Selective production of novel ACE-inhibitory peptides from brewer’s spent grain via two-stage enzymatic hydrolysis and integrated fractionation

Abstract

Brewer's spent grain represents an underutilized protein source capable of yielding bioactive peptides with potential health benefits, supporting food system sustainability, aligning with an environmental management system, and contributing to environment and health. This study aimed to optimize the release of Angiotensin-I Converting Enzyme (ACE)-inhibitory peptides from brewer's spent grain proteins through enzymatic hydrolysis using Flavourzyme and Alcalase, both individually and in a sequential manner. Peptides generated via selective Alcalase-Flavourzyme hydrolysis exhibited significant bioactivities (p < 0.05), including ABTS radical scavenging (381.91 ± 6.93 μmoL Trolox equivalent/g sample), metal chelation (1128.34 ± 19.45 μmoL EDTA/g sample), and ACE-inhibitory activity (IC₅₀, 0.11 ± 0.01 mg/mL). Peptides were further separated using membrane ultrafiltration into four fractions based on molecular weight: <3 kDa, 3–5 kDa, 5–10 kDa, and >10 kDa. The <3 kDa fraction showed the highest ACE-inhibitory activity (IC₅₀, 74.68 ± 0.28 μg/mL, p < 0.05). The most active fraction underwent size exclusion chromatography and Liquid Chromatography-Tandem Mass Spectrometry to determine amino acid sequences. Hydrophobic residues, including valine, proline, glycine, and leucine, were prevalent in four identified peptides (GDRLL, LPTHL, VPFVVPGSE, and VPFVPGSVE), contributing to ACE inhibition. The combined two-stage enzymatic hydrolysis and integrated fractionation within the manufacturing process significantly enhanced ACE-inhibitory activity, achieving approximately 1500-fold improvement compared to non-hydrolyzed protein. Bioactive peptides derived from brewer's spent grain protein hydrolysates may serve as sustainable, high-value ingredients, supporting healthy diets and innovative applications in food and nutraceutical products.