*3.3. Color Observation*

In addition to its high antioxidant activity and many health-beneficial effects, betanin is a natural food colorant [26,27]. Therefore, observing the changes in the color of pastes could help foresee the visual quality of pastes during storage, which plays an important role in determining the acceptability and desirability of the products for consumers. Figure 3 displays the appearance of starch and starch–betanin pastes during retrogradation, and the lightness (*L*\*), redness (*a*\*) and yellowness (*b*\*) of these samples are listed in Table 3. The fresh PoS paste was transparent, and RS and PeS pastes tended to be white. The *L*\* value for PoS (29.67) was the lowest, and those of RS and PeS pastes were 40.17 and 57.85, respectively. Nevertheless, fresh starch–betanin pastes exhibited a vivid and attractive red color, which indicated that betanin could endow starch paste with a good visual image. As shown in Table 3, the values of *a*\* for fresh starch–betanin pastes were also significantly higher than that of the corresponding fresh starch pastes. After retrogradation for 7 days, *L*\* values of pure starch pastes were higher than that of the corresponding fresh starch paste. Starch molecules were rearranged to form crosslinking during the retrogradation, resulting in the color to gradually become cloudy. The transparency was decreased and the whiteness was increased, giving rise to the increasing of lightness. In regard to the starch–betanin pastes, similar alterations as occurred to the pure starch pastes after retrogradation were found. Starch–betanin pastes after retrogradation for 7 days presented higher *L\** and *a\** values than fresh starch–betanin pastes. In light of the analysis of retrogradation properties in the next sections, betanin inhibited the long-term retrogradation of all starches. In other words, betanin delayed the transformation to cloudy; thus, the redness *a*\* of starch– betanin pastes was increased after storage. According to the appearance of the pastes, the retrograded starch–betanin pastes seemed to slightly fade, which was mainly related to the phenomenon of becoming cloudy during retrogradation. Overall, the retrograded starch–betanin pastes maintained a bright red color, which indicates that betanin can also be applied as a good colorant during the storage of starches.

**Figure 3.** The appearance of starches and starch–betanin samples after retrogradation for 0 days and 7 days. RS: rice starch; PoS: potato starch; PeS: pea starch.


**Table 3.** The color of starches and starch–betanin samples after retrogradation for 0 days and 7 days.

Differences between values, indicated by different letters in the same columns, are significant at 0.05 level of confidence. RS: rice starch; PoS: potato starch; PeS: pea starch; *L*\*: brightness value; *a*\*: red-green value; *b*\*: yellowish-blue value.

#### *3.4. XRD Analysis*

The X-ray diffraction patterns of the retrograded starch and starch–betanin samples were displayed in Figure 4. Native rice starch (NRS), native potato starch (NPoS) and native pea starch (NPeS) exhibited typical A-, B- and C-type diffraction patterns, respectively. After retrogradation for 7 days, RS with an A-type diffraction pattern was transformed to a V-type pattern, and B-type patterns for the retrograded PoS and PeS were shown. The relative crystallinity of the retrograded RS, PoS and PeS was 16.8%, 10.9% and 9.5%. The lowest crystallinity of the retrograded PeS may be related to its low amylopectin content, because the crystallinity of the processed starch was considered to be associated with retrogradation of amylopectin [28]. In regard to all the retrograded starch–betanin samples, their diffraction intensities were observably weakened in comparison with the retrograded starch. For example, the diffraction peak at 19.6◦ for the retrograded RS– betanin almost disappeared, and peaks at 17.3◦ for the retrograded PoS–betanin and PeS–betanin narrowed significantly. The relative crystallinity was reduced by 14.2%, 8.8% and 4.4% for the retrograded RS–betanin, PoS–betanin and PeS–betanin, respectively. These results imply that betanin impeded the rearrangement of starch molecules during long-term retrogradation. The presence of betanin may alter the distribution and rearrangement of starch molecules, and retrogradation was restrained by steric hindrance. The degree of inhibiting crystallinity by betanin was the highest for PeS, and it is likely that betanin and the high amylose content of PeS occupied the space to interfere with the association of amylopectin.

**Figure 4.** XRD diagrams of starches and starch–betanin samples after retrogradation; (**A**) rice starch, (**B**) potato starch, and (**C**) pea starch. NRS: native rice starch; NPoS: native potato starch; NPeS: native pea starch. RS: retrograded rice starch; PoS: retrograded potato starch; PeS: retrograded pea starch.

#### *3.5. Short-Range Ordered Structure Analysis*

Internal structural changes of starches after retrogradation could also be determined by a FTIR spectrometer that was used to analyze the short-range ordered structure, and the FITR spectra of starch and starch–betanin after retrogradation for 7 days were displayed in Figure 5A. Compared to the retrograded starches, no new characteristic absorption peaks appeared in the spectra of the retrograded starch–betanin samples, and just some of the wavenumbers of peaks were shifted, which implied that the primary structure of the retrograded starches was maintained and no covalent bond was formed. As demonstrated by Sevenou et al. [29], the absorbance at approximately 995 cm−1, 1047 cm−<sup>1</sup> and 1022 cm−<sup>1</sup> could be associated with the structural order of starch chains near the granule surface. Meanwhile, the ratio of 1047/1022 cm−<sup>1</sup> and 995/1022 cm−<sup>1</sup> could be used to determine a degree of short-range order for the retrograded starches [30]. As displayed in Figure 5B, the retrograded starch–betanin behaved with lower ratio values of 1047/1022 cm−<sup>1</sup> and 995/1022 cm−<sup>1</sup> as compared to the retrograded starch. For example, the ratios of 995/1022 cm−<sup>1</sup> for RS, PoS and PeS were 0.974, 0.967 and 0.972, respectively. However, the ratios of RS–betanin, PoS–betanin and PeS–betanin were decreased to 0.908, 0.937 and 0.946, respectively. These results suggest that betanin reduced the short-range order structure of the retrograded starches, which was in accord with the effect of betanin on crystallinity in the XRD analysis.

**Figure 5.** FTIR spectra (**A**) and 1047 cm−1/1022 cm−<sup>1</sup> ratios from deconvoluted FTIR spectra (**B**) of starches and starch–betanin samples after retrogradation for 7 days. RS: rice starch; PoS: potato starch; PeS: pea starch.
