Methods

WEAX GelationA WEAX solution (3 % w/v) was prepared in 0.05 M citrate phos-phate buffer pH 5.5. Laccase (1.675 nkat per mg WEAX) was added to WEAX solu-tion as cross-linking agent. Gels were allowed to develop for 2 hr at 25°C [10].

Laccase ActivityLaccase activity was measured at 25°C from a laccase solution at 0.125 mg/ml dissolved in 0.05 M citrate-phosphate buffer pH 5.5 as previously reported [4].

RHEOLOGY

Small Deformation MeasurementsSmall amplitude oscillatory shear was used to fol-low the gelation process of WEAX solution. Cold (4°C) WEAX solution (3% w/v)

in 0.05 M citrate phosphate buffer pH 5.5 was mixed with laccase and immediately poured on cone-plate geometry (5.0 cm in diameter and 0.04 rad in cone angle) of a strain controlled rheometer (Discovery Hybrid Rheometer, TA Instruments). Exposed edges were recovered with silicone to prevent evaporation. WEAX gelation was start-ed by a sudden increase of temperature from 4 to 25°C and monitorstart-ed at 25°C for 2 hr by recording the storage (G’) and loss (G”) moduli. Measurements were carried out at 1.0 Hz frequency and 10% strain. From strain sweep tests, WEAX gels showed a linear behavior from 0.02 to 100% strain. 10% strain was used in all the rheological measurements. The mechanical spectra of gels were obtained by frequency sweep from 0.01 to 10.0 Hz with a 10% strain at 25°C [3,10].

LARGE DEFORMATION MEASUREMENTS

The hardness of 3% (w/v) WEAX 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 cylin-drical plunger (diameter 15 mm). The maximum force obtained from the force versus distance curve was recorded as a measure of gel hardness [11].

STRUCTURE OF FREEZE DRIED WEAX GELS

The WEAX gels at 3% (w/v) were frozen at −20°C and lyophilized at −37°C/0.133 mbar overnight in a Freezone 6 freeze drier (Labconco, Kansas, MO). The external structure of the freeze-dried WEAX gel was analyzed with a stereo light microscope (Leica CLS 150 XE Leica Microsystems®, Switzerland) at a low magnification (10×).

The internal structure of freeze-dried WEAX gel was studied by SEM (JEOL 5410LV) at low voltage (20 kV). The SEM image was obtained in secondary electrons image mode.

9.3 DISCUSSION AND RESULTS 9.3.1 WEAX GELATION

The cross-linking process of WEAX was rheologically investigated by small ampli-tude oscillatory shear. Figure 2 shows the development of storage (G’) and loss (G’’) moduli versus time of 3% (w/v) WEAX solution undergoing oxidative gelation by laccase. G’ and G’’ moduli rise to reach a pseudo plateau region. This behavior reflects an initial formation of covalent linkages between FA of adjacent WEAX molecules producing a three-dimensional network. Once sufficient cross-links have formed, movement of chains is impeded by the rigidity of the gel. The final G’ and G’’ values of 3% (w/v) were 71 and 16 Pa, respectively. These results are in the range reported

for WEAX gelled by laccase [3,4]. The gelation time (tg), calculated from the cross-over of the G’ and G” curves (G’ >G”) was 3 min. The tg value indicates the sol/gel transition point and at this point G’ = G” or tan δ = G”/G’ = 1 [12]. The mechanical spectra of WEAX after 2 hr gelation (Figure 3) was typical of solid-like materials with a linear G’ independent of frequency and G’’ much smaller than G’ and dependent of frequency [13]. This behavior is similar to that previously reported for WEAX cross-linked by laccase or peroxidase/H₂O₂ system [14-16].

The WEAX solutions at 3% (w/v) produced firm and brittle gels in presence of laccase. The hardness of the WEAX gels is presented in Figure 4. Hardness is related to the strength of the gel structure under compression. The WEAX gel registered a hardness value of 3.5 N, which is higher than those previously reported for laccase induced wheat flour and maize bran arabinoxylan gels (0.5–1 N) prepared at different concentrations (1–8% w/v) [1,4].Such behavior might have its origin in the structural and/or conformational characteristics of these macromolecules. Clearly, further stud-ies on the distribution of arabinose and feruloyl groups along the polymer chain back-bone are needed to establish relationships between the molecular structure and gelling ability of WEAX.

FIGURE 2 Rheological kinetics of 3% (w/v) WEAX solution gelation by laccase. G’ (○), G’’

(●), and tan δ (×). Measurements at 25°C, 1 Hz and 10% strain.

FIGURE 3 Mechanical spectrum of WEAX gel at 3% (w/v). G’ (○), G’’ (●). Measurements at 25°C, 1 Hz and 10% strain.

FIGURE 4 Experimental curve obtained from compression test of gels at 3% (w/v) in WEAX.

9.3.2 FREEZE DRIED WEAX GEL

In Figure 5 is shown a WEAX gel image after freeze drying (A). Figure 5 (B) shows a stereomicrograph of the freeze dried WEAX gel external structure. It is possible that frozen caused the crust formation of the sample. The internal structure of the lyophi-lized WEAX gel was observed by SEM (Figures 5 (C) and (D)). The WEAX gel net-work presents many connections and can be compared with an irregular honeycomb structure. In this study the average inner dimensions of the cell were approximately 100 x 200 µm (Figure 5 (D)). This morphological microstructure is similar to that re-ported before for lyophilized wheat and maize AX gels [7,17-20]. However, the SEM microstructure of freeze dried WEAX gels are different to that recently reported for supercritical CO₂-dried WEAX aerogels which present more spongy network structure [21].

FIGURE 5 (Continued)

FIGURE 5 (Continued)

FIGURE 5 Lyophilized WEAX gel (A), stereomicrograph of lyophilized WEAX gel (B), SEM micrographs of lyophilized WEAX gel at 35× magnification (C), and 200× magnification (D).

9.4 CONCLUSION

The WEAX from the spring wheat variety Tacupeto F2001 are able to form gel in the presence of laccase as shown by dynamic rheometry. Freeze dried arabinoxylan gels present a porous network constituted by an irregular honeycomb structure. Under-standing Tacupeto F2001 arabinoxylan gels characteristics can be useful to propose alternative uses of this improved wheat cultivar.

9.5 FUTURE CONSIDERATIONS

The WEAX gels continue to be investigated and functional properties are being pro-posed. Several questions remained to be elucidated, especially those concerning the effect of food process on health benefits of these compounds. In addition, arabinox-ylan gels could be matrices suited for bioactive compounds or microorganisms.

KEYWORDS

• Ferulated arabinoxylans

• Gelation

• Macromolecule

• Scanning electron microscopy

• Trametes Versicolor

ACKNOWLEDGMENT

This research was supported by Fondo Proyectos y fortalecimiento de Redes Temáticas CONACYT de investigación formadas en 2009 (Grant 193949 to E. Carvajal-Millan).

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FREE AND ESTER-LINKED FERULIC

ACID CONTENT IN A HARD-TO-COOK PINTO BEAN (PHASEOLUS VULGARIS L.) VARIETY

AGUSTÍN RASCON-CHU, KARLA ESCARCEGA-LOYA, ELIZABETH CARVAJAL-MILLAN, and ALFONSO SÁNCHEZ

CONTENTS

10.1 Introduction ...182

10.2 Materials and Methods ...182

10.2.1 Materials ...182

10.2.2 Cooking Process...182

10.2.3 Free and Ester-linked Ferulic Acid Content ...183

10.2.4 Color ...183

10.2.5 Breaking Strength ...183

10.3 Discussion and Results ...183

10.4 Conclusion ...185

Keywords ...185

Acknowledgment ...185

References ...185