Physicochemical and antioxidant properties of Gracilaria changii hydrolysate produced via hydrothermal acid pretreatment and dual-enzymatic hydrolysis

Gracilaria changii is a rich source of bioactive compounds, yet its utilization in Malaysia remains limited. Its rigid cell wall restricts bioavailability, necessitating effective strategies to explore its functional potential for food and nutraceutical applications. This study investigated the effects of hydrothermal acid pretreatment and dual-enzymatic hydrolysis on the physicochemical and antioxidant properties of G. changii hydrolysate. Mild hydrothermal pretreatment using citric acid (S1; 0–0.25 M, 15–60 min) was followed by sequential enzymatic hydrolysis with pectinase (S2; 0–20%, 2–24 h) and Flavourzyme (S3; 0–2%, 0–4 h). Total reducing sugar (TRS) and soluble protein content (SPC) increased across processing steps, reaching 50.28% (31.03-fold in S1 and 1.65-fold in S2) and 9.25% (1.78-fold in S3), respectively. The overall fold increase in total phenolic content (TPC) reached 14.32-fold (28.45 mg GAE/g), while total flavonoid content (TFC) increased by 19.50-fold to 1.56 mg QE/g. Antioxidant activity measured by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays increased by 15.68-fold (75.42 mM TE/g) and 7.10-fold (28.13 mM AAE/g), respectively. Monosaccharide analysis revealed elevated levels of galactose (35.43%), glucose (11.95%), and galacturonic acid (9.16%) in the S2 treatment, whereas amino acid profiling showed a marked increase in essential (4.34%) and non-essential (5.51%) amino acids, particularly glutamic acid, aspartic acid, alanine, and glycine, following the S3 treatment. Overall, the combined strategy enhanced the biochemical composition and antioxidant properties of G. changii by employing technically feasible processing steps, thereby supporting its scalability for functional food development and production.