*3.6. Storage Stability of HS-Loaded Liposomes and Chitosomes*

Regarding our previous research on the effect of temperature on the physical stability of vitamin D3-loaded liposomes and the instability and aggregation of the formulation at ambient temperature due to higher fluidity of the lipid bilayer and higher loss of encapsulated bioactive material, refrigerator temperature (4 ◦C) was selected to examine the physical stability of selected formulations [7]. Liposomes as vesicular droplets have permeable and flexible bilayers and a high tendency to fuse and aggregate, resulting in the release of encapsulated bioactive materials during storage. In this study, stabilizing agents (cholesterol and γ-oryzanol) and coating material (chitosan) were tested to enhance the physical stability of liposomes. HS-loaded γ-oryzanol-liposomes and the corresponding chitosomes had no marked difference (*p* < 0.05) in magnitude, PDI, and ζ after 30 days of storage at 4 ◦C (Table 3). Moreover, both samples preserved more than 90% of their initial antioxidant activity in ABTS radical scavenging activity (*p* < 0.05). When the storage time was expanded, slight lipid oxidation may have occurred in unsaturated fatty acids of the phospholipid bilayers, leading to decreased antioxidative activity of the tested formulations. On the other hand, no significant precipitate was observed, especially in the HS-loaded chitosome system, during storage. When storage time was expanded to 2 months, the chitosome system showed higher physical stability compared to the uncoated liposomes. In contrast, the stability of HS-loaded cholesterol-liposomes in terms of mean diameter, PDI, and ζ was much lower than that of HS-loaded γ-oryzanol-liposomes at 4 ◦C. When storage time was expanded, the mean diameter and PDI of these formulations significantly increased (*p* < 0.05), and observable precipitates were separated into round-bottomed Falcon tubes.


**Table 3.** Physical stability of hydrolysate-loaded γ-oryzanol-liposomes and chitosomes during one month at 4 ◦C.

Means in same column with different superscripts (a, b, c, d) are statistically different (*p* < 0.05).

On the other hand, the residual antioxidant activity of HS-loaded cholesterol-liposomes was 50% of its initial antioxidant activity. This observed instability of HS-loaded cholesterolliposomes could have resulted from the poor coating of liposomal space cores with cholesterol stabilizing agents, and possible replacement of empty spaces with hydrophilic domain residues (glycine, arginine, and lysine) of hydrolysates. Nevertheless, some hydrophobic domains of *Spirulina* hydrolysates could interact with acyl chains of the lipid bilayer of liposome through hydrophobic binding, leading to more flexibility and fluidity of liposomes [32]. HS-loaded γ-oryzanol-chitosomes showed better results in terms of particle size, PDI, aggregation, and antioxidant activity compared to HS-loaded cholesterolliposomes during storage. In summary, HS-loaded γ-oryzanol-liposomes and HS-loaded γ-oryzanol-chitosomes showed promising storage stability with no precipitate or change in mean diameter and a slight decrease in antioxidant activity at 4 ◦C. Thus, γ-oryzanol as a stabilizing agent and chitosan polymer as a coating material were able to reduce membrane fluidity and flexibility, contributing to stability.

In another study, different stabilizers (cholesterol and glycerol) were used to encapsulate peptides obtained from defatted Asian sea bass skin. Regarding the results, both formulations showed small particle size and high encapsulation efficiency. However, after the lyophilization process, hydrolysate-loaded cholesterol-liposomes showed higher stability and higher antioxidant activity in the gastrointestinal tract than lyophilized glycineliposomes during storage at 25 ◦C for 28 days [24]. It was reported that curcumin-loaded chitosomes showed higher physical stability than curcumin-loaded uncoated liposomes due to the lower flexibility of chitosan-coated liposomes and, as a result, lower membrane fusion between droplets [33].

#### *3.7. Production Yield and Physicochemical Properties of Spray-Dried Chitosomes*

The powder moisture content was found to be 4.65 ± 0.61%, which is lower than the specified minimum moisture content of many powders used in food applications to inhibit microbiological spoilage and lipid oxidation and extend the product's shelf life.

The bulk density of the powder was 0.31 ± 0.01 g cm−3. A higher bulk density has several advantages, including more convenient storage conditions due to lower space requirements for storage and greater protection against oxidation during storage due to the presence of less air in powder. In addition, the solubility and production yield of powder were found to be 96% and 57%, respectively.

## *3.8. Microstructure and Particle Size Distribution*

The morphology of dried particles is mostly affected by the evaporation rate and viscoelastic properties of shell material. SEM images (Figure 4) show mostly spherical particles with little evidence of roughness or fracturing in the microcapsules. Moreover, most of the spray-dried powders showed a well-defined spherical shape with a particle size of around 1–3 μm, which is smaller than the threshold diameter used in food fortification (10–50 μm) [34]. The coarse powder results in a sandy and unfavorable mouthfeel in the fortified food products. These results are in accordance with those reported by Sarabandi et al. [13].

**Figure 4.** (**a**) SEM image of spray-dried hydrolysate-loaded γ-oryzanol-chitosomes; (**b**) particle size result of reconstituted γ-oryzanol-chitosome powder.
