*2.10. Data Analysis*

All tests were repeated three times with mean ± standard deviation results. Duncan's test was used to analyze the data in SPSS software (Version 21, IBM SPSS Inc, New York, NY, USA), and the differences were considered significant if *p* < 0.05.

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

### *3.1. Characterization of the BAL Liposomes*

The results of different liposomes with average particle sizes, Zeta potentials, PDI values, microstructures, and EE values are shown in Table 1. With the addition of lecithin, the average particle sizes of the liposomes obviously increased from 131.39 nm to 311.42 nm, which was attributed to the amount of hydrogen and van der Waals force between the anthocyanins and lecithin [18]. Zeta potential is an important parameter to characterize the stability of liposomes. The higher value of Zeta potential, the greater repulsion strength required to settle and coagulate liposomes [19]. The Zeta potentials of BAL1 and BAL2 were −48.23 mV and −40.16 mV, respectively, indicating the stable dispersion of liposome particles in the solution. PDI is an index that reflects the particle size distribution [20]. The smaller the PDI, the better the regularity of dispersion of the particles. A PDI < 0.4 indicates a homogenous particle size distribution in the system [21]. With the addition of lecithin, the PDI increased from 23.96% to 29.51%, indicating the heterogeneous size distribution. This was consistent with the Zeta potential results. These structure formations can also be observed in the microstructures of the multi-compartmental but obvious core–shell structures. Thus, the EE increased from 36.06% to 46.99% with the increasing ratio of lecithin. The above results indicated that the ratio of lecithin was one of the key factors in the characterization of anthocyanin-loaded liposomes.

**Table 1.** The sizes, Zeta potentials, PDI values, EE values, and microscope pictures of the liposomes.


Note: the superscripted characters a, b, c represent significant differences (*p* < 0.05).

#### *3.2. The pH Response of Anthocyanin-Loaded Liposomes*

As shown in Figure 1A, both anthocyanins and liposomes showed obvious color changes in different pH values. The color of BA changed from pink to purple, then blue, and finally blue-green. The color of BAL1 changed from pink to purple-green, then cyan, and finally green. The different color changes of anthocyanin were caused by structural transformations, which were found in a previous study [13]. In fact, the different color changes between BA and BAL were attributed to the cavity structure of liposomes, which

decreases the structural transformation rate in anthocyanins [22]. As shown in Figure 1B, 2 characteristic absorption peaks can be observed around 574 nm and 620 nm for BA and BAL. At pH 2, the absorption peak of BA was at 552 nm and gradually red-shifted to 574 nm at pH 3–8. With the pH increasing to 9–10, the absorption peak disappeared due to the destroyed structure of the anthocyanin molecular center ring under strong alkaline conditions [23]. The response mechanism of BAL to pH was consistent with that of the anthocyanin solution. However, the peak at 574 nm disappeared at pH 8 for the BAL1 spectrum while occurring at pH 7 for the BAL2 and BAL3 spectrums, respectively. This is mainly because of the encapsulation difference. The ratio of A620 to A574 reflected the shift changes of the absorption peaks in Figure 1C. This was clearly observed in the variation of the maximum values of the BA and BAL spectra. The above results showed that the coloration degree of the solution obviously decreased after being encapsulated by liposomes, while the color sensor function of the anthocyanins was not hindered.

**Figure 1.** Color (**A**) and ultraviolet-visible spectra (**B**), and the ratio of A620 and A574 (**C**) of BA and BAL at different pH values.

#### *3.3. The Structural Analysis of the Bi-Layer Films*

#### 3.3.1. SEM Analysis of Indicator Films

The film compatibility can be observed in the cross-section of a bi-layer film. As can be seen in Figure 2, all the films presented an obvious two-layer structure, which was attributed to the hydrogel thermal irreversibility processes between agar and carrageenan. Meanwhile, hydrogen bonding, cross-linked agar, and carrageenan prevented the bi-layer films from separating. The agar outer layers appeared relatively uniform except for the parts that were contaminated by the inner anthocyanin layers. In Figure 2A, the A-CBA film with free anthocyanins displays a homogeneous and compact structure. Compared with the free anthocyanins, the liposomes with hydrophobic structures of the A-CBAL film caused a reduction in the cross-linking between the film-forming solution and water molecules. Therefore, the liposomes in the film-forming matrix presented a lower homogeneous dispersion. However, there were no obvious differences between the A-CBAL films, indicating that the anthocyanin encapsulation of liposomes hardly presented a negative effect on the film morphologies. Importantly, the above results indicated that bi-layer films were satisfactorily prepared.

**Figure 2.** Cross-section SEM images of A-CBA (**A**), A-CBAL1 (**B**), A-CBAL2 (**C**), and A-CBAL3 films (**D**), and FTIR spectra (**E**) of colorimetric films.
