Optimization of hydrolysis conditions to enhance solubility of hempseed (Cannabis sativa L.) protein isolate using response surface methodology for functional beverage application

Hempseed protein isolate (HPI) is a non-allergenic, nutritionally balanced protein source with promising potential in food applications. However, its poor solubility presents a major limitation, particularly in food systems requiring high protein dispersion and stability. Enzymatic hydrolysis, a process that cleaves proteins into smaller peptides, has been shown to modify protein structure and enhance functional properties such as solubility. In this study, response surface methodology (RSM) was employed to optimize enzymatic hydrolysis conditions for HPI, targeting maximum solubility. Independent variables included treatment time (120-240 min), enzyme concentration (0.5-2.5 mg/mL), pH (7.0-9.0), and substrate concentration (0.07 g/mL). The optimal hydrolysis conditions were identified as a treatment time of 215 min, enzyme concentration of 1.75 mg/mL, and pH 8.7, achieving a solubility of 53.23%. The enzymatic hydrolyzed HPI (EH) exhibited a degree of hydrolysis (DH) of 35.44% and ABTS radical scavenging activity of 94.28%, significantly outperforming untreated HPI (7.63% DH and 23.81% scavenging activity). The optimized formulation was tested in a beverage product, where the beverage made with EH showed improved stability and color retention (ΔE: 0.71-4.60) compared to beverages made with untreated HPI (ΔE: 1.50-15.17). These findings provide a theoretical basis for the enzymatic hydrolysis of HPI and expand the application potential of HPI in food development and industrialization by establishing conditions for optimizing the solubility.