No abstract

To investigate the importance of substrate selectivity for xylanase functionality in bread making, the secondary binding site (SBS) of xylanases from Bacillus subtilis (XBS) and Pseudoalteromonas haloplanktis was modified. This resulted in two xylanases with increased relative activity toward water-unextractable wheat arabinoxylan (WU-AX) compared to water-extractable wheat arabinoxylan, i.e., an increased substrate selectivity, without changing other biochemical properties. Addition of both modified xylanases in bread making resulted in increased loaf volumes compared to the wild types when using weak flour. Moreover, maximal volume increase was reached at a lower dosage of the mutant compared to wild-type XBS. The modified xylanases were able to solubilize more WU-AX and decreased the average degree of polymerization of soluble arabinoxylan in dough more during fermentation. This possibly allowed for additional water release, which might be responsible for increased loaf volumes. Altered SBS functionality and, as a result, enhanced substrate selectivity most probably caused these differences.

The molecular mobility of water and biopolymers in wheat dough and the influence of xylanases thereon was investigated with time domain proton nuclear magnetic resonance relaxometry. To reduce the complexity, model systems containing starch, gluten and/or water-unextractable arabinoxylan (WU-AX) were used. In the starch-WU-AX-water model, starch binds water fast but less strong compared to WU-AX, resulting in water withdrawal from starch during resting. In contrary, WU-AX did not affect the water distribution in a gluten-WU-AX-water system, despite the higher water retention capacity (WRC) of WU-AX compared to gluten. In a starch-gluten-WU-AX-water model and in wheat flour, water was distributed over the different constituents including WU-AX. Addition of xylanase reduced the WRC of WU-AX, resulting in a release of water. Therefore, the beneficial effect of xylanase on dough and bread quality can, in part, be attributed to the redistribution of water, initially bound by WU-AX, between the other flour constituents.

The importance of inhibition sensitivity for xylanase functionality in bread making was investigated using mutants of the wild-type Bacillus subtilis xylanase (XBSTAXI), sensitive to Triticum aestivum xylanase inhibitor (TAXI). XBSNI, a mutant with reduced sensitivity to TAXI, and XBSTI, a mutant sensitive to all wheat endogenous proteinaceous inhibitors (TAXI, Xylanase Inhibiting Protein and Thaumatin-like Xylanase Inhibitor) were used. The higher inhibition sensitivity of XBSTAXI and XBSTI compared to XBSNI was associated with a respective 7- and 53-fold increase in enzyme dosage required for a maximal increase in bread loaf volume. XBSTI and XBSTAXI were only active during the mixing phase and the beginning of fermentation, while XBSNI was able to hydrolyze arabinoxylan until the end of fermentation. In spite of this difference in activity profile, no differences in loaf volume were observed for the different xylanases at optimal concentrations. Dough extensional viscosity analysis suggests that increased water availability as a result of xylanase activity favors starch-starch and starch-gluten interactions and drives the improvement in bread loaf volume.