Membrane-based separation and enzymatic hydrolysis of Whey protein concentrate for antioxidant peptide production

Whey production is estimated at 190 million tons/year, with about 50% discarded as effluent into soil or rivers, causing soil alterations and negatively impacting aquatic life. An alternative to reduce the environmental impact of this by-product is producing protein isolate (PI) with bioactive properties. This study aimed to evaluate the relationship between the degree of hydrolysis (%DH) of whey protein and the antioxidant activity (AA) of peptides obtained using Trypsin, Bromelain, and Papain under optimal enzymatic conditions. A multi-step separation process—including skimming, microfiltration, ultrafiltration, and diafiltration—was implemented to obtain a purified protein isolate (PI) before enzymatic hydrolysis. The PI was then hydrolyzed at a constant pH. The %DH was determined using both the pH-stat and OPA methods. AA was assessed using the ABTS method. Microfiltration and ultrafiltration effectively concentrated and purified the PI, reaching a soluble protein concentration of 23.24 ± 0.05 mg/mL after ultrafiltration. However, diafiltration was impacted by membrane fouling and selectivity alterations, reducing the concentration to 11.42 ± 0.11 mg/mL. Our findings demonstrate that Trypsin yields the highest %DH (17.80%) and AA (0.93 mg ascorbic acid equivalents/mL), establishing a strong correlation between %DH and AA. No significant difference in %DH was observed between the pH-stat and OPA methods, validating OPA as a viable alternative for industrial applications. This study integrates an enzyme selection process with filtration techniques, providing valuable insights into bioactive peptide production. The findings support a scalable strategy for the dairy industry to enhance whey utilization while mitigating environmental impact.