Bioactive peptides from blackberry (Rubus L.) seed proteins: enzymatic production, fractionation, and multi-functional properties

Abstract

Blackberry (Rubus L.) seeds are an underutilized processing byproduct containing 20–25% protein, yet their protein fraction has received limited attention. This study aimed to optimize enzymatic hydrolysis of blackberry seed protein isolate (BSPI), fractionate the resulting hydrolysate, and comprehensively evaluate the bioactivities of peptide fractions. Response surface methodology optimized alkaline protease hydrolysis conditions (2.5% substrate, 16,500 U/g enzyme, pH 9, 3 h), achieving a 22.98% degree of hydrolysis. Ultrafiltration fractionation yielded four molecular weight fractions (< 3, 3–5, 5–10, > 10 kDa). The < 3 kDa fraction (F1) exhibited the strongest antioxidant activity (DPPH IC50: 15.38 µg/mL), significant α-glucosidase (50.99%) and α-amylase (63.87%) inhibition, and potent pancreatic lipase inhibition (62.55%). Notably, F1 demonstrated exceptional cytotoxicity against HepG2 cells (IC50: 0.12 µg/mL) with remarkably low toxicity toward normal HUVEC cells (IC50: 288.24 µg/mL), yielding a selectivity index of 2402. In vivo xenograft studies confirmed significant tumor growth inhibition (48.95%) without observable organ toxicity. This study provides the first systematic optimization of hydrolysis for blackberry seed glutelin-rich proteins, comprehensive multi-functional bioactivity characterization, and in vivo anticancer validation. These findings demonstrate that blackberry seed-derived peptides, particularly F1, possess significant potential as functional food ingredients or nutraceuticals, supporting the sustainable valorization of agricultural byproducts.