Enzymatic hydrolysis altered the physicochemical and immunogenic profile of protein-based ingredients derived from industrial hempseed (Cannabis sativa L.)

Authors: RODRIGUEZ, ABBY - University Of Tennessee; CHEN, BINGQI - University Of Tennessee; PANGLOLI, PHILIPUS - University Of Tennessee; D'SOUZA, DORIS - University Of Tennessee; Krishnan, Hari; DIA, VERMONT - University Of Tennessee

Submitted to: Food Bioscience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2025
Publication Date: 6/2/2025
Citation: Rodriguez, A., Chen, B., Pangloli, P., D'Souza, D., Krishnan, H.B., Dia, V.P. 2025. Enzymatic hydrolysis altered the physicochemical and immunogenic profile of protein-based ingredients derived from industrial hempseed (Cannabis sativa L.). Food Bioscience. 69. https://doi.org/10.1016/j.fbio.2025.106965.
DOI: https://doi.org/10.1016/j.fbio.2025.106965

Interpretive Summary: Recent studies have emphasized the nutritional, economic, and social significance of industrial hemp as a functional food ingredient. Hemp proteins exhibit optimal levels of essential amino acids, exceptional digestibility, and a nutritional profile comparable to soy and ovalbumin. However, increasing public exposure to industrial hempseed has led to reported allergies involving various parts of the plant, including pollen, leaves, and seeds. While enzymatic hydrolysis is widely utilized to reduce the allergenicity of food proteins, such as those derived from soy, it remains unexplored in hemp-based ingredients. In this study, we examined the physicochemical and immunogenic properties of industrial hemp protein isolate that was subjected to enzymatic hydrolysis using bromelain, alcalase, and trypsin. Our findings indicate that hydrolyzed hemp seed proteins significantly reduced allergenicity, positioning them as a promising alternative protein source for functional food applications. Our research holds potential to benefit U.S. hemp growers by expanding the market for their products, enabling them to better compete within the food industry through the development of hypoallergenic diets utilizing hemp seed proteins.

Technical Abstract: Industrial hempseed (Cannabis sativa L.) is gaining recognition as a sustainable plant-based protein source, yet its allergenic potential remains largely unexplored. In addition, analyzing the potential cross-reactivity of hemp ingredients against the biggest 9 global allergens is important in understanding HPI's nutritional safety and accessibility for food allergic individuals. Thus, this study aimed to investigate the physicochemical properties and immunogenicity of hemp protein isolate (HPI) extracted at pH 10 and 12, with and without 30-min enzymatic hydrolysis using bromelain, Alcalase, and trypsin. Results found that enzymatic hydrolysis significantly altered the protein structure of HPI-10, redistributing the molecular weight via an increase in <10 kDa peptides, significantly increasing the degree of hydrolysis for all 3 hydrolysates (p < 0.05), and disrupting native folded conformations, as assessed by SDS-PAGE, Orthophtaldialdehyde, and fluorescence spectroscopy, respectively. Immunogenicity was evaluated through enzyme-linked immunosorbent assay (ELISA) and Western blot using human allergic IgE antibodies corresponding to the “Big 9' food allergens, as well as hempseed. Results demonstrated that hydrolysis effectively minimized immunogenic responses with hemp-allergic plasma while revealing potential immunogenic interactions with unhydrolyzed HPI-10 and wheat and soy allergic individuals. LC/MS-MS analysis of immunoreactive bands showed peptide fragments associated with storage proteins in industrial hempseed including edestin-2, cupin-type 1, and edestin-3. These findings suggest that enzymatically hydrolyzed HPI may serve as a hypoallergenic, high protein ingredient, warranting further investigation into its application in functional food formulations.