Background, Context, or Rationale Buttermilk, a by‐product of butter production, contains proteins with encrypted bioactive peptides that may display antioxidant activity when released. Enzymatic hydrolysis combined with membrane separation offers a sustainable approach to enhance the functional properties of dairy by‐products and promote their valorisation. Aim This study aimed to obtain low‐molecular‐weight peptides with improved antioxidant activity from buttermilk through sequential enzymatic hydrolysis coupled with ultrafiltration in a membrane bioreactor. Methods Buttermilk was hydrolysed using Alcalase®, Prolyve®, and Neutrase®, individually and in sequence. The hydrolysates were processed in a membrane bioreactor with a tubular ultrafiltration membrane (MWCO 30–80 kDa) operated at 100 kPa and 40°C. The degree of hydrolysis, protein distribution, peptide size and antioxidant activity were determined. Peptide size was assessed by electrophoresis and theoretical calculations, and antioxidant activity by the ABTS assay. Statistical analysis was performed using ANOVA and Tukey's test ( P < 0.05). Major Findings Sequential hydrolysis with Alcalase® and Neutrase® achieved the highest degree of hydrolysis (34%), which resulted in short peptides with an average length of approximately three amino acids and a molar mass of 0.33 kDa. Ultrafiltration separated these low‐molecular‐weight peptides into the permeate fraction, which exhibited the highest antioxidant activity (971 μmol Trolox equivalents/g protein). Electrophoresis confirmed the absence of proteins above 5 kDa in the permeate, and both permeate and retentate displayed higher bioactivity than untreated buttermilk. Scientific or Industrial Implications The combined enzymatic and membrane process enabled the recovery of bioactive peptides from buttermilk, adding value to this underutilised by‐product. The results highlight a feasible strategy for producing functional food ingredients with antioxidant properties and contribute to a more sustainable and circular dairy industry.
Fortuna et al. (Wed,) studied this question.