3.1. Materials
Ferulated arabinoxylans were isolated from wheat bran by-product kindly provided by a commercial wheat milling industry in Northern Mexico (Molinos La Fama). Laccase (benzenediol: oxygen oxidoreductase, E.C.1.10.3.2) from Trametes versicolor, sodium hydroxide, citric acid, sodium phosphate dibasic, hydrochloric acid and sulfuric acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
3.2. Methods
3.2.1. WBAX Extraction
Wheat bran was milled to a 20-mesh particle size using a CyclotecTM 1093 Mill (FOSS, Sweden). Wheat bran (500 g) was suspended in ethanol (2,500 mL) over 12 h at 25 °C and 100 rpm to remove lipophilic components. The ethanol treated bran was then filtered and subjected to starch gelatinization and enzyme inactivation (boiling for 30 min in 3500 mL of water). After boiling, wheat bran was recovered by filtration and treated with 2,500 mL of 0.5 N NaOH solution at 25 °C in darkness for 0, 0.5, 1.0, 1.5 or 2.0 hours with shaking (100 rpm). Residual bran was eliminated by filtration and the filtrate was centrifuged (12,096 g, 20 °C, 15 min). Supernatant was acidified to pH 4 with 3N HCl and then centrifuged (12,096 g, 20 °C, 15 min). Supernatant was then recovered and precipitated in 65% (v/v) ethanol for 4 h at 4 °C. Precipitate was recovered and dried by solvent exchange (80% (v/v) ethanol, absolute ethanol and acetone) to render wheat bran ferulated arabinoxylans (WBAX) powder.
3.2.2. Laccase Activity
Laccase activity was measured at 25 °C from a laccase solution at 0.125 mg/mL in 0.1 M citrate-phosphate buffer pH 5.5 [10,12]. Syringaldazine 0.0216 mM in methanol was used as substrate. The enzymatic reaction was followed for 4 min at 530 nm.
3.2.3. Sugar Composition
Sugar composition was determined after WBAX hydrolysis with 2 N trifluoroacetic acid at 120 °C for 2 h. The reaction was stopped on ice, the extract was evaporated under air at 40 °C and rinsed twice with water (200 μL) and resuspended in water (500 μL). All samples were filtered through 0.45 μm (Whatman) and analyzed by high performance liquid chromatography (HPLC) using a Supelcogel Pb column (300 × 7.8 mm; Supelco, Inc., Bellefont, PA, USA) eluted with 5 mM H2SO4 (filtered 0.2 μm, Whatman) at 0.6 mL/min and 50 °C [17]. A Varian 9012 HPLC with Varian 9,040 refractive index detector (Varian, St. Helens, Australia) and a Star Chromatography Workstation system control version 5.50 were used. Series of sugar calibration standards were prepared in HPLC grade water at appropriate concentrations for creating a calibration curve for each sugar of interest (xylose, arabinose, galactose, mannose and glucose) in the range of 0.2–12.0 mg/mL. Inositol was used as internal standard.
3.2.4. Ferulic Acid
FA was quantified by reverse phase high-performance liquid chromatography (RP-HPLC) after a deesterification step, as described elsewhere [11]. An Alltima (Alltech, Deerfield, IL, USA) C18 5 μm column (250 × 4.6 mm) was used. Detection was by UV absorbance at 320 nm. Gradient elution was performed using acetonitrile and 0.05 M sodium acetate buffer, pH 4.0, at 1 mL/ min at 35 °C, in linear gradients from 15:85 to 35:65 in 30 min, from 35:65 to 60:40 in 0.5 min, from 60:40 to 15:85 in 4.5 min, and finally maintained at 15:85 for 5 min. A Waters 996 (Millipore Co., Milford, MA, USA) photodiode array detector was used to record the ferulic acid spectrum.
3.2.5. Intrinsic Viscosity and Viscosimetric Molecular Weight
Specific viscosity (ηsp) of WBAX solutions was measured by registering WBAX solutions flow time in an Ubbelohde capillary viscometer at 25 ± 0.1 °C, immersed in a temperature controlled water bath. WBAX solutions were filtered using 0.45 μm membrane filters before viscosity measurements. The intrinsic viscosity ([η]) was estimated from relative viscosity measurements (ηrel) of WBAX solutions by extrapolation of Kraemer and Mead and Fouss curves to “zero” concentration [20,21]. The viscosimetric molecular weight (Mv) was calculated from the Mark-Houwink relationship, Mv = ([η]/k)1/α.
3.2.6. WBAX Enzymatic Cross-Linking
WBAX solutions at 5 and 6% (w/v) were prepared in 0.1 M citrate phosphate buffer pH 5.5. WBAX solutions were mixed with 50 μL of laccase (1.675 nkat/mg WBAX). Gels were allowed to form for 6 h at 25 °C.
3.2.7. Rheology
The hardness of 5% and 6% (w/v) WBAX gels made in 6 mL glass flasks of 30 mm height and 25 mm internal diameter was analyzed with a TA.XT2 Texture Analyzer (Stable Micro Systems, Godalming, England). The gels were deformed by compression at a constant speed of 1.0 mm/s to a distance of 4 mm from the gel surface using a cylindrical plunger (diameter 15 mm). The maximum force obtained from the force vs. distance curve was recorded as a measure of gel hardness [17].
3.2.8. Gel Structure
After laccase addition, 2 mL WBAX solutions were quickly transferred to a 5 mL tip-cut-off syringe (diameter 1.5 cm) and allowed to gel for 6 h at 25 °C. After gelation, the gels were removed from the syringes, placed in glass vials and weighed. The gels were allowed to swell in 20 mL of 0.02% (w/v) sodium azide solution to prevent microbial contamination. During 10 h the samples were blotted and weighed. After weighing, a new aliquot of sodium azide solution was added to the gels. Gels were maintained at 25 °C during the test. The equilibrium swelling was reached when the weight of the samples changed by no more than 3%. The swelling ratio (q) was calculated as:
q
=
(
W
s
-
W
d
)
/
W
d
where Ws is the weight of swollen gels and Wd is the weight of WBAX in the gel.
From swelling measurements, the molecular weight between two cross-links (Mc), the cross-linking density (ρc) and the mesh size (ξ) values of the different WBAX gels were obtained as described before [18].