Identification and biofunctionality profiling of peptides from Arthrospira platensis and Tetraselmis chuii: In silico predictions and in vitro validation

To address the growing demand for sustainable sources of health-promoting biomolecules, this study aimed to identify and characterize bioactive peptides from two protein-rich and commercially relevant microalgae: Arthrospira platensis and Tetraselmis chuii. These species were selected based on their high protein content, ecological sustainability, and potential as underexplored reservoirs of functional peptides. Proteins were extracted from dried biomass using an optimized ultrasonication and hydrolyzed with pepsin at an enzyme-to-substrate (E/S) ratio of 1:4. The resulting protein hydrolysates of A. platensis (APPH) and T. chuii (TCPH) were analyzed using RP-HPLC-UV and fractionated into nine fractions (F₁ to F₉). Peptides were identified using RP-HPLC-MS/MS, revealing 261 unique peptides in APPH and 99 in TCPH. In silico predictions of physicochemical properties, sensory attributes (umami, bitterness), and biological activities (antioxidant, antihypertensive, anti-inflammatory, antimicrobial, and neuroactive) were performed using machine learning tools. These were validated by in vitro assays, including DPPH radical-scavenging, ferrous-ion chelation, ACE and DPP-IV inhibition, hyaluronidase inhibition, and antibacterial testing. Peptide fractions from APPH consistently exhibited higher bioactivities than those from TCPH. In particular, APPH fractions F₄ and F₅ demonstrated the most potent activities, with 67.8 ± 0.8 % ACE inhibition, 50.33 ± 0.80 % DPP-IV inhibition, 37.90 ± 1.57 % anti-inflammatory activity, and 66.2 ± 3.7 % DPPH scavenging. These findings highlight A. platensis as a particularly promising source of multifunctional peptides. This integrated proteomic, computational, and functional framework offers a scalable pipeline for microalgal peptide discovery and supports the development of innovative functional foods and nutraceuticals.