*2.5. Oxidative Stability*

The anti-oxidation capability to avoid CBLO oxidation is an important indicator for CS@CBLO NPs. CBLO is highly susceptible to oxidation due to its large number of PUFAs. In the early stage of oil oxidation, oxygen directly interacts with conjugated diene to form hydroperoxides [44]. FTIR spectroscopy has been used in the past to identify changes in functional groups in samples that have undergone lipid oxidation [45]. Non-oxidized oils show a narrow weak band in the region of 3400–3500 cm−1, with maximum absorbance wavenumbers around 3470 cm<sup>−</sup>1, which are assigned to the overtone of the glyceride ester carbonyl absorption [46]. However, the hydroperoxides generated in the oxidation process cause the maximum absorption to shift to lower wavenumbers [47]. The oxidative stability of CBLO and CS@CBLO NPs during the storage period was observed by FTIR. As Figure 7a shows, the FTIR spectra of CBLO obviously change with the storage time. The maximum absorbance wavenumbers shifted from 3473 to 3421 cm−<sup>1</sup> as the storage time increased from 1 to 28 d. The peak shift of CBLO was due to the formation of hydroperoxides during storage. In contrast, the FTIR spectra of CS@CBLO NPs (Figure 7b,c) in the region of 3400–3500 cm−<sup>1</sup> were more stable and without significant shifts or changes. This result indicated that CS@CBLO NPs had a lower hydroperoxide formation during storage compared to CBLO. The ratio of maximum absorbance wavenumber changes in the region of 3400–3500 cm−<sup>1</sup> during storage can be seen in Figure 8. During the first two weeks of storage, CBLO showed little change, but by day 16 the formation of hydroperoxides caused a significant decrease in the ratio of the maximum absorbance wavenumber. At the same time, the ratio of maximum absorbance wavenumber for CS@CBLO NPs was only slightly decreased, supporting the notion that it had better antioxidative ability. On the other hand, the wavenumber at 967 cm−<sup>1</sup> was associated with the bending vibrations of CH functional groups of isolated trans-olefins; the increase in trans double bonds during thermal oxidation can be observed by the increasing peak intensities at 967 cm−<sup>1</sup> [48]. The wavenumbers at 973 and 976 cm−<sup>1</sup> can be assigned to secondary oxidation products, such as aldehydes or ketones, supporting isolated trans-double bonds [49]. As Figure 9a shows, the peak intensities of CBLO at 967, 973, and 976 cm−<sup>1</sup> increase with storage time, while the peak intensities of CS@CBLO NPs are almost flat with storage time. These results suggest that CBLO produced more trans double bonds and secondary oxidation

products. Several studies have demonstrated that chitosan has good antioxidant properties, especially antioxidant activity, scavenging ability on hydroxyl radicals and chelating ability on ferrous ions [50–52]. In this paper, we clearly found that CS@CBLO NPs showed lower lipid hydroperoxides and secondary oxidation products, which might be attributed to chitosan providing good protection against CBLO oxidation.

**Figure 7.** The changes of FTIR spectra at the region around 3400 to 3500 cm−<sup>1</sup> during storage at room temperature for four weeks: (**a**) CBLO, (**b**,**c**) CS@CBLO NPs prepared using the weight ratios of CBLO to chitosan of (**b**) 1.00:1, and (**c**) 1.25:1.

**Figure 8.** The ratio of maximum absorbance wavenumber in the region of 3400–3500 cm−<sup>1</sup> during storage at room temperature for four weeks.

**Figure 9.** The changes in the FTIR spectra at the wavenumber of 967 and 976 cm−<sup>1</sup> during storage at room temperature for four weeks: (**a**) CBLO, (**b**,**c**) CS@CBLO NPs prepared using the weight ratios of CBLO to chitosan of (**b**) 1.00:1, and (**c**) 1.25:1.
