*2.2. Effect on Bread Properties*

Baking experiments with up to 8% addition of α-, β-, and γ-CD were made on a domestic bread maker (Breadmaking I) to investigate the effect on the bread volume. The results can be seen in Figure 3. Although the addition of CDs affected the water absorption, we decided to apply constant water addition in the bread doughs to minimize the number of variables. For the different types of CD, the largest specific bread volumes were observed for 4% α-CD, 2% β-CD, and 2% γ-CD, which resulted in an increase of 14%, 9%, and 7% in specific bread volume, respectively. However, only the bread with 4% α-CD were significantly larger than the control sample. When higher concentrations of the three types of CDs were added, the specific bread volume decreased, especially when β-CD was applied. All bread with the exception of the 8% α-CD, and 4% and 8% β-CD supplemented version, displayed acceptable crust and crumb structure as perceived by visual and manual inspection (see Supplementary Materials). In contrast, for the exceptions, it was observed that the crumb of the bread had partially collapsed, had an irregular crumb, and was very dense at the bottom. An irregular and uneven crust was observed at the top of the bread as if air had escaped. This indicates that the gluten network had been adversely effected by the addition of CD, preventing the development of a suitable gluten network with sufficient stability from supporting the dough foam.

**Figure 3.** Specific bread volume for wheat bread with the addition of different types and concentrations of CD (Breadmaking I). The error bars indicate the standard deviation. Different letters indicate the significant difference between the treatments (*p* < 0.05).

Similar bread was produced using a kitchen mixer (Breadmaking II). 8% addition of CD was omitted, as this concentration resulted in a significant decrease in specific bread volume. During the preparation of the dough pieces for the analysis, it was noted that the dough stickiness increased with increasing CD concentration. In contrast to the bread produced in the domestic bread maker (Breadmaking I), no significant difference in specific bread volume between the bread with and without CD could be observed. The specific bread volumes in this trial ranged from 3.6–3.9 mL/g.

These results only partly confirm the results presented in other studies, where, in general, significant increases in bread volume could be observed in the range of 1–3% added CD. Kim and Hill [11] showed that an increase in bread loaf volume of 12% could be obtained in the range of 0.8 to 1.4% β-CD added for wheat bread. Mutsaers and Eijk [34]

reported a 14–20% increase in loaf volume for two types of wheat bread supplemented with 1.5–2% β-CD. The addition of β-CD was found to be on par with the addition of shortening (3%), CGTase, and amylase in an American straight dough process judged from bread loaf volume [34]. Zhou et al. [12] found a slight increase in specific loaf volume until 1.5% β-CD after which the specific volume decreased below the specific volume of the control without β-CD. Zhou et al. [13] found a maximum increase in a specific volume at 2% α-CD and 3% γ-CD in a study in which the range of added CD was 0.5 to 3%. Both Zhou et al. [12] and Zhou et al. [13] observed a change in bread crumb pore distribution towards smaller and more uniform pores for the bread supplemented with either 2% α-CD, 1.5% β-CD, or 3% γ-CD. Furthermore, multiple studies have used CGTases in the production of wheat bread and found bread volume increments, which is assigned to the production of CDs [34,35,37]. Although our results, at least for the Breadmaking II data set, did not fully corroborate the data obtained on a domestic bread maker (Breadmaking I), we can partly confirm the tendency that the addition of small amounts (1–2%) CD may lead to an increase in bread volume. On the other hand, our results clearly demonstrate that the addition of larger amounts of CD, e.g., >4%, leads to a loss of bread volume compared to the control. However, the changes in bread volume are, as shown, somewhat dependent on production conditions and procedure. Although significant increases in bread volume based on the addition of CDs could not be unequivocally verified, our results underline that acceptable bread with respect to bread volume and quality may be achieved for additions of all three native CDs up to at least 4%.

The results obtained for the effects of CDs on bread volume, in essence, corroborates the tendencies obtained from the alveograph analysis of the doughs, including minor increases in parameters correlated to bread quality (volume; e.g., *Ie*) at low CD concentrations, followed by large decreases at high CD concentration. This is to some extent expected since both alveograph analysis and the foam producing step in breadmaking involves bubble inflation causing biaxial extension of the dough matrix [52]. This substantiates that the tendencies found for the effects of CD on bread volume are caused by the interaction of the CD with the proteins in the gluten network, facilitating minor improvements of the network quality (as judged by bread volume) at low concentration and larger adverse effects at high CD concentration.

To evaluate the effects of the CDs on the staling of the bread, the bread crumb from bread stored at room temperature were analyzed by texture analysis and LF-NMR to detect changes in the firmness and in the water distribution, respectively.

The result of the texture analysis can be seen in Figure 4. The firmness of the bread crumb gradually increased with longer storage time for all the bread. Increasing firmness of bread crumb is often used as a measure of bread staling [53]. No significant difference in the firmness measurements between the bread with and without CD during the storage could be found, indicating that the three types of CD did not retard the staling of the bread as judged by firmness.

To further elucidate a potential effect on staling, LF-NMR analysis on breadcrumb was conducted. The use of LF-NMR in food science is well established [54]. The LF-NMR data were analyzed to label discrete exponential decays, representing distinct water populations. Three populations of protons were identified in all bread samples with relaxation time T<sup>21</sup> varying between 0.5–2.1 ms, T<sup>22</sup> 2.8–7.7 ms, and T<sup>23</sup> 17.4–36.6 ms (see Supplementary Materials). The ranges of T2n values are similar to those presented in the literature [55,56]. The relaxation time T<sup>21</sup> represents the least-mobile proton population and, therefore, the most tightly bound, and vice versa, T<sup>23</sup> represents the most mobile proton population. No apparent systematic development of the T2n values during storage was found. The corresponding Mn-values, which are the abundances of the three proton populations, presented in Figure 5 indicate the relative concentration of the different proton populations. The figures show a stable distribution up until 7 days of storage for all treatments with a reproducible signal. After 7 days, the samples display considerable variation among the triplicate measurements within each treatment, suggesting variations within the bread

crumb. A weak tendency of proton exchange between the two faster relaxation times, T<sup>21</sup> and T22, is also observed, in which it should be noted that M<sup>n</sup> is a relative indicator. Selective loss of water during the staling process will thus give the same impression.

**Figure 4.** Firmness of bread without and with the addition of α-CD (**a**), β-CD (**b**), and γ-CD (**c**) for different storage time. The error bars indicate the standard deviation.

No systematic change in the distribution of water populations could be observed between the control and the CD supplemented bread. This corroborates the firmness studies carried out on the same series of bread (Figure 4). However, the literature suggests that an anti-staling effects may be achieved by the addition of CDs to wheat bread. β- and γ-CD have been found to have a small (but significant) retarding effect on the staling rate of wheat bread stored at room temperature, while no significant decrease in staling has been observed for the addition of α-CD [14,15]. Tian et al. [14] suggested that retarding effect of β-CD on staling was caused by the formation of an amylose-lipid-β-CD complex, which retard the transformation of the crystalline starch types in the crumb. Furthermore, the retarding effect of β-CD on the retrogradation of various starches has been presented in several studies [43,44,57–59]. The addition of CGTases has also in multiple studies been

shown to inhibit the staling in bread, but it is also presumed to be linked to the amylolytic activity of the enzymes and not solely the effects of the CD produced [35–38].

**Figure 5.** The relative abundances (Mn) of the three different proton populations in the breads with different types and concentrations of α-CD (**a**), β-CD (**b**) and γ-CD (**c**) for different storage time. The error bars indicate the standard deviation.

Despite that several studies have found an anti-staling effect by the addition of CDs in bread, no such effect could be identified in this study. This discrepancy may originate in differences in ingredients, water addition, processing methods, and method for assessing a potential anti-staling effect, as several of the abovementioned studies apply DSC measurements together with texture analysis. It may also underline that an anti-staling effect of CDs, if any, may be small and lower than the random variation of the experiments.
