Journal of the Science of Food and Agriculture

J Sci Food Agric 79 :396402 (1999)

Review Ferulic acid and diferulic acids as components of sugar-beet pectins and maize bran
heteroxylans
Luc Saulnier* and Jean-Francois Thibault
Unite de Recherche s ur les Polys accharides , leurs Organis ations et leurs Interactions , Ins titut National de la Recherche Agronomique , BP 71627 -44316 Nantes Cedex 03 , France

Abstract : Enzymatic hydrolysis of sugar-beet pulp, and subsequent isolation of feruloylated oligosaccharides, has shown that ferulic acid groups are ester-linked mainly on O-2 of arabinose residues and on O-6 of galactose residues in the pectin side-chains. After saponication of sugar-beet pulp enzymatic digests, dehydrodiferulic acids (0.14% , w/w) have also been identied and characterised as 8-5, 5-5, 8-8 and 8-O-4 isomers, suggesting that covalent cross-linking of pectic polysaccharides through diferulic bridges occurs in sugar-beet pulp. Feruloylated oligosaccharides from the side-chains of heteroxylans have been isolated from maize bran by acid hydrolysis. Ferulic acid is esteried on O-5 of arabinofuranose residues. 8-8, 8-5, 8-O-4 and 5-5 coupled dimers, which represent 2.5% (w/w) of the bran, have also been detected. It has been calculated that, in the cell wall, each heteroxylan macromolecule bore 75 esteried ferulic acid groups and could be cross-linked through 30 diferulic bridges. This result suggests a high degree of cross-linking of heteroxylans chains through ferulic acid in maize bran cell walls. ( 1999 Society of Chemical Industry

Keywords : sugar-beet pulp ; maize bran ; pectins ; heteroxylans ; ferulic acid ; diferulic acids ; dehydrodiferulic acids

INTRODUCTION It is now well established that many plant cell walls contain phenolic acids that are ester-linked to polysaccharides.1 In all tissues of monocots, such as cereals and grasses, p-coumaric acid and ferulic acid are associated with heteroxylans. In dicots, phenolic acids are generally not components of the cell walls, except in a few plants, such as the Chenopodiaceae family. Indeed, ferulic acid was found ester-linked to pectic polysaccharides in spinach,2 sugar-beet,3 glasswort4 or quinoa.5 These phenolic acids are very important components for the biology of the cell wall, as well as for its structure, because they can potentially cross-link polysaccharide chains through a dimerisation reaction.1,2 Two mechanisms have been reported. One is the cycloaddition between the ethylenic carbons of two phenolic acids which is catalysed by UV light and leads to the formation of the so-called truxillic and truxinic acids.6 In vitro and in vivo, this mecha Bas ed on a paper pres ented at Ferulate 98, IFR, Norwich, 811 July 1998. * Corres pondence to : Luc Saulnier, Unite de Recherche s ur les Polys accharides , leurs Organis ations et leurs

nism occurs preferentially with p-coumaric acid. The other mechanism is an oxidative coupling reaction which can be catalysed by various systems, such as peroxidase/hydrogen peroxide,7h9 polyphenol oxidase, including laccases,9,10 as well as some purely chemical systems.11 In these reactions, the phenoxyradicals may react together to form various dehydrodimer isomers : 5-5@, 8-O-4@, 8-5@ and 8-8@ (Fig 1).8 In model systems using cell walls12 or feruloylated arabinoxylans9 the 8-5@ derivative is preferentially formed, although other dimers are also observed. The present paper will be focused on the results we have recently obtained on two substrates of economic interest, namely maize bran and sugar-beet pulp. They are quite dierent as sugar-beet pulp is constituted of primary walls rich in pectins, whereas maize bran contains secondary walls rich in heteroxylans. However, neither tissue is lignied and ferulic acid is the main phenolic acid ester-linked to

Interactions , Ins titut National de la Recherche Agronomique, BP 71627-44316 Nantes Cedex 03, France (Received 12 Augus t 1998 ; revis ed vers ion received 7 September 1998 ; accepted 15 October 1998 )

( 1999 Society of Chemical Industry. J Sci Food Agric 0022-5142/99/$17.50

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Feruloylated pectins and arabinoxylans

Figure 1. Structure of the different dehydrodimers of ferulic acid.

wall polysaccharides. We will describe the isolation of feruloylated oligosaccharides from sugar-beet pulp and maize bran, as well as dimers of ferulic acid, and we will discuss the possible role of ferulic acid in the cell wall organisation. In order to demonstrate that polysaccharides and phenolic acids are linked together by ester bonds, ferulic or diferulic acids have to be obtained with the ester bond to polysaccharide fragments still intact. For this purpose, cell wall materials have been submitted to enzymic degradation or to controlled acid hydrolysis. The mixtures of oligomers have been separated by various chromatographies and the structure of the pure oligomers determined by NMR and methylation analysis. Furthermore, the amounts of ester-linked ferulic and diferulic acids were quantied by GC or HPLC after their release from the cell wall by an alkaline treatment. Sugar-beet pulp Sugar-beet pulp is the main by-product of the sugar renery industry. It is rich in cellulose (D20% of the pulp) but the main polysaccharide is pectin : galacturonic acid, rhamnose, arabinose, galactose, and also methanol and acetic acid, accounting for approximately 50% of the pulp. Phenolic acids represent approximately 1%, ferulic acid being the major one (Table 1).13,14 Location of ferulic acid on the pectins It is already known that ferulic acid is linked to pectins as it is possible to extract ferulic acid containing pectins from sugar-beet pulp and to make gels with these pectins in oxidising conditions.7 The
J Sci Food Agric 79 :396402 (1999)

exact location of ferulic acid has been studied. Pectins are complex molecules comprising smooth regions (or homogalacturonans) composed of a-(1,4) linked galacturonic acid residues partially methylesteried on the carboxyl groups and, in the beet, also acetylesteried on O-2 and/or O-3. These smooth regions are interspersed with hairy regions composed of rhamnogalacturonans bearing arabinan and/or (arabino)galactan side-chains linked to rhamnose residues.15 In order to know the location of ferulic acid, the smooth regions of the pectins have been rst submitted to degradation by pectolytic enzymes. Ferulic

Table 1. Compos ition of the s ugar-beet pulp and maize bran (% w/w of material)

Sugar -beet pulp a

Galacturonic acid Glucuronic acid Rhamnos e Arabinos e Galactos e Xylos e Glucos e Methanol Acetic acid Phenolic acids Proteins (N ] 6.25) Lignin As h 21.1 2.4 21.0 5.1 1.7 21.1 1.8 3.9 1.0 11.3 \1.0 3.6

Maize bran b
0.0 4.2 0.0 22.8 5.6 34.3 22.5 0.0 4.2 5.5 2.4 1.0 0.5

a Values from Ref 13. b Des tarched bran values from Ref 14.

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acid was not found in the low molecular weight fraction, indicating that it was linked to the hairy regions and not to the smooth regions.16 Therefore, enzymes were used to degrade the hairy regions.17,18 The small oligomers containing ferulic acid were extracted by ethanol and fractionated on a DEAE-Sephacel column. The ferulic acid residues were found in the neutral fractions, indicating that they were not linked to the galacturonic acid residues. The neutral fraction was then chromatographed on Sephadex LH20 column (eluted by water). Fractions devoid of ferulic acid eluted in the fractionation range of the column, in contrast to fractions containing both ferulic acid and neutral sugars, which eluted after the total volume of the column. After refractionation on the same column or on Biogel P2 column, pure oligomers containing arabinose or galactose residues, and ferulic acid were obtained. They were comprised of a ferulic acid linked to a dimer of arabinose, to a trimer or arabinose, and to a dimer of galactose. Their structure (Fig 2) was established by NMR.18,19 Ishii and Tobita,20 using a commercial enzyme preparation (Driselase), have isolated similar feruloylated oligosaccharides from spinach-leaf cell walls. Structural and compositional analysis of the feruloylated arabinose oligomers derived from arabinan side-chains of pectins indicated that ferulic acid was esteried to O-2 of arabinofuranose residues which were part of a-(1,5) linked arabinan chains.21,22 This main chain is also substituted on O-3 by arabinofuranose residues, indicating that the feruloyl groups are linked to the main core of arabinan and not to the arabinose residues substituting this backbone. For the feruloylated galactose oligomers

derived from galactan side-chains of pectins, ferulic acid is linked to O-6 of galactopyranose residue linked to another galactose by a b-(1,4) linkage. This feature suggests that the feruloyl groups are linked to the main core of the b-(1,4)-linked type I galactan.21,22 Figure 3 gives an overview of the location of ferulic acid in sugar-beet pectin. It was calculated that approximately 50% of the feruloyl groups in the beet pectins are linked to arabinose residues, and 50% to galactose residues. Dehydrodiferulic acids The presence of dehydrodiferulic acids, but not cyclodimers, has been shown in sugar-beet pulp or in enzymic digests of sugar-beet pulp.23,24 Dimers were identied and quantied by GC and GC-MS8 after alkaline deesterication of cell wall material or enzymic digests of sugar-beet pulp. The four main dehydrodimers were 5-5@, 8-O-4@, 8-5@ and 8-8@ dehydrodiferulic acids, the 8-5@ derivative being the most abundant (Table 2). The sum of the dehydrodimers represented [0.1% of the wall. It can be concluded that the ferulic acids may be coupled into a variety of dehydrodimers suggesting that cross-linking of arabinans and/or arabinogalactans side-chains may occur in the cell wall. Upon cross-linking of pectins with a mixture of hydrogen peroxide/peroxidase,24 the concentration of the dimers increased, particularly the 8-5@ and the 8-O-4@ isomers. A model for the sugar-beet cell wall A schematic model of the structure of cell walls in sugar-beet pulp is proposed in Fig 3. Only cellulose microbrils and pectins are represented, the pres-

Figure 2. Structure of the main feruloylated oligos accharides derived from s ugar-beet pectins and maize bran heteroxylans (F, ferulic acid ; A, arabinos e ; X, xylos e ; G, galactos e).

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Feruloylated pectins and arabinoxylans

Figure 3. Model for the s ugar-beet cell walls .

Table 2. Amount of the main phenolic acids in s ugar-beet pulp and maize bran (% w/w of the cell wall)

Sugar -beet pulp a p -Coumaric acid Ferulic acid Dehydrodiferulic acids 5-5@ dimerb 8-O-4@ dimerb 8-5@ dimerb 8-8@ dimerb
0.80 0.14 9 29 51 11

Maize bran
0.3 2.9 2.5 23 35 31 11

ence of hemicelluloses (especially xyloglucans) is still an open question in these cell walls. Besides ionic interactions through calcium bridges,25 it is likely that covalent linkages involving dehydroferulic acids exist in pectins. The resulting network may embed the microbrils of cellulose. A positive role for dehydroferulic acids in the texture and thermal stability of beet tissues has been proposed.26 MAIZE BRAN Maize bran, one of the by-products of the maize industry, is comprised mainly of pericarp tissues of secondary cell walls. It contains cellulose (D22% of

a Values are from Ref 23. b Values are expres s ed as % of total dimers .

Figure 4. Schematic s tructure of maize bran heteroxylan.

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the bran) but the major polysaccharide is a heteroxylan composed primarily of arabinose and xylose with minor amounts of galactose and glucuronic acid. It represents more than 50% of the wall. A relatively large amount of phenolic acids (D5%), mainly ferulic acid, is present. The bran also contains some proteins and traces of lignin (Table 1). Location of ferulic acid on the heteroxylans The backbone of heteroxylans is composed of b-(1,4) -linked xylose residues. This backbone is highly substituted with monomeric side-chains of arabinofuranose or glucuronic acid linked to O-2 and/or O-3 of xylose residues, and also by oligomeric sidechains containing arabinose, xylose and sometimes galactose residues (Fig 4).14,27 About 80% of the xylose residues in the backbone are substituted by side-chains. This is rather high compared to heteroxylans from other sources.28 The native heteroxylan is markedly insoluble in the cell wall and only alkaline treatment can extract signicant amounts of the polymer. Typically the use of 1 M KOH at 95C allows almost complete extraction (92%) of the heteroxylan.27 In these conditions, however, all the ester linkages, including the ferulic esters, are broken.14 In order to solubilise feruloylated heteroxylan fragments, other conditions that preserve ester linkages were investigated. Various enzyme mixtures were tested but none were able to solubilise signicant amounts of heteroxylan fragments. Therefore, controlled mild acid hydrolysis (50 mM triuoroacetic acid, 100C, 2 h) was used in order to split glycosidic linkages while leaving ester linkages, notably those involving phenolic acids, intact.29 After acid hydrolysis, Amberlite XAD-2, which is a polymeric adsorbent binding aromatic compounds, was used for purication of the extract. The column was rst eluted with water in order to release sugar rich material not adsorbed on the column, while a mixture of methanol and water (1/1, v/v) eluted a material enriched in ferulic acid and still containing sugars. This material was submitted to chromatography on Sephadex LH20 and was eluted by a mixture of methanol and water (1/3, v/v). Fractions containing various ratios of neutral sugars to ferulic acid were separated. Three main fractions were isolated, corresponding to a feruloylated arabinose, a feruloylated disaccharide containing arabinose and xylose, and a feruloylated trisaccharide containing arabinose, xylose and galactose.29 These oligomers were isolated and submitted to structural analysis by methylation and NMR. The oligomers have the same basic unit composed of one arabinofuranose residue esteried on position O-5 by ferulic acid. This type of structure has been found in all feruloylated oligomers isolated from monocots.1 From this unit, the other oligomers were built by adding one xylose residue on position 2 of the arabinose, and one galactose residue on O-4 of
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the xylose residue (Fig 2). It can be observed that the oligosaccharide moieties of these oligomers have the same structure as the side-chains of the xylan backbone (Fig 4). This can be explained by the fact that the acid hydrolysis has preferentially split the (weak) glycosidic bond between arabinofuranose in the sidechain and the xylan backbone. If we assume that all ferulic acid is esteried to arabinose residues in the bran, it is calculated that each heteroxylan (mean degree of polymerisation D2000)30 bears 75 ferulic acid esters. Dehydrodiferulic acids The high amount of ferulic acid linked to heteroxylan suggested that maize bran might contain a high level of phenolic dimers. The dehydrodimers were quantied by HPLC and GC after alkaline treatment of the bran.8 Both methods gave similar results. Ferulic acid was the main constituent (2.9%) but p-coumaric acid was also detected. The bran contains high amounts (2.5%) of the dierent dehydrodiferulic acids, but no cyclodimers. The balance between the dierent isomers was dierent from that found in sugar-beet pulp, 5-5@, 8-O-4@ and 8-5@ being in fairly similar amounts, with a minor proportion of 8-8@. If all these dimers cross-link heteroxylan chains, each heteroxylan molecule should be crosslinked, on average, through 30 diferulic bridges. A model for the maize bran cell wall Figure 5 shows possible associations of polysaccharides in maize bran cell walls. The heteroxylans which are probably highly cross-linked through diferulic bridges, constitute a network in which the cellulosic microbrils may be imbedded. However, these diferulic molecules cannot be the unique bridges insolubilising the heteroxylans in the cell wall, since it is possible to release all ferulic acid and its dimers from cell walls in conditions which solubilise only a minor part of the heteroxylans.14 A general assumption is that hemicelluloses are closely associated to cellulose microbrills through hydro-

Figure 5. Model for the maize bran cell walls .

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Feruloylated pectins and arabinoxylans

gen bonding, but these linkages are not present for maize bran heteroxylans, due to the highly branched character of the macromolecule which prevents noncovalent associations.14 Therefore other linkages probably occur in the cell wall. Structural wall proteins, which are present in maize pericarp,31 might be cross-linked together by isodityrosine bridges and with feruloylated heteroxylans, thus forming an insoluble network. However, the nature of the linkage between heteroxylans and proteins has still to be determined. CONCLUSIONS Ferulic acids may participate to the structure of nonlignied cell walls. It is a unique bifunctional element which can cross-link heteroxylans in cereal tissues and pectins in beet root tissues. By this way, mechanical properties of the walls may be altered26,32 as well as the resistance to degradation by exogenous enzymes. However, isolation of dehydrodiferulic acids linked to neutral sugars has to be carried out in order to strengthen these hypotheses.33,34 The maize bran cell walls are very resistant to enzymatic degradation probably due to the highly branched character of the heteroxylan, but also to the high level of cross-linkings through diferulic bridges as well as the high level of ferulic acid esteried to arabinoxylan which probably restricts enzyme accessibility.35 The pectins in sugar-beet pulp are easily degraded by enzymes possibly due to their much lower contents in ferulic and diferulic acids.36 However, it has been shown that cross-linking of the pectins may result in an altered texture.26 Knowledge of cell wall structure is also very important to nd valorisation of these by-products rich in ferulic acid. Gels with feruloylated polysaccharides from beet or from maize could be obtained and these gels may lead to interesting applications.7,37,38 On the other hand, ferulic acid may be extracted by chemicals or by enzymes, and used as antioxidant39 or as a precursor for many useful aromatic chemicals,40 including vanillin.41 REFERENCES
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