barrier lter BA420) of the microscope. 2.4. Moisture content Weighed samples of the sieved fractions were dried in a vacuum oven at 55 1C, 0.099 Torr for 42 h over calcium oxide. The oven was cooled to 20 1C, re-pressurised with dry air, and the samples reweighed immediately. 2.5. Neutral sugar composition Sugars were released from the fractions by hydrolysis with 72% H2SO4 for 3 h, followed by dilution to 1 M (Saeman hydrolysis). An internal standard of 2-deoxyglucose was added prior to neutralisation with ammonia. The monosaccharides were analysed as their alditol acetates by GLC (Blakeney et al., 1983), on a cross-bonded 50% cyanopropyl methyl-, 50% phenyl methyl-polysiloxane column (Thames Chromatography, Maidenhead, UK) using a ame ionisation detector. The oven temperature programme used was: 140 1C (0 min), +2.5 1C min1 (5 min), 210 1C (45 min). A second set of samples was hydrolysed with 1M H2SO4 acid only (100 1C, 2.5 h), but otherwise treated the same as the rst set, so as to quantify non-cellulosic glucose. 2.6. Starch analysis

2.1. Materials Brewers spent grain was provided by Scottish Courage Ltd. (Edinburgh, Scotland), and frozen to 20 1C within 6 h of collection from the mash tun in order to avoid microbial spoilage. It was then lyophilised and stored at ambient temperature and humidity in sealed plastic containers. All chemicals were of analytical grade. 2.2. Milling and sieving Using a vibrating feeder, dried BSG was fed into an ultracentrifugal mill (Retsch, Germany). Three batches Starch content was determined using a starch assay kit supplied by Megazyme (Bray, Republic of Ireland), according to the manufacturers instructions. 2.7. Methylation analysis Linkage positions for the milled material and sieve fractions were determined by methylation analysis. Freezedried samples (56 mg) were freeze-milled in a SPEX 6700 freeze mill, and then dispersed in dry dimethyl-sulfoxide (DMSO) at 20 1C for 16 h after ushing with argon. They were methylated by sequential addition of freshly powdered

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sodium hydroxide (0.5 g) and iodomethane (4 ml) (Ciucanu and Kerek, 1984; Needs and Selvendran, 1993). After elutionextraction on a C18-bonded cartridge (Sep-Pak, Waters, Watford, UK), the methylated carbohydrates were dried, extracted into CHCl3/CH3OH (50/50, v/v), and evaporated to dryness. The samples were hydrolysed using 2 M triuoroacetic acid (Blakeney et al., 1983), and converted to partially methylated alditol acetates (PMAAs) by NaBD4 reduction and acetylation with acetic anhydride and N-methylimidazole (Albersheim et al., 1967). The PMAAs were analysed using the same GC as above, with a temperature programme, 55 1C (2 min), +45 1C min1 (1.9 min), 140 1C (2 min), +2 1C min1 (35 min), 210 1C (40 min). The PMAAs were identied by measuring their retention times relative to myo-inositol hexaacetate, and comparing the relative retention times with those of external standards. A mixture of standards for each sugar was prepared by deliberate under-methylation of methyl glycosides (Doares et al., 1991). Peak areas were represented as relative molar quantities using effective carbon response factors (Sweet et al., 1975). Identities of PMAAs were conrmed by their electronionisation mass spectra (Carpita and Shea, 1989). GCMS analysis was performed on an identical GC in series with an Analytical Trio 1S Mass Spectrometer (Fisons Instruments, San Jose, CA, USA), using a source temperature of 200 1C and an ionisation potential of 70 eV. 2.8. Uronic acid content Total uronic acid content was determined by the method of Blumenkranz and Asboe-Hansen (1973) using glucuronic acid as a standard. 2.9. Phenolic acid analysis The total alkali-extractable hydroxycinnamate content of BSG fractions was determined by hydrolysis with 4 M sodium hydroxide (deoxygenated), under nitrogen for 24 h in the dark at room temperature. Samples were centrifuged (1000 rpm, 5 min) and 800 ml of supernatant was taken for analysis. trans-Cinnamic acid (10 mg/50 ml) was added and the solution adjusted to pH 2 with 6 M hydrochloric acid. Phenolic acids were extracted with ethyl acetate (3 3 ml) which was evaporated to dryness. The dry extract was redissolved in aqueous methanol (MeOH/H2O, 50/50, v/v, 500 ml), ltered (0.22 mm uoropore membrane) and injected onto a LUNA C18 reverse-phase HPLC column (Phenonomex, Maccleseld, UK). Ferulic and p-coumaric acid levels were quantied against standard curves. Ferulic acid dehydrodimers were quantied according to the method 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 from Theander and Westerlund (1986). Samples (50 mg) were treated with 72% (w/v) sulphuric acid (0.75 ml) for 3 h at 20 1C. Water (9 ml) was added, mixed and incubation continued for 2.5 h at 100 1C. Residues were recovered by ltration through pre-weighed sintered glass funnels under vacuum. The solid was washed three times with warm water until the residue was free of acid. The glass lters were dried at 50 1C in an oven until a constant weight was obtained. 2.12. Lipid analysis Total lipid content and fatty acids were determined using the method described by Mondello et al. (2000). 3. Results and discussion 3.1. Fractionation proles BSG contains owering glume material, rachilla and lodicule remains, and parts of the embryonic axis, in addition to bran layers, and partially degraded fragments of the endosperm. In comparison to wheat bran, this makes the composition of BSG physically more heterogeneous and chemically more complex. Initial milling involved the evaluation of three mill mesh sizes and their impact on the subsequent distribution of particles after sieving for 1 h (Fig. 1). A 1 mm mill mesh resulted in a sieved prole in which the rst major fraction (30%) was retained by the 250-mm sieve. A further 40% was retained by the 150-mm sieve and 19% by the 106-mm sieve. Less than 10% was retained by the 53-mm sieve and less than 0.3% passed through (results not shown). In contrast, a 0.5 mm mill mesh resulted in a signicant shift in this prole towards the smaller sieve size, with a decrease in material retained by the 250 and 150 mm sieves, and an increase to 40% of material retained by the 106-mm sieve. However, milling through a 0.25 mm mill mesh did not result in a further shift in the prole, but resulted in an increase in material retained by the 150-mm sieve. Low-power microscopy revealed that this was due to blockage of that sieve with clumps of particles which individually would be expected to pass through (blinding). Prolonged sieving of this material (2 and 16 h) failed to improve on this. As a result, the sieve fractions derived from material milled through the 0.5-mm mesh were chosen for further study. The production of ve separated fractions using this method improves on the three fraction separation described previously by Ishiwaki et al. (2000). 3.2. Light microscopy Examination of whole, thawed BSG under low magnication revealed large, at fragments consisting essentially

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70.0

60.0 Quantity Retained (% w/w)

50.0

40.0

30.0

20.0

10.0

0.0 500m 250m 150m Sieve Fraction

Fig. 1. Distribution of particle sizes after milling and sieving BSG. After milling lyophilised BSG through 1 mm (E), 0.5 mm () and 0.25 mm (m) mesh screens, samples were separated by a vibratory sieve shaker for 1 h. Samples of the 0.25 mm 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.

106m

53m

of grain enveloped in owering 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 magnication, the dried, milled BSG sample showed that, even after milling, fragments of many sizes and tissue types are present (Fig. 2c). The use of autouorescence enhanced differentiation of the tissue types (Fig. 2d). Low magnication imaging of the sieve fractions showed that the rst three fractions (retained by 500, 250 and 150 mm sieves) comprised predominantly different sized fragments of lemma and palea, and attached material (Figs. 2eg). The last two fractions (retained by 106 and 53 mm sieves) comprised much ner, crumb-like material (Figs. 2hi). Using autouorescence microscopy, material derived from the lignin-rich glume appears blue, consistent with the presence of p-coumaric acid (see below). Material of aleurone origin autouoresces green, probably due to the presence of ferulic acid (see below). Both types of material were found to be present in the rst three fractions (Figs. 2jm). A closer examination of the third fraction (150 mm) showed signicant amounts of outer epidermis of the glume (Fig. 2n); bran layers, i.e. pericarp (green) and inner 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 of tissue types (Figs. 2ru). In addition to small fragments of those types already identied, a greater number of lipid containing fragments were seen (e.g. Figs. 2v), and also granular fragments of starch (Fig. 2wx). These stained reddish-blue with iodine and appeared slightly gelatinised

(Fig. 2). Other elements were visible in small numbers, e.g. brovascular bundles from the glume ribs (green) and thin walled bres (blue) (not shown). 3.3. Total chemical composition The initial BSG and each sieved fraction was analysed for protein, starch, cell-wall carbohydrate, lignin, phenolic acids and moisture content (there was insufcient material from the 500 mm fraction, preventing moisture determination). The initial material was also analysed for lipid content, but insufcient material was available to do this for the sieved fractions. The percentage total recoveries of these components are shown in Fig. 3. The results show that there was a segregation of components between the sieved fractions, resulting in a considerable enrichment in the proportion of protein and starch, and a concomitant decrease in cell-wall polymers, in the small particle size fractions (retained by the 106 and 53 mm sieves) as compared with the original milled BSG. Klason lignin remained relatively constant within all fractions. The presence of residual starch in the BSG was surprising as it is generally thought that most of the starch in the raw material had been removed and converted to fermentable sugars during the mashing process (Tang et al., 2005). The moisture level ($8%) does not vary greatly between the sieved fractions and is probably similar in the 500 mm fraction. However, the amount of material not accounted for by these analyses is clearly greater in the rst three fractions. This may be due to unquantied lipidic and cuticular components, and possibly due to the difculty in quantifying polysaccharide components that are cross-linked with lignin.

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Fig. 2. Light, bright eld and autouorescence micrographs of total and sieved fractions of BSG. Bars: (ab) bar 1 mm; (ei) bar 500 mm; (cd, jm, ru) bar 200 mm; (nq, vw) bar 100 mm; (x) bar 50 mm. (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 autouorescence, pH 10; (ei) light micrographs of milled BSG retained by 500, 250, 150, 106 and 53 mm sieves, respectively; (jm) autouorescence micrographs (pH 10) of mixtures of aleurone and glumes in 500, 250 and 150 mm fractions; (n) autouorescence micrograph (pH 10) of the outer epidermis of the glume; (o) autouorescence micrograph (pH 10) of the pericarp and inner layers of the glume; (p) autouorescence micrograph (pH 10) of the outer aleurone layer; (q) autouorescence micrograph (pH 10) of the lipidic material from the embryo; (ru) light and uorescence micrographs of mixtures of cell types from 106 and 53 mm fractions; (vx) light and autouorescence micrographs of lipidic components and granular starch (wx).

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100%

80% Total weight composition Not determined Water Lipid Phenolics Lignin Protein Carbohydr. (non-starch) Starch

60%

40%

20%

0% BG1 500m 250m 150m 106m 53m Sieve fraction

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 BSG 500 mm 250 mm 150 mm 106 mm 53 mm 0.20 0.24 0.29 0.21 0.30 0.22 Fuc 0.11 0.14 0.08 0.09 0.13 0.13 Ara 15.36 9.18 14.33 17.06 15.54 12.76 Xyl 29.84 47.03 41.06 32.87 20.42 14.44 Man 1.24 0.34 0.72 0.99 1.61 1.72 Gal 2.00 1.04 1.51 1.83 2.44 2.25 Glc (non-c.) 25.71 8.96 12.97 16.79 44.56 32.57 Glc (cel.) 12.60 19.57 17.53 14.69 5.33 20.52 UA 12.95 13.50 11.51 15.47 9.67 15.40 Total (m/mga) 444 488 450 472 419 516 Ara/Xyl ratio 0.515 0.195 0.349 0.519 0.76 0.883

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

3.4. Polysaccharide composition The sugar composition of the milled BSG and sieved fractions is shown in Table 1. Information on the linkages of the component sugars in all samples except for the 500 mm fraction are shown in Table 2. The non-starch glucose (i.e. hydrolysed by 1 M sulphuric acid) is accounted for principally by the starch component which was assayed diagnostically (Fig. 3; see Section 2.1). The small quantities of (13)-linked Glcp are probably derived from mixedlinkage glucans which are present in BSG in only minor quantities (Robertson, J.A., et al., unpublished results). The ratio of the (starch-derived) non-cellulose glucans to cellulose exhibits a considerable increase across the 500 to 106 mm fractions, and then decreases in the 53 mm fraction, reecting the partitioning of starch shown in Figs. 2 and 3. The high proportion of cellulosic glucose in the 500, 250 and 150 mm fractions, as inferred from the release of Glc by acid hydrolysis conditions (Table 1) is in keeping with the dominant palea and lemma fragments (Figs. 2eg). The majority of the glucose was (14)-linked with small quantities of 1,4-6-linked glucose consistent with the

presence of starch-derived amylopectin (Table 2). Interestingly, signicant quantities of terminal glucose was also present. The bulk of the xylose component comprised (14)linked xylose, indicative of xylan hemicellulose. Only trace quantities of (1,3-4)- and (1,2-4)-linked Xylp could be detected. However, the presence of terminal Araf, and the ratio of ara:xyl shown in Table 2 is indicative of arabinoxylan hemicelluloses from which one would expect to see larger quantities of branched xylose residues. It is probable that the arabinoxylans of the palea and lemma are difcult to evaluate by methylation analysis. This may reect their insolubility in the highly-lignied palea and lemma fragments, resulting in their removal from the analytical process during ltration. Such insolubility may well be enhanced through the cross-linking of ester-linked phenolics present on terminal arabinose branches. This may explain discrepancies between the relative levels of sugars in Table 1 compared with levels in Table 2, and the presence of small amounts of under-methylated xylose and glucose. The signicant levels of uronic acid probably derive from glucuronic acid as found in other cereal cell

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A.J. Jay et al. / Journal of Cereal Science 47 (2008) 357364 Table 2 Methylation analysis of milled and sieved BSG Sugar linkage residue Fraction BSG t-Araf 1,4-Arap/1,5-Araf t-Xylp 1,4-Xylp (+ t-Galp) 1,3,4-Xylp (+ 1,2,4-Xylp) 1,2,3,4-Xylp t-Glcp 1,3-Glcp 1,4-Glcp 1,4,6-Glcp 2,3,4,6-Glcp 1,4-Manp 1,6-Galp 1.34 1.1 0.64 6.33 T 1.5 9.47 1.48 66.69 5.39 2.35 1.46 2.23 250 mm 1.64 1.15 0.9 9.75 3.42 1.83 6.02 0.82 64.64 3.53 3.83 0.69 1.78 150 mm 1.47 1.31 0.84 9.97 2.39 1.56 7.3 1.13 66.2 4.29 1.53 0.88 1.12 106 mm 2.08 0.69 0.69 4.94 t 1.27 8.61 1.33 74.24 4.15 0.27 1.13 0.6 53 mm 2.62 0.79 0.79 5.21 t 1.18 9.25 0.94 73.46 3.64 0.29 1.18 0.65 363

Values are expressed as mol%. n.d. signies linkage not detected. Ara, arabinose; Gal, galactose; Glc, glucose; Man, mannose; Xyl, xylose.

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 Vanillin trans-p-Coumaric acid 8,80 :6,70 -Diferulic acid (AT) trans-Ferulic acid cis-p-Coumaric acid 8,50 -Diferulic acid cis-Ferulic acid 5,50 -Diferulic acid 8-0-40 -Diferulic acid 8,50 :7-O-40 -Diferulic acid (BF) n.d. 7.58 n.d. 1.16 27.72 35.41 n.d. 28.11 51.00 15.70 4.52 3.17 2.21 0.44 6.16 2.03 2.03 1.44 6.36 2.63 n.d. 2.32 0.19 0.83 40.17 n.d. 38.91 6.44 1.81 4.90 1.49 5.26 n.d. n.d. n.d. 22.22 n.d. 52.19 5.50 2.54 7.72 1.89 7.94 n.d. n.d. n.d. 15.98 n.d. 55.63 4.26 3.49 8.98 3.16 8.50 n.d. n.d. n.d. 14.58 n.d. 56.72 3.83 4.36 9.44 3.43 7.64 n.d.

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

walls (Brett and Waldron, 1996; Waldron and Faulds, 2007). The amount of pectin-derived galacturonic acid is likely to be low in the lignied tissues, but may be signicant in the growing embryonic tissues, components of which are present in the small particle-size fractions. Signicant differences were observed in the phenolic compositions of the fractions (Table 3). The proportion of trans-p-coumaric acid decreased with particle size, and is probably associated with lignied palea and lemma tissues which are enriched in the larger particle-sized fractions. Interestingly, the trans-ferulic acid component shows an opposite trend to that of trans-p-coumaric acid, and is highest in the smaller particle-sized fractions. This indicates that it is associated with poorly lignied cell walls including the remnants of endosperm and growing embryonic tissues where, as indicated by the similar trends of the 8-0-40 -, 5,50 and 8,50 -diferulic acids, it is involved in interpolymeric cross-linking, probably of arabinoxylan hemicelluloses.

However, p-hydroxybenzaldehyde, vanillin and the two polycyclic diferulic acids, 8-80 :6,70 (aryltetralin) diferulate and 8,50 :8-O-40 (benzofuran) diferulate, are seen only in the rst and second fractions; these have been associated with the outermost, highly lignied layers of BSG, and may be breakdown 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 the extraction of feruloylated arabinoxylan-rich extracts. The use of alkali caused the removal of most of the ferulate and diferulate moieties from this arabinoxylan and no other polymer was isolated. The hydrothermal treatment of BSG resulted in the degradation of certain sugars, mainly arabinose, with a conversion of this sugar to furfural, formic and levulinic acid (Kabel et al., 2002). By the combined milling and vibratory sieving method described in this paper, together with economic drying regimes, such as thin layer drying in superheated steam (Tang et al., 2005), enriched fractions of phenolic acid-containing arabinoxylan or protein/starch can be produced. This would allow a cleaner and much wider utilisation of all the spent grain components for a number of potential food and non-food industrial applications, such as prebiotics (Fooks et al., 1999), plant-based protein source for animal feed, emulsifying and thickening agents, pulp paper substitutes (Ishiwaki et al., 2000), or direct acid hydrolysis to produce pentose sugars for microbial fermentations (Carvalheiro et al., 2004). In summary, this investigation demonstrates that the components of BSG can be fractionated effectively by simple milling and sieving. The fractionation involves separation of palea and lemma lignied glumes from the more protein- and starch-rich embryonic tissues. It is possible that with further physical treatments, starch-rich endosperm that is attached to the lignied tissues could also be separated and recovered. Further characterisation of the starch and non-carbohydrate polymers will lead to further information on the properties and exploitation of value-added components in BSG. Acknowledgements This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), UK, and the Department of the Environment, Food and Rural Affairs (DEFRA), UK, through a BridgeLINK grant (AFM201). We would like to express our gratitude to Scottish Courage Limited, Edinburgh, UK, for supplying the brewers spent grain. References
Albersheim, P.D., Nevins, D.J., English, P.D., Karr, A., 1967. A method for the analysis of sugars on plant cell-wall polysaccharides by gasliquid chromatography. Carbohydrate Research 5, 340345.

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