*3.6. Thermal Properties Analysis*

The thermal and retrogradation properties of starch and starch–betanin determined by DSC are presented in Table 4. The presence of betanin increased the gelatinization transition temperatures (To, Tp and Tc) of all starches, and it was implied that starch gelatinization was delayed by betanin, which might be related to the strong hydrophilicity of betanin and its ability to hinder starch from absorbing water. Δ*Hg* represents the required thermal energy for melting the double-helix structure and destroying crystallinity within the starch granule [31]. In comparison to pure starches, a lower Δ*Hg* for starch–betanin was found. Similar results were reported in our previous paper [14], in which the addition of polymeric proanthocyanidin decreased the gelatinization enthalpy of RS, PoS and PeS. Simultaneously, betanin significantly delayed the long-term retrogradation of the three starches, resulting in a reduction in the retrogradation enthalpy (Δ*Hr*) and retrogradation rate (*R*). After incorporating betanin, the *R* of RS, PoS and PeS declined from 34.13%, 42.95% and 31.82% to 23.24%, 31.48% and 25.80, respectively. It was indicated that betanin affected the formation and weakened the order degree of crystallinity during the long-term retrogradation of starches, which conformed to the results of the XRD and FITR analyses. In light of these results, it is known that betanin could be used as an excellent inhibitor of retrogradation in products such as bread and pastry.


**Table 4.** Thermal properties of starches and starch–betanin samples after retrogradation for 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. To: onset temperature; Tp: peak temperature; Tc: conclusion temperature; Δ*Hg*: gelatinization enthalpy; Δ*Hr*: retrogradation enthalpy; *R*: the degree of retrogradation (Δ*Hr*/Δ*Hg*) \* 100.

#### *3.7. Morphology Analysis*

Microstructures of the retrograded starches and starch–betanin samples were examined through SEM to observe the changes from adding betanin, and images are depicted in Figure 6. The retrograded RS displayed a dense surface with a uniformly distributed cavity, and the retrograded PoS and PeS presented relatively rough network-like structures with some fragments. Nevertheless, the incorporation of betanin altered the formation of the gel network of the retrograded starches, and the retrograded starch–betanin samples displayed more porous and loose structures. In comparison with the morphology of the retrograded starches, RS–betanin and PoS–betanin showed an increased cavity volume, and porous spongy-like morphology was observed for PeS–betanin. This phenomenon was similar to the report by Xu et al. [32], who found *Vaccinium bracteatum Thunb. leaf* pigment loosened matrices of rice starch gels. The microstructural changes reflected that the addition of betanin affected the gel network of the retrograded starches. That is, betanin might inhibit the long-term retrogradation of starches, which coincides with the results of the aforementioned investigations.

**Figure 6.** SEM photographs of starches and starch–betanin samples after retrogradation for 7 days. Magnification was 100. RS: rice starch; PoS: potato starch; PeS: pea starch.

#### **4. Conclusions**

The pasting, rheology and retrogradation properties of rice, potato and pea starches were changed by the presence of betanin. Betanin decreased the peak, trough and final viscosity of rice and potato starches, but increased these of pea starch. Rheological properties including thixotropy, the dynamic modulus and the loss factor of the three starches were varied by different degrees after incorporating betanin. Betanin endowed starch pastes with a vivid red appearance and maintained color during storage. Furthermore, the poor short-range molecular order, low crystallinity and low retrogradation enthalpy of starches were induced by betanin during retrogradation, suggesting that betanin could inhibit the retrogradation of starches. The micromorphology of the retrograded starches was also altered by betanin. These findings provide guidance for the application of betanin in developing foods that require colorant and an inhibitor of starch retrogradation.

**Author Contributions:** Conceptualization, T.D., J.C. and C.L. (Changhong Li); methodology, X.H. (Xiaohong He), J.X. and T.D.; investigation, X.H. (Xuemei He), Q.G. and X.H. (Xiaohong He); data curation, X.H. (Xuemei He) and T.D.; writing—original draft preparation, X.H. (Xiaohong He), T.D. and C.L. (Changhong Li); writing—review and editing, J.S., C.L. (Chengmei Liu) and J.C.; visualization, J.S. and X.H. (Xuemei He); supervision, T.D. and C.L. (Chengmei Liu); funding acquisition, J.S. and T.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the China Postdoctoral Science Foundation [Grant No. 2020M6832211], Postdoctoral Foundation of Guangxi Academy of Agricultural Sciences (Grant No. 2020037), Guangxi Natural Science Foundation (Grant No. 2022GXNSFBA035522), Guangxi Key Laboratory of Fruits and Vegetables Storage-processing Technology Project (20-065-68), National Natural Science Foundation of China (Grant No. 32101948), China Agriculture Research System (CARS-31-12), and Special Fund for Guangxi Bagui Scholars (Grant No. (2016)21).

**Data Availability Statement:** The data presented in this study are available in this article.

**Acknowledgments:** The authors would like to thank the Centre of Analysis and Testing of Nanchang University and State Key Laboratory of Food Science and Technology for their expert technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.
