Enhancement of Protein Hydrolysis and Bioactivity in Hempseed Cake via Solid-State Fermentation Using Aspergillus niger, Bacillus subtilis, and Lactobacillus rhamnosus

Abstract Hempseed cake, a by-product of hempseed oil extraction, contains proteins and other compounds whose bioactivity can be enhanced through solid-state fermentation (SSF). Thus, this study investigated the effects of SSF using Aspergillus niger , Bacillus subtilis , and Lactobacillus rhamnosus on the degree of protein hydrolysis (DH), protein composition, and bioactive properties of the fermented hempseed cake. An orthogonal design was employed to determine the optimal fermentation conditions of each microorganism. Fermentation significantly ( P < 0.05) increased DH and soluble protein and free amino acid (FAA) contents, with B. subtilis showing the highest increase (DH, 2.77% → 48.72%; soluble protein, 28.12 mg/g dw → 208.21 mg/g dw; FAA, 4.09 mg/g dw → 52.81 mg/g dw). SDS-PAGE confirmed the hydrolysis of macromolecular proteins into peptides. SSF also enhanced the antioxidant capacity of fermented hempseed cake as measured by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity, as well as increased inhibition against ACE (angiotensin-converting enzyme), AChE (acetylcholinesterase), and intestinal α-glucosidase activity. The highest ABTS radical scavenging activity was observed with L. rhamnosus fermentation (4.98 mg TE/g), while B. subtilis fermentation yielded the strongest ACE (88.22%) and AChE (84.39%) inhibitory activities. A. niger exhibited the highest α-glucosidase inhibitory activity (26.04%). Pearson correlation analyses supported that these enhancements were linked to protein hydrolysis. Among the tested strains, B. subtilis generally exhibited superior performance in protein hydrolysis. These findings highlight SSF as a promising approach to enhance the nutritional and bioactive properties of hempseed cake, offering potential applications in functional food and nutraceutical development.