*2.10. Characterization of Spray-Dried Powder*

Production yield was measured by calculating the mass ratio of the produced powder to the total solid content in the feed. Other physical properties of the spray-dried powder, such as moisture content, bulk density, and solubility, were computed using the methods described by Sarabandi et al. [20].

The particle morphology of spray-dried powder was evaluated by scanning electron microscopy (SEM; MIRA3, TESCAN, Brno, Czech Republic).

To determine the size, polydispersity, and ζ of reconstituted nanoliposomes, the powder was dissolved in an appropriate concentration and its particle size and ζ potential were determined by a zetasizer.

#### *2.11. Statistical Analysis*

Statistical analysis was performed using SPSS software (version 24.0, IBM, Chicago, IL, USA). Normal distribution and variance homogeneity had been previously tested (Shapiro– Wilk). Data of 3 repetitions were subjected to analysis of variance (ANOVA), followed by Tukey's test at a 5% significance level.

## **3. Results and Discussion**

#### *3.1. Characterization of Hydrolyzed Spirulina Protein (HS)*

Extracted *Spirulina* isolate was hydrolyzed by pepsin enzyme. The hydrolysis degree was found to be 16.5% over 4 h hydrolysis time. The solubility of hydrolysate under harsh acidic conditions was improved after enzymatic hydrolysis, but the highest solubility was obtained under alkaline pH conditions.

Figure 1 shows the amino acid composition of pepsin-hydrolyzed peptides. According to the obtained profile, the hydrolysate was rich in acidic amino acids (aspartic and glutamic acid), arginine, valine, lysine, alanine, glycine, threonine, and leucine. All the essential amino acids (except sulfur-containing amino acids) were present in the hydrolysate at concentrations higher than the FAO recommended levels for adults. Moreover, the hydrolysate showed good nutritional value as determined by amino acid score (72%) and biological value (78%), and good antioxidant activity (IC50 1 mg/mL).

**Figure 1.** Amino acid composition of pepsin-hydrolyzed peptides: (1) aspartic acid; (2) glutamic acid; (3) asparagine; (4) histidine; (5) serine; (6) glutamine; (7) arginine; (8) glycine; (9) threonine; (10) alanine; (11) tyrosine; (12) methionine; (13) valine; (14) phenylalanine; (15) isoleucine; (16) leucine; (17) lysine; (18) tryptophan.

Compared to native protein (with a DPPH IC50 value of 3 mg/mL), the hydrolysates had a significantly lower IC50 value of 1.0 mg/mL, indicating their effectiveness in scavenging DPPH radicals. The ABTS IC50 of hydrolysate was estimated to be 2 mg/mL. Compared with native protein (with an IC50 value of 4.5 mg/mL), the hydrolysates had lower IC50 values, indicating their effectiveness against ABTS radicals.

Encapsulating these bioactive compounds in lipid-based nanocarriers such as liposomes can be an appropriate solution to cover all of the mentioned problems and increase their efficacy under different conditions.
