SELF-ASSEMBLED ZEIN NANOCARRIERS FOR THE STABILIZATION AND DELIVERY OF PLANT-DERIVED BIOACTIVE PEPTIDES

Bioactive peptides obtained from plant proteins are emerging as promising ingredients for food, nutraceutical and agricultural applications. However, their intrinsic conformational flexibility and susceptibility to proteolytic and environmental degradation strongly limit their functional exploitation. Developing biodegradable delivery systems able to preserve peptide integrity represents a major challenge in protein biotechnology [1]. Within the 4EU+ BIOnanoDELY project, we developed a fully bio-based nanocarrier platform exploiting the self-assembly properties of zein, the major maize storage protein [2], to encapsulate peptides from two sustainable legume sources: cowpea (Vigna unguiculata) β-vignin and soybean (Glycine max) okara by-products. LC-MS/MS of trypsin-digested β-vignin identified 240 peptides (64% between 1000–2000 Da, 33% between 500–1000 Da), confirming efficient generation of short peptides; among the 15 most abundant, 11 were predicted as anti-inflammatory (PReTP-Stack) [3]. Okara-derived peptides (OPID), obtained by pancreatin hydrolysis, were selected for their known antifungal potential [4]. Both peptide pools were incorporated into zein nanoparticles through a one-step anti-solvent nanoprecipitation approach [2]. SDS-PAGE analysis of pellet and supernatant fractions showed efficient association of both zein and peptide fractions with the nanoparticle phase, supporting successful nanostructure formation and peptide entrapment. Peptide incorporation reduced the mean hydrodynamic diameter from ~240 to ~160 nm (DLS), while SEM analysis confirmed preserved spherical morphology and reduced aggregation. Building on this platform, functional validation is now underway: encapsulated OPID is being tested against phytopathogenic fungi, extending the antifungal spectrum beyond that previously assessed for free OPID [4] to include Alternaria alternata, Botrytis cinerea, and Zymoseptoria tritici, while gastrointestinal stability and bioaccessibility of encapsulated β-vignin peptides are being evaluated via the INFOGEST model. These ongoing assessments will establish whether nanocarrier stabilization translates into preserved peptide functionality, supporting the platform's potential as a sustainable strategy for delivering bioactive peptides across nutritional and crop-protection applications. References [1] Zhang X, Li X, Zhao Y, Zheng Q, Wu Q, Yu Y. Nanocarrier system: an emerging strategy for bioactive peptide delivery. Frontiers in Nutrition. 2022; 9: 1050647. [2] Massironi A, Toccaceli M, Marinelli A, Maggioni D, Scapuzzi C, Emide D, Scarafoni A, Verotta L, Petroni K, Marzorati S. Extraction and encapsulation of purple corn cob anthocyanins into starch- and zein-based delivery systems. Journal of Drug Delivery Science and Technology. 2025; 105: 106624. [3] K. Yan et al., IEEE/ACM Transactions on Computational Biology and Bioinformatics; 20 (2023), pp 1337–1344. [4] De Benedetti S, Girlando V, Pasquali M, Scarafoni A. Valorization of okara by enzymatic production of anti-fungal compounds for plant protection. Molecules. 2021; 26: 4858.