## 4 Discussion
### Strategies to Improve Gluten-Free Dough Rheology and Bread Characteristics
#### 4.1.6 Enzymes  
Like hydrocolloids, enzymes are widely used in both gluten-free doughs and to improve bread, and they have been widely investigated in the literature (see Section 3.3.4). The most comprehensive study, by [PERSON] and [PERSON] (2016) [36], summarized the role of enzymes in improving the functionality of gluten-free doughs, bread, and bakery products. The authors noted that the main purpose of the addition of enzymes is to create protein aggregates that mimic (or try to mimic) gluten functionality, and to modify proteins, with the aim of changing their functionality. They highlighted that transglutaminase (TGase), oxidases, and proteases are the most interesting options. As reported in [PERSON] and [PERSON] (2016) [36], TGase is a protein-glutamine \(\gamma\)-glutamyl-transferase, which catalyzes an acyl-transfer reaction between the \(\gamma\)-carboxamide group of peptide-bound glutamine residues, and a variety of primary amines. Its use could significantly improve gluten-free doughs. The results of their batter rheological analysis and bread quality evaluation confirmed the improving effect of TGase on buckwheat and brown rice doughs and breads [36]. The authors argued that this finding was due to protein cross-linking, and the formation of large protein complexes in both breads [36]. Conversely, TGase had a detrimental effect on the elastic-like behavior of corn batters, but increased specific volume and lowered crumb hardness in corn breads [36]. The latter study also found that it was not effective in improving gluten-free doughs and breads made from oat, sorghum, and teff. With respect to oxidases, [PERSON] and [PERSON] (2016) [36] and [PERSON] and [PERSON] (2004) [37] evaluated several types (lipoxygenase, sulfhydryl oxidase, glucose oxidase, and peroxidase). Improvements were related to their dough strengthening and stabilization effect, and their activity as bleaching agents. Another interesting enzyme is glucose oxidase [36; 37]. When added to gluten-free dough formulations based on rice flour, it increased bread specific volume and reduced crumb hardness [36]. The authors of the latter studies suggested that this was due to an increase in dough consistency related to protein cross-linking, connected to the ability of hydrogen peroxide to form disulfide bonds, and gelation of water-soluble pentosans in rice flours. Last but not least, we turn to the proteases. Proteinases and peptidases hydrolyze peptide bonds in proteins. Proteases are reported to significantly improve gluten-free dough rheology and bread characteristics, and induce changes in protein-protein and protein-starch interactions in rice batters, due to the creation of interlinked protein-starch aggregates after protein degradation [36; 38; 39]. Moreover, protease treatments are reported to decrease peak viscosity, and the breakdown of starchy-based gluten-free doughs, independent of the type of base flour used [36; 38; 39]. The findings reported above are widely supported by other authors. In particular, [PERSON] and [PERSON] (2004) [37] tested the efficacy of glucose oxidase in the improvement of gluten-free dough rheology and bread characteristics. The results showed that it was able to modify proteins in rice flour, by lowering thiol and amino group concentrations. Furthermore, its addition increased both the elastic and viscous moduli of gluten-free doughs [37]. Finally, its addition to rice flour resulted in bread with increased specific volume and improved texture, minimizing the addition of HPMC [37]. Similarly, the addition of protease (0.01%), in [PERSON] and [PERSON] (2009) [38], improved the rheological properties of gluten-free doughs made with brown rice flour, notably a decrease in viscosity and breakdown. The same study highlighted an increase in loaf specific volume, and a decrease in crumb hardness and chewiness. The effectiveness of protease was confirmed by [PERSON] et al. (2013) [39], who tested pre-fermented (with _Aspergillus oryzae_) rice flour.