*2.8. Scanning Electron Microscopy (SEM)*

Morphological properties of the retrograded starches and starch–betanin samples were observed using SEM (JSM 6701F, JEOL, Japan). The longitudinal section of each freeze-dried sample was fixed on a metal sample holder using double-backed cellophane tape, and then sprayed with a layer of gold to a level of 250–500 nm at an operating voltage of 5 kV. The magnification was 100 times.

#### *2.9. Statistical Analyses*

The results were expressed as means ± standard deviation (SD) of triplicate analyses for each sample. Data analysis adopted Duncan's test using SPSS 24.0 statistical software, and differences were considered to be significant at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Pasting Properties of Starches*

The pasting properties of different starches and starch–betanin samples were shown in Figure 1 and Table 1. RS and PoS exhibited typical pasting curves with significant convex and concave peaks. Upon heating, the starches swelled early and leached out amylose, which resulted in an increase in viscosity and displaying PV. The PV value of RS and PoS was 1032 and 3839 mPa·s, respectively. When constantly shearing at 95 ◦C, starch granules were disintegrated, and the BD value represented the degree of disintegration. The BD value of RS and PoS was 124 and 2353 mPa·s, respectively. Subsequently, cooling promoted the rearrangement of amylose molecules, and short-term retrogradation occurred. However, in the pasting patterns of PeS, the concave peak was extremely weak (Figure 1) and the TV value was close to that of the PV, accompanied by a very small BD value. Generally, pea starches were characterized by a high amylose content [22]. As reported by Han et al. [23,24], pasting properties of starches are related to their amylose content. The discrepancy of pasting patterns between RS, PoS and PeS may be affected by their amylose content, resources and other inherent characteristics of starch.

For starch–betanin samples, the combination with betanin affected pasting properties in varying degrees, and the change in pasting parameters for PoS was the biggest. Meanwhile, betanin lowered the PV, TV, BD and FV of RS and PoS. On the contrary, these parameters of PeS were slightly increased. The carboxylate groups in betanin interacted with phosphate groups in PoS, which impeded the absorption of water, swelling of PoS, and leaching out of amylose, thus significantly reducing the viscosity parameters of PoS. In comparison with PeS, RS, with a relatively high content of lipids, was prone to form a complex with betanin; this situation was also not conducive for RS to absorb water and gelatinize. Nevertheless, PeS had a high amylose content and steric hindrance owing to the presence of betanin was beneficial for the interactions between amylose molecules during gelatinization. Despite the increasing or decreasing of the viscosity parameters of starches, the combination with betanin just slightly elevated the SB value of the three starches, indicating the weak ability of betanin to inhibit the short-term retrogradation of starches.

**Figure 1.** RVA pasting profiles of starches and starch–betanin samples. RS: rice starch; PoS: potato starch; PeS: pea starch.


**Table 1.** Pasting properties of starches and starch–betanin samples.

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.

#### *3.2. Rheological Properties of Starches*

#### 3.2.1. Steady Shear Rheological Properties

The steady shear rheology curves of starch and starch–betanin pastes are shown in Figure 2A. RS and PoS exhibited typical upward and downward curves as a function of shear rate, and hysteresis loops were observed. However, PeS showed an irregular profile of shear stress, and it was difficult to obtain a hysteresis loop. It is likely that PeS pastes easily and rapidly formed a fragile gel during measurement due to their high content of amylose. The shear stress of RS–betanin and PoS–betanin pastes were significantly lower than that of RS and PoS at the shear rate ranges of 0.01~1000 s−<sup>1</sup> and 1000~0.01 s−1; that is, the corresponding apparent viscosity of RS and PoS was lowered when combined with betanin. As for PeS–betanin, typical curves of shear stress and a small hysteresis loop (8943.9 Pa·s<sup>−</sup>1) were present, indicating that betanin improved the shear stability of PeS. Additionally, the hysteresis loop area of RS–betanin (30,553.2 Pa·s<sup>−</sup>1) and PoS–betanin (25,944.7 Pa·s<sup>−</sup>1) was smaller than RS (31,483.2 Pa·s<sup>−</sup>1) and PoS (52,972.6 Pa·s<sup>−</sup>1), respectively (Table 2). Betanin had the biggest effect on the hysteresis loop area of PoS, as the hysteresis loop area was reduced by 40.6%, which indicated that betanin reduced the thixotropy and enhanced the shear stability of starch paste, especially for PoS. The fitting results of the shear stress curves to the power law model are shown in Table 2. Owing to an irregular shear stress curve, PeS was unable to fit the model. Except for PeS, the *R*<sup>2</sup> of shear stress curves for other samples was greater than 0.97, indicating the high fitting accuracy to the power law

model. The fluid indexes *n* of the fitted starches and starch–betanin pastes were all less than 1, indicating that they were typical pseudoplastic fluids with shear thinning behavior. Betanin seemed to have no effect on the *n* of RS and PoS, but reduced their consistency coefficient *K*. It was implied that betanin weakened the thickening property and enhanced the pseudoplasticity of RS and PoS. Meanwhile, betanin endowed PeS with shear thinning behavior, and the biggest *K* and the smallest *n* were presented for PeS–betanin.

**Figure 2.** Shear rheology curves (**A**), storage moduli (G- ) (**B**), loss moduli (G") (**C**) and loss tangents (tanδ) (**D**) of starches and starch–betanin samples determined by rheological measurements. RS: rice starch; PoS: potato starch; PeS: pea starch.



RS: rice starch; PoS: potato starch; PeS: pea starch; *K*: the consistency coefficient; *n*: the flow behavior index.

#### 3.2.2. Dynamic Rheological Properties

A dynamic frequency sweep range from 0.1 to 20.8 Hz was employed to investigate the viscoelastic properties of starch and starch–betanin pastes, and their storage modulus (G- ), loss modulus (G") and loss tangent (tan δ) as functions of frequency were depicted in Figure 2B–D, respectively. Owing to the unstable state in the measurements of initial small frequency, the results were recorded from 0.6~20.8 Hz. G and G" represent the elasticity and viscosity of the tested samples, and tanδ represents the ratio of G" and G- , which can be used to explain the viscoelastic behavior [25]. G and G" of all samples were increased along with the frequency, and G was bigger than G", indicating that starch and starch–betanin were typical weak gels. Compared to RS and PoS, PeS had the highest moduli, with the reason possibly being that the high content of amylose contributed to promoting the crosslinking of gel networks. The combination with betanin affected the dynamic rheological properties of starches, and the change in the degree of PoS was the most significant, which was similar to that of the pasting properties. Furthermore, the effect of betanin on G was stronger than that of G"; thus, betanin performed a more effective influence on the elasticity than on the viscous properties of RS, PoS and PeS. The tanδ values of RS, PoS and PeS were less than 1 over the whole frequency range, and PeS exhibited the lowest tanδ values, showing the strongest elastic behavior. The addition of betanin decreased the tanδ value of starches; that is, starch–betanin exhibited lower tanδ values than that of the corresponding starch. The effect of betanin on the tanδ of PoS was the biggest, and the smallest effect was observed on the tanδ value of PeS. The decreased tanδ values implied that the structure of the gel network was enhanced, which was probably attributed to increasing the junctions or crosslinking between amylose and swollen fragments caused by the presence of betanin. The result demonstrated that betanin changed the network structure of PoS pastes to a greater extent compared to RS and PeS, which was consistent with the result of the steady shear rheological properties.
