*2.4. Characterization XRD and FTIR Spectroscopy*

The crystal structure of chitosan powder, CS NPs, and CS@CBLO NPs was analyzed using the XRD technique. As Figure 5a shows, chitosan exhibits the main diffraction peak at 20.3◦, indicating the high degree of crystallinity. After the electrostatic interaction with TPP, no peak was found in the diffractograms of CS NPs (Figure 5b). A quite flat diffraction pattern was obtained, indicating an amorphous structure. The width of the peaks in the XRD pattern is related to the grain size of the crystallites, and the broadened peaks are

usually caused by imperfect crystals [42]. Therefore, the broad peak of CS NPs might be caused by ionic gelation with TPP, which did not allow a regular arrangement of the polymer network, leading to its amorphous structure [43]. Compared to chitosan and CS NPs, the characteristic peak of CS@CBLO NPs (Figure 5c) slightly shifted to 18.8◦ and was markedly sharp, confirming the presence of CBLO within CS NPs. This result also confirmed that the incorporation of CBLO caused a change in the packaging structure of chitosan-TPP.

**Figure 5.** The XRD patterns of (**a**) chitosan powder, (**b**) CS NPs, and (**c**) CS@CBLO NPs.

The results of the FTIR showing the characteristic spectra of CBLO, CS NPs, and CS@CBLO NPs are presented in Figure 6. Figure 6a shows the characteristic peaks of CBLO appearing at 3473 cm−<sup>1</sup> (C=O overtone), 3008 cm−<sup>1</sup> (=CH stretching), 3000–2800 cm−<sup>1</sup> (CH stretching), 1743 cm−<sup>1</sup> (C=O stretching band), 1464 cm−<sup>1</sup> (–CH2–bending), 1377 cm−<sup>1</sup> (–CH3 bending), and 964 cm−<sup>1</sup> (C=C stretching band). On the other hand, CS NPs shows the characteristic peaks of chitosan and TPP in Figure 6b. The characteristic peaks of CS NPs were found at 3467 cm−<sup>1</sup> (OH stretching), 1655 cm−<sup>1</sup> (amide I stretching), 1541 cm−<sup>1</sup> (amide II stretching), 1155 cm−<sup>1</sup> (P=O stretching), 1095 cm−<sup>1</sup> (C–O–C stretching), and 899 cm−<sup>1</sup> (P-O-P stretching). Compared to CBLO and CS NPs, all the characteristic peaks of both appeared in the FTIR spectra of CS@CBLO NPs (Figure 6c–g), indicating no modification or interaction between the CBLO and chitosan or TPP. In particular, the characteristic peaks of CS@CBLO NPs located at 2924–2854 cm−<sup>1</sup> (CH stretching), and 1743 cm−<sup>1</sup> (C=O stretching band), assigned to the methylene and carbonyl group of triglycerides, significantly increased in intensity with an increased ratio of CBLO to chitosan. This result not only reflected the presence of CBLO in CS@CBLO NPs but also showed the content of CBLO in CS@CBLO NPs. Compared to the results of LC in Table 1, the LC of CBLO increased with the increased ratio of CBLO to chitosan. Therefore, the peaks of CH stretching at 2924–2854 cm−<sup>1</sup> and the C=O stretching band at 1743 cm−<sup>1</sup> can be used as an indicator to represent the content of CBLO loaded into chitosan nanoparticles. With the analysis of XRD and FTIR, the two-step method through emulsification and ionic gelation is shown to be suitable for encapsulating CBLO in CS@CBLO NPs.

**Figure 6.** FTIR spectra of (**a**) CBLO, (**b**) CS NPs, and (**c**–**g**) CS@CBLO NPs prepared using different weight ratios of CBLO to chitosan of (**c**) 0.25:1, (**d**) 0.50:1, (**e**) 0.75:1, (**f**) 1.00:1, and (**g**) 1.25:1.
