MOLECULAR STRUCTURE OF ARABINOXYLANS

7.4 MOLECULAR STRUCTURE OF ARABINOXYLANS

7.4.1 M ONOSACCHARIDE R ESIDUES AND G LYCOSIDIC L INKAGES IN A RABINOXYLAN S TRUCTURES

Arabinoxylans consist of linear (1 →4)-β-D-xylopyranosyl chains to which α-L- arabinofuranosyl residues are attached as side branches. Arabinose residues can be attached to xylose units at the O-2, O-3, or both O-2,3 positions, resulting in four structural elements in the molecular structure of arabinoxylans: monosubstituted Xylp at O-2 or O-3, disubstituted Xylp at O-2,3, and unsubstituted Xylp (Figure 7.4). The relative amount and the sequence of distribution of these structural elements vary depending on the source of arabinoxylans. The majority of arabinofuranosyl residues in arabinoxylans are present as monomeric substituents; however, a small proportion of oligomeric side chains consisting of two or more arabinosyl residues

linked via 1 →2, 1→3, and 1→5 linkages have been reported. 7 The molecular structure of arabinoxylans from rice, sorghum, finger millet, and maize bran is more

complex than that from wheat, rye, and barley, since the side branches contain, in addition to arabinose residues, small amounts of xylopyranose, galactopyranose, and α-D-glucuronic acid or 4-O-methyl-α-D-glucuronic residues (Figure 7.5). 45–48 Glu- curonopyranosyl residues constitute about 4% of the arabinoxylans from barley

husk 49 and are also present in arabinoxylans from wheat bran. 50

A recent investigation of the molecular structure of water-soluble wheat endosperm arabinoxylans using atomic force microscopy has confirmed the gener-

ally linear structure of arabinoxylans as described above. However, it also revealed that a small fraction (~15%) of the polymers might, in fact, be branched. 51 It was reported that the branches were composed of β-(1→4)-linked xylose residues, and they appeared to be randomly located along the chain. The likelihood of their presence increased with the increasing length of the molecules. Only about 1% of the branched chains contained more than one branch.

The physiologically active gel-forming polysaccharides of psyllium husk (Plan- tago ovata Forsk) have recently been found to consist of neutral arabinoxylans,

Functional Food Carbohydrates

Xylp Xylp

Araf Araf (a)

(b)

Xylp Xylp

HO Araf

Araf

(c) (d) FIGURE 7.4 Structural elements present in arabinoxylans: (a) monosubstituted Xylp at O-

3, (b) monosubstituted Xylp at O-2, (c) disubstituted Xylp at O-2,3, (d) unsubstituted Xylp.

β(1 4) linkage Xyl

β(1 4) linkage Ara

Gal

Xyl

-Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl -Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - Xyl - GlcA GlcA

OO 9 7 8 Ester linkage 1 6 2

FIGURE 7.5 General structure of arabinoxylans. containing mainly arabinose (22.6%) and xylose (74.6%) residues, with only traces

of other sugars. However, their structure is significantly different from that of arabinoxylans in common cereals. Psyllium arabinoxylans, despite their low Ara/Xyl

ratio (0.30), are highly branched polymers with the main chain of densely substituted

Arabinoxylans

(1 →4)-linked xylopyranosyl residues, some carrying single xylose units at position O-2 and others bearing trisaccharide branches (Araf- α-(1→3)-Xylp-β-(1→3)-Araf)

at position O-3. 8 This unique molecular structure is associated with some unusual physicochemical and physiological properties of psyllium arabinoxylans, such as strong gelling potential and low fermentability by the intestinal microflora.

7.4.2 F ERULIC A CID R ESIDUES AND I NTERMOLECULAR C ROSS -L INKING

An unusual feature of the structure of arabinoxylans is the presence of ferulic acid residues covalently linked via an ester linkage to O-5 of the arabinose residue (Figure 7.5). Digestion of wheat aleurone cell walls and detailed analysis of the hydrolyzates led to identification of two feruloylated arabinoxylosides: (5-O-feruloyl-α-L-ara- binofuranosyl)-(1 →3)-β-D-xylopyranosyl-(1→4)-β-D-xylanopyranose (FAXX) and β-D-xylanopyranosyl-(1→4)-(5-O-feruloyl-α-L-arabinofuranosyl)-(1→3)-β-D-

xylopyranosyl-(1 →4)-β-D-xylanopyranose (XFAXX). 52 Ferulic acid is usually reported in its trans-isomeric form; however, the exposure of grain to UV light at the later stages of development may cause trans to cis isomerization, resulting in up to 30 to 40% of cis-ferulic acid, especially in arabinoxylans originating from the

outer grain tissues. Smith and Hartley 53 estimated that the cell walls of wheat bran contained approximately 34.0 μmol of feruloyl groups per gram of walls, whereas the endosperm cell walls contained 5.6

μmol/g of walls. Rattan et al. 54 reported that arabinoxylans from flours of several Canadian wheat varieties contained 0.63 to 1.37

mg of ferulic acid per gram of isolated and purified polymers. Dervilly-Pinel et al. 55 compared the contents of ferulic acid in arabinoxylans isolated from the endosperm

of wheat, barley, rye, and triticale. It was reported that the amounts ranged from 18 to 60 ferulic acid residues per 10,000 xylose residues, and that purified arabinoxylans

from wheat and barley contained more ferulic acid than those from rye and triticale.

A greater concentration of ferulic acid was found in the wheat aleurone preparations (0.71%) than in the pericarp preparations (0.31%). 36,56 Ferulic acid residues can act as cross-linking agents between polysaccharides or between polysaccharides and lignin. The cross-linking is effected by ferulate dimerization by either photochemical or, more importantly, free radical coupling reactions of ferulate–polysaccharide esters. Ferulate esters dimerize via phenoxy radicals to form dehydrodiferulate esters. Three electron-delocalized phenoxy rad- icals can be induced at position 4-O, C-5, or C-8 of ferulic acid residue, giving rise to at least five known diferulate esters coupled via 8-5', 8-O-4', 5-5', 8-8', and 4-O-

5' linkages (Figure 7.6). Bunzel and coworkers 57 have recently isolated and measured the amount of different diferulates in cereals. The distribution patterns of various diferulates in water-insoluble arabinoxylans were similar among the different cere- als. The 8-5'-coupled diferulate predominated, followed by the 8-O-4'-linked dimer. The diferulate distribution patterns in water-soluble arabinoxylans in various cereals were very different from those in water-insoluble fiber. The amount of 8-8' dimers increased substantially, whereas the amounts of 5-5' and 8-O-4' dimers decreased. It has to be pointed out that the identification of dehydrodiferulic acids released upon saponification of plant material does not provide sufficient evidence of

Functional Food Carbohydrates

OO Polysaccharide

8-8'-DFA

8-5'-DFA

5-5'-DFA

8-O-4'-DFA 4-O-5'-DFA

FIGURE 7.6 Three electron-delocalized phenoxy radicals generated from ferulic acid (FA) by a free radical-generating system (peroxidase–H 2 O 2 ) and molecular structure of five known dehydrodiferulic acids.

polysaccharide cross-linking, since these diferulate bridges can theoretically be formed intramolecularly. Evidence for polysaccharide cross-linking via dehy- drodiferulates was provided, however, by isolation and identification of feruloylated

saccharide fragments. The isolation of 5-5'-diferuloyl saccharides 58 and diarabinosyl ester of 8-O-4'-dehydrodiferulate 59 provided more satisfactory evidence for inter- molecular cross-linking, although the molecular modeling experiments did not exclude the possibility that 5-5'-diferulate may be tethered to the same arabinoxylan chains as long as the arabinose residues bearing ferulate units are at least three

xylose residues apart. 60 Fry et al. 61 speculated that free radical polymerization of ferulates does not stop at the dimer stage in vivo, but proceeds to form higher oligomers. Indeed, in 2003 the first 4-0-8'/5-'5''-coupled dehydrotriferulic acid was

8-O-4′/8′-O-4"dehydro-triferulic acid 8-8′/4′-O-8"dehydro-triferulic acid

4-O-8′/5′-5"dehydro-triferulic acid 5-3′-dehydro-ferulic acid-tyrosine

4-O-3′-dehydro-ferulic acid-tyrosine 5-O-4′-dehydro-ferulic acid-tyrosine

FIGURE 7.7 Structures of new ferulic acid dehydrotrimers and speculative structures of dehydroferulic acid–tyrosine heterodimers.

isolated from maize (Figure 7.7). 62,63 It was suggested, however, that this trimer originates from initial intramolecular 5-5' linkage, further coupled with ferulate on another chain, rather than from the interaction of three ferulates on three separate chains. 62,64 With the isolation and identification of another two trimers coupled by 8-O-4'/8'-O-4'' and 8-8'/4'-O-8'' linkages, it became clear that higher oligomers of ferulate may play a significant role in cross-linking of cell wall polysaccharides (Figure 7.7). 65

Cross-linking of cell wall polymers via diferulate bridges is of importance to plant physiologists, food chemists, and technologists. Thermal stability of cell adhesion and

Functional Food Carbohydrates

maintenance of crispness of plant-based foods (e.g., water chestnut after cooking), 66 gelling properties of cereal arabinoxylans and sugar beet pectins, 67 insolubility of cereal dietary fibers, 57 and limited cell wall degradability by ruminants 68 are related to the formation of ferulate cross-links. Bunzel et al. 57 estimated the degree of cross-linking in arabinoxylans from the ratio of diferulic acid to xylose residues and proposed that the diferulate bridges are partly responsible for the insolubility of these polymers. It was found that the degree of cross-linking in insoluble arabinoxylans was 8 to 39 times higher than in their water-soluble counterparts. Theoretically, in the presence of proteins, ferulic acid residues could be linked to the N-terminal of the protein amino

group or to tyrosine. 69 Recent studies by Piber and Koehler 70 provided evidence for a covalent linkage between arabinoxylans and proteins. Dehydroferulic acid–tyrosine dimers were isolated from wheat and rye dough preparations and identified based on mass spectrometric data (Figure 7.7).

7.4.3 S TRUCTURAL H ETEROGENEITY AND P OLYDISPERSITY OF A RABINOXYLANS

Arabinoxylans from various cereals and different plant tissues share the same general molecular structures; however, they differ drastically in fine structural features,

which may affect their physicochemical properties. These differences are reflected in the degree of polymerization, in the ratio of arabinose to xylose residues, in the

relative proportions and sequence of various glycosidic linkages, in the pattern of substitution of the xylan backbone with arabinose residues, and in the presence and amount of other substituents, such as feruloyl groups or glucuronic acid residues. Since arabinoxylans are not under strict genetic control, even polymers isolated from

a single plant or tissue exhibit structural microheterogeneity. To obtain a better insight into the structural characteristics of these polymers, arabinoxylans have been fractionated into more homogeneous populations by chromatographic or chemical means. Perhaps the most successful fractionation practices utilize a stepwise pre- cipitation with alcohol or ammonium sulfate. The fractionation is based on differ- ential solubilities of arabinoxylans with different molecular weights and structures in solutions containing various amounts of ethanol or ammonium sulfate. Arabinox- ylans precipitating at increasing concentrations of ethanol or ammonium sulfate exhibit an increasing ratio of Ara/Xyl but decreasing weight-average molecular weight (Figure 7.8).

The ratio of Ara/Xyl residues indicates a degree of branching in these polysac- charides. Depending on the origin of arabinoxylans, the ratio of Ara/Xyl may vary from 0.3 to 1.1, although some minor fractions with the Ara/Xyl ratio outside this common range have also been reported (e.g., corncob arabinoxylans with Ara/Xyl

= 0.07, 71 rye bran arabinoxylans with Ara/Xyl = 0.14, 72 water-soluble wheat arabi- noxylans with Ara/Xyl = 1.28, 73 water-soluble rye arabinoxylans with Ara/Xyl =

1.42 74 ). The ratio of Ara/Xyl in arabinoxylans from wheat endosperm generally varies from 0.50 to 0.71, and it is usually slightly lower than in arabinoxylans from wheat bran (0.82 to 1.07). 23,54,75–77 Antoine and coworkers 56 reported Ara/Xyl ratios of 0.33, 0.37, and 1.13 for arabinoxylans in the walls of aleurone, intermediate, and pericarp fractions, respectively. A study of the water-soluble and -insoluble arabi-

A/X = 0.50 esp

A/X = 0.80 F80

Mw = 360,000

A/X = 0.88 F100

Mw = 290,000

A/X = 0.91 8 12 16

Mw = 220,000

Elution volume (ml)

FIGURE 7.8 High performance size exclusion chromatography (HPSEC) profiles and molec- ular weight of arabinoxylan fractions obtained by sequential precipitation of arabinoxylan

solution with increasing saturation level of ammonium sulfate. F55, F70, F80, F100, fractions obtained with 55, 70, 80, and 100% saturation of ammonium sulfate; Mw, weight-average molecular weight of arabinoxylan fractions determined with multiangle light-scattering detec- tor after elution from the size exclusion column.

noxylans from wheat endosperm found a lot of similarities between these two types of polymers, 78 but significant differences between the structures of water- and alkali- extractable arabinoxylans from bran were also found. 77 The water-extractable ara- binoxylans from wheat bran were found to have a lower degree of substitution (Ara/Xyl = 0.45) and lower molecular weights (20- and 5-kDa fractions), compared to their alkali-extractable counterparts (Ara/Xyl = 0.82; 100- to 120-kDa and 5- to 10-kDa fractions). In rye, the ratio of Ara/Xyl falls between 0.48 and 0.78, 9,79 and

a distinction can be made between the structures of arabinoxylans from the rye starchy endosperm and those from the bran. The ratio of Ara/Xyl indicates the degree of branching in arabinoxylans; how- ever, it does not reveal detailed structural features of these polymers. The relative amounts of unsubstituted, monosubstituted (at O-3 or O-2), and doubly substituted xylose residues, as well as the sequences of these four structural elements, are better indicators of the molecular structures of cereal arabinoxylans. However, the relative proportions of the four structural elements in arabinoxylan chains may be related to the Ara/Xyl ratio, and some trends have been reported. For example, in water- extractable rye and wheat arabinoxylans, a higher Ara/Xyl ratio was associated with

a higher content of 2-monosubstituted and disubstituted xylose residues, and a lower content of 3-monosubstituted and unsubstituted xylose residues. 9,78,80 Compared to rye, water-extractable arabinoxylans from wheat exhibit a higher proportion of unsubstituted xylose residues (50 to 80% vs. 22 to 54%), a somewhat lower pro- portion of monosubstituted xylose residues (<20% vs. up to 40%), and a higher proportion of arabinose residues in 2-, 3-, or 5-linked short chains. 78,81

Functional Food Carbohydrates

The barley grain water-soluble arabinoxylans generally contain 47 to 65% unsub- stituted, 20 to 25% monosubstituted, and 19 to 26% disubstituted xylose residues. 82–84 In barley, more arabinoxylans are found in the aleurone than in the endosperm cell walls. Recent studies have identified significant differences in the molecular structure between arabinoxylans isolated from the pearling by-products (PBPs) (enriched in the aleurone layer) and fiber-rich fractions (FRFs) (enriched in the endosperm cell

walls) of hull-less barley samples with various starch types. 85 In general, the water- extractable arabinoxylans from PBPs were more substituted than those from the FRFs. This was observed for all barley types and evidenced by a higher Ara/Xyl ratio and a lower content of unsubstituted Xylp residues in PBPs than in FRFs. The mode of substitution varied, however, depending on the barley type and tissue (Figure 7.9). While the water-extractable arabinoxylans from PBPs of high-amylose and normal barley had almost the same amount of mono- and disubstituted Xylp residues, those from waxy barley had twice as many singly as doubly substituted xylose

High amylose

2-Xyl 3-Xyl

Normal 2,3-Xy l U-Xyl

Waxy

WE-AX PBP WE-AX FRF FIGURE 7.9 Substitution patterns in water-extractable (WE) arabinoxylans from pearling

by-products (PBPs, 10% abraded) and fiber-rich fractions (FRFs, obtained by roller milling of 10% pearled grain) of barley with high-amylose, normal, and waxy starch characteristics.

(Adapted from Izydorczyk, M.S. et al., Cereal Chem., 80, 645, 2003.)

Arabinoxylans

residues. An interesting feature of arabinoxylans from PBPs, common to all barley types, was a very high content of Xylp residues substituted at the O-2 position.

It is generally agreed that cereal arabinoxylans are highly heterogeneous, consisting of a range of structures with different degrees and patterns of substi- tution. However, despite the structural heterogeneity of arabinoxylans, most studies point to a nonrandom distribution of Araf residues along the xylan backbone. 73,86,87 Detailed structural analysis of isolated and purified arabinoxylans from various cereals resulted in tentative structural models for these polysaccharides. For the water-extractable arabinoxylans from wheat endosperm, it was shown that the highly substituted regions (or entire chains) of the fraction obtained with 100% saturation of ammonium sulfate are enriched in Xylp residues doubly substituted at C(O)-2,3, Xylp monosubstituted at C(O)-2, and short arabinose side chains. 88 Furthermore, these regions contain sequences of up to four contiguously substi- tuted Xylp residues, with three contiguously substituted Xylp residues occurring

most frequently. More recently, Dervilly-Pinel et al. 73 isolated homogeneous frac- tions of wheat arabinoxylans using both graded ethanol precipitation and size exclusion chromatography (SEC) fractionation and found blocks up to six contig- uously substituted xylosyl residues in highly branched populations of these poly- mers. In contrast, the less substituted fractions of wheat arabinoxylans, obtainable by precipitation at a relatively low concentration of ammonium sulfate or ethanol, are built up mainly of less densely substituted regions, containing sequences of contiguously (at least up to six, but possibly more) unsubstituted xylose residues. The branched Xylp in these regions are more frequently mono- than disubstituted and occur mostly as single or blocks of two substituted residues. 88

Bengtsson et al. 89 proposed that in rye arabinoxylans, the mono- and disubsti- tuted xylose residues were present in different polymers or in different regions of the same polymer chain; it was hypothesized that the major polymer structure (arabinoxylans I) contained only un- and monosubstituted (~46%) xylose residues, whereas the minor structure (arabinoxylans II) contained un- and disubstituted (57%) Xylp. The ratio of arabinoxylans I/II could vary from 1.1 to 2.8 for different rye

varieties grown in different countries. 90 Vinkx et al. 74 isolated a highly branched rye arabinoxylan (Ara/Xyl = 1.42) with a very low proportion of 3-monosubstituted Xylp. However, this polymer differed from arabinoxylans II, hypothesized by

Bengtsson et al., 89 in that it contained a relatively high level of 2-monosubstituted Xylp (14%) in addition to disubstituted Xylp residues (60%). It was therefore concluded that a range of polymer structures exists in rye arabinoxylans, rather than two classes as initially suggested. 80,91

7.4.4 M OLECULAR W EIGHT

The molecular weight (Mw) of arabinoxylans varies depending on their origin and the method used for its determination. The early studies using sedimentation techniques 92,93 reported relatively low Mw values (65,000 to 66,000) for water- extractable wheat arabinoxylans. However, very high Mw values (800,000 to 5,000,000) were reported when gel filtration chromatography was used, and the molecular weight of arabinoxylans was estimated by comparing their elution volume

Functional Food Carbohydrates

with that of standards with known Mw. 20,94 The latter approach overestimates the molecular weight and highlights the difficulties in accurately measuring the molec-

ular weight of polymers with different conformations than those of commonly used gel filtration standards (pullulans, dextrans). More recent studies, utilizing high-

performance size exclusion chromatography, combined with a multiangle light- scattering detector, have determined that the weight-average molecular weight of wheat arabinoxylans ranges from 220,000 to 700,000 (Figure 7.8). 78,95 Higher molec- ular weight values were reported for alkali-extractable arabinoxylans from hull-less

barley (850,000 to 2,430,000). 85 The size exclusion chromatography profiles of arabinoxylans indicate a very broad distribution of molecular weights. High ratios of weight-average molecular weight to number-average molecular weight (Mw/Mn) reported for alkali-extractable wheat arabinoxylans (1.3 to 2.5), as well as for water-

extractable wheat (4.1) and rye (8.5) arabinoxylans, 7 indicate the inherent polydis- perse nature of these polymers. Warrand et al. 5 reported that the main fraction of the mucilage extracted from seeds of Linum usitassinum contained highly polydis- persed arabinoxylans with three different populations of these polymers with molec-

ular weights of 5 × 10 6 (~10%), 1 × 10 6 (40%), and 0.2 × 10 6 (~50%).