*4.1. C. vulgaris Hydrolysates Production*

*C. vulgaris* was supplied, as powder, by AllMicroalgae—Natural Products, SA (Pataias, Portugal). The *C. vulgaris* hydrolysates were produced by an acid pre-treatment followed by an enzymatic hydrolysis, using a previously optimised method [41]. Briefly, C. vulgaris (Table 7) was mixed with an acetic acid solution (2% in deionised water) in a ratio of microalgae:water of 1:3 (*w*/*v*). The mixture was incubated for 1 h at 50 ◦C and 125 rpm in an orbital shaker (ThermoFisher Scientific, Waltham, MA, USA, MaxQ™ 6000). Then, deionised water was added until microalgae:water ratio reached 1:10 and the pH was adjusted to 7.5. For the enzymatic hydrolysis, first, 5% cellulase was added and the mixture was incubated for 2 h at 50 ◦C and 125 rpm. Secondly, 3.9% subtilisin was added and the mixture was incubated for 2 h at 40 ◦C at 125 rpm. During the enzymatic hydrolysis, pH was constantly verified and adjusted to 7.5, mainly in the subtilisin hydrolysis step. To stop the hydrolysis reaction, the mixture was incubated at 90 ◦C for 10 min to inactivate the enzymes. The resulting solution was centrifuged at 5000× *g* for 20 min, and both the water-soluble peptide-enriched supernatant (Table 8) and the pellet were collected and freeze-dried for further analysis.


**Table 7.** Microalgae *Chlorella vulgaris* biomass composition (prior to extraction).

**Table 8.** *Chlorella vulgaris* soluble extract protein concentration and in vitro bioactivities.

