PHYSICOCHEMICAL PROPERTIES OF ARABINOXYLANS

7.6 PHYSICOCHEMICAL PROPERTIES OF ARABINOXYLANS

7.6.1 C ONFORMATION OF A RABINOXYLAN C HAINS IN S OLIDS AND S OLUTIONS

The conformation of an unsubstituted xylan bears some resemblance to that of other β-(1→4)-linked polysaccharides, such as cellulose or mannan. However, the single hydrogen bond between two adjacent xylosyl residues, compared to two hydrogen bonds between two adjacent glycosyl residues in cellulose, has an important effect on the capacity of the xylan chain to form cooperative intramolecular hydrogen bonds, and hence on its conformation. As a result, xylans form twisted threefold ribbon-like strands that are more flexible than the rigid twofold helices of cellulose. 101

The β-(1→4)-linked xylan in the unsubstituted form aggregates into insoluble com- plexes stabilized by numerous intermolecular H bonds. The presence of side groups protruding from the xylan backbone drastically suppresses the interchain linking system, thus making the polymer partially soluble in water, but apparently does not substantially change the basic backbone conformation. 101 Andrewartha and

coworkers 92 postulated that the presence of arabinosyl substituents stiffens the chain by maintaining a more extended xylan backbone. The origin of the viscous behavior of arabinoxylans was associated with the formation of rod-like structures in solution. 92 However, the evidence presented by other workers indicates that arabinoxylans behave in solution as semiflexible coils. Ebring- erová et al. 71 reported the Mark–Houwink exponent ‘a’ of 0.50 ([ η] = KM w a ) for corncob arabinoxylans with an unusually low degree of substitution (Ara/Xyl = 0.07),

a value characteristic for molecules in unperturbed coil-shaped structures. Recent studies, based on the light-scattering measurements, also indicate that in solution, arabinoxylans behave as locally stiff, semiflexible random coils. 95,102,103 Dervilly-Pinel et al. 102 reported that the conformational parameters, such as the exponent ‘a’ (0.74)

and the hydrodynamic parameter v (0.47) (R g 0.5 = KM w v ) determined for the water- soluble wheat arabinoxylans, were typical for a random coil conformation. However, the persistence length (q = 6 to 8 nm) representing the chain rigidity indicated that arabinoxylan chains are semiflexible, in comparison with very flexible polysaccharides (q = 1.70), such as amylose or pullulans.

The solubility of arabinoxylans is closely related to the presence of the arabi- nosyl substituents along the xylan backbone. Andrewartha and coworkers 92 prepared

a series of arabinoxylan chains with various degrees of branching by partially removing the arabinofuranosyl side branches with α-L-arabinofuranosidase. At Ara/Xyl ~ 0.43, the solubility of arabinoxylans abruptly declined. The amount of arabinose substituents as well as their distribution along the xylan backbone affects the potential of arabinoxylan chains to interact with each other or with other polysac- charides. It is prudent to predict that the presence of segments of unsubstituted xylose residues in the polymer chains will increase the potential of arabinoxylans

Arabinoxylans

to form intermolecular aggregates. This may lead to either an increase in viscosity or precipitation of polymer chains if the interactions are numerous. Izydorczyk and MacGregor 104 provided empirical evidence of noncovalent interactions between sparsely substituted arabinoxylan chains (Ara/Xyl = 0.18 to 0.32) and cellulose-like fragments of β-glucans. In the plant cell wall material, the noncovalent topological associations between β-glucans and arabinoxylans might contribute to poor water solubility or to low enzymic digestibility of these polymers.

7.6.2 V ISCOSITY OF A RABINOXYLAN S OLUTIONS

As a result of the high molecular weight and locally stiff, semiflexible random coil conformation, arabinoxylans exhibit a very high viscosity in aqueous solutions. An

intrinsic viscosity of 5.9 dl/g was reported for rye arabinoxylans, whereas for wheat arabinoxylans the values ranged from 2.75 to 5.48 dl/g depending on cultivars. 9,81

In dilute solutions, the zero-shear-rate specific viscosity (η sp ) 0 increases linearly with increasing arabinoxylan concentration (c) with a slope of log ( η sp ) 0 vs. log c ≈1.

Above the so-called critical concentration (c*), marking the onset of physical entan- glements and coil overlap, the concentration dependence of ( η sp ) 0 increases and the slope increases to ≈3.7 to 3.9 (Table 7.3). The critical concentration values for arabinoxylans from wheat endosperm are relatively low, ranging from 0.2 to 0.4% (w/v) depending on the chain length and fine molecular structure of these polymers (Table 7.3). 105,106

The apparent viscosity of aqueous solutions of arabinoxylans is strongly depen- dent on their concentration and the rate of shear at which the viscosity measurements are taken. At low shear, arabinoxylan solutions behave like Newtonian fluids and exhibit very little shear rate dependence (Figure 7.11a). The apparent viscosity increases with the polymer concentration. With the increasing shear rates, arabinox- ylans display a reduction in the apparent viscosity, commonly known as shear thinning. The molecular size of arabinoxylans is an important determinant of the solution behavior of these polymers. Izydorczyk and Biliaderis 105 reported that the high molecular weight fractions of wheat arabinoxylans (with intrinsic viscosities of 8.5 and 6.2 dl/g) exhibited weakly elastic properties in solutions (Figure 7.11b). The dynamic rheological measurements indicated that with increasing polymer con- centration, the viscoelastic behavior of arabinoxylan fractions changed from that of viscous solution (G" > G' at all frequencies) to weakly elastic (G' > G" at higher frequencies) (Figure 7.11b).

Warrand et al. 107 showed that arabinoxylans have a tendency to form macro- structures in aqueous solutions via chain aggregation and physical entanglements, and emphasized the importance of hydrogen bonds in stabilizing these aggregates. This aggregation tendency may be responsible for pseudo-gel behavior of arabi- noxylan solutions under some conditions. For example, it was shown that 2% (w/v) solutions of high molecular weight arabinoxylans isolated from flaxseed mucilage exhibited preponderantly elastic properties, with the elastic modulus, G', exceeding the viscous modulus, G'' (Figure 7.12). 107 These weak gel properties were, however, greatly diminished in the presence of chaotropic salts, known for weakening the hydrogen bonds between solute molecules. The behavior of arabinoxylans in

Functional Food Carbohydrates

TABLE 7.3 Values for Intrinsic Viscosity, Critical Concentration (c*), Coil Overlap, and Slopes of Dilute and Entangled Domains of Arabinoxylan Fractions Obtained by Stepwise Precipitation with Ammonium Sulfate or Size Exclusion Chromatography Fractionation

Slope Arabinoxylan

Dilute Entangled Fraction

c*

Coil Overlap

(dl/g) (g/100 ml)

(c*[ η ])

Domain Domain

F60 a 4.70 0.26 1.24 1.13 2.19 F70

nd nd F1 b 8.5 0.17 1.44 1.1 3.9

F2 6.2 0.20 1.24 1.1 3.7 F3 4.3 0.28 1.20 1.1 3.8 F4 3.8 0.29 1.10 1.1 3.8 F5 3.4 0.30 1.02 1.1 3.9

Note : nd = no data. a Fractions obtained by stepwise precipitation of isolated and purified water-soluble wheat flour ara- binoxylans with ammonium sulfate; the numbers 60 to 95 indicate the saturation of ammonium sulfate.

b Isolated and purified water-soluble wheat flour arabinoxylans were fractionated by size exclusion chromatography.

Source : Adapted from Izydorczyk, M.S. and Biliaderis, C.G., J. Agric. Food Sci., 40, 561, 1992; Izydorczyk, M.S. and Biliaderis, C.G., Carbohydr. Polym., 17, 237, 1992.

solutions and their viscosity-building properties are probably the most important characteristics responsible for the functional properties of arabinoxylans in the human digestive tract.

7.6.3 O XIDATIVE C ROSS -L INKING

Arabinoxylan solutions possess a unique capacity to form hydrogels in the presence of free radical-generating agents, such as peroxidase–H 2 O 2 , laccase, linoleic acid–lipoxygenase, ammonium persulfate, or ferric chloride. 108–112 Covalent cross- linking of arabinoxylan chains through dimerization of ferulic acid substituents is responsible for this unusual property of arabinoxylans (Figure 7.6). Although five dimers of ferulic acid (identical to those found in the cell walls) have been identified in gelled arabinoxylans, the 8-5' and 8-O-4' forms are preponderant, suggesting that both the aromatic ring and the double bond in the structure of ferulic acid serve as cross-linking sites. 112 Recently, a very small amount of a trimer of ferulic acid (4- O-8'/5'-5") has been detected in the laccase cross-linked wheat arabinoxylan gels. 64

Small-amplitude oscillatory measurements have been useful in following the development of three-dimensional networks in solutions of arabinoxylans undergo-

Viscosity (Pa.s)

0.01 0.1 1 10 100 Shear rate (s −1 )

Frequency (Hz)

FIGURE 7.11 Effects of shear rate and polymer concentration of the apparent viscosity(a) and viscoelastic properties (b) of arabinoxylan solutions. (Adapted from Izydorczyk, M.S. and Biliaderis, C.G., J. Agric. Food Sci., 40, 561, 1992. With permission.)

100.0 10.00 .s) (Pa

″ ( ) (Pa) sity 10.00 co is

1.000 ′( ) and G G ynamic v D

Frequency (Hz)

FIGURE 7.12 Frequency dependence of the storage modulus, G' (•), loss modulus, G" ( 䡩 ), and dynamic viscosity (䡲) of the solution (20 g/l, 25˚C) of arabinoxylan isolated from flaxseed

mucilage. (Adapted from Warrand, J. et al., Biomacromolecules, 6, 1871, 2005. With permission.) of arabinoxylans, there is usually a rapid rise in the elastic modulus (G') followed

by a plateau region with very little further increase of G' (Figure 7.13). This behavior has been attributed to the initial formation of covalent cross-linkages between ferulic acid residues of adjacent arabinoxylan chains that, once created in sufficient amount, could impede chain mobility and thus prevent further formation of cross-links. 7,54,55 The central role of feruloyl groups in gelation of arabinoxylan solutions is evidenced by disappearance of ferulic acid residues with a simultaneous formation of ferulic acid dimers and trimers during the initial stage of the gelation process. 64,110 Carvajal- Millan et al. 112 prepared arabinoxylan samples having the same molecular structure

Functional Food Carbohydrates

1 40 G′′ dulus G

100 Frequency (Hz)

stic mo

Ela 20 10 G′′ 1 G′

0.1 1 10 100 Frequency (Hz)

Time (min)

FIGURE 7.13 Development of storage modulus, G', with time of a water-soluble arabinox- ylan (2.3% (w/v)) isolated from wheat flour and treated with horseradish peroxidase (0.11

purpurogallin units/ml) and H 2 O 2 (3 mg/ml). Insets represent the molecular spectra of arabi- noxylans before (left) and after (1 h) (right) addition of the oxidant.

but varying ferulic acid content, and showed that the elasticity of arabinoxylan gels increased linearly with the increasing ferulic acid content (in the range of 1.4 to 2.4 μg of ferulic acid per mg of arabinoxylan). It was also shown that arabinoxylans with ferulic acid content lower than 0.4 μg/mg of arabinoxylan (i.e., less than 1 ferulic acid residue per 2000 Xylp) are not capable of forming gel structures. 112

Izydorczyk and Biliaderis 7 and Rattan et al. 54 proposed that in addition to the pivotal role of ferulic acid in the gelation of arabinoxylans, the molecular structure (amount and distribution of Araf residues along the xylan backbone) and molecular weight

of arabinoxylan chains also affected their gelation capacity. Rattan et al. 54 found a positive relationship between the elastic modulus of the gels from wheat arabinox- ylans and the intrinsic viscosity and ferulic acid content in native polymers. It was also established that the rate and extent of gelation were dependent on the concen- tration of arabinoxylans and oxidizing agents. 54,110 Recently, Vansteenkiste et al. 113 and Carvajal-Millan et al. 112 have shown that cross-linking densities of gels, deter- mined from swelling experiments, were higher than those calculated theoretically from the amount of dimers and trimers formed during gelation; these results suggest that in addition to the covalent cross-links, concomitant formation of noncovalent bonds between adjacent arabinoxylan chains might also occur and contribute to the final gel structure of arabinoxylans.

7.6.4 P HYSICOCHEMICAL P ROPERTIES OF A RABINOXYLAN G ELS

Arabinoxylan gels have neutral taste and odor, very high water absorption capacity (up to 100 g of water per gram of dry polymer), and are not susceptible to changes

Arabinoxylans

in pH or electrolyte concentrations. 7 These properties, together with the macroporous texture of gels (mesh sizes varying from 200 to 400 nm) and the dietary fiber nature

of arabinoxylans, give them potential to be used as matrices with controlled releases of active agents in the food, cosmetic, and pharmaceutical industries. Vansteenkiste et al. 113 demonstrated that proteins embedded in the arabinoxylan gel network are protected against enzymic hydrolysis. Carvajal-Millan et al. 114 modified the rheo- logical properties of arabinoxylan gels by altering either the initial ferulic acid content or the concentration of polymers before gelation. Consequently, the differ- ences in rheological properties of gels were shown to affect their capacity to load and release proteins with various molar masses. The possibility to modulate protein release from arabinoxylan gels makes them useful for controlled delivery of thera- peutic proteins.