Probiotic-Enriched Bilayer Edible Films from Pumpkin Seed Flour: Effects of Pectin and Glycerol on Barrier Properties and Probiotic Stability

Edible films derived from agro-industrial by-products have gained increasing attention as sustainable and functional materials for food applications, particularly as delivery systems for probiotic microorganisms. In this study, mono- and bilayer edible films were developed from shelled and unshelled pumpkin ( Cucurbita moschata ) seed flours as protein-rich, bioprocessed raw materials and enriched with Lactiplantibacillus plantarum ATCC 8014. The effects of key formulation variables, including glycerol concentration and the incorporation of a pectin-based outer layer, on the structural, mechanical, barrier, antioxidant, and probiotic-related properties of the films were systematically investigated. The results demonstrated that formulation-driven modifications significantly altered polymer–plasticizer interactions and matrix organization. Compared with monolayer systems, bilayer films exhibited improved structural integrity and functional performance, highlighting the protective role of spatially separated layers. Films prepared from shelled pumpkin seed flour showed higher protein content and antioxidant activity, whereas those produced from unshelled flour displayed enhanced structural homogeneity and solubility. Probiotic viability was strongly influenced by the glycerol–pectin ratio; however, viable cell counts remained above the recommended threshold of 6 log CFU/g across all film formulations during storage. This study provides new insights into formulation–structure–function relationships in pumpkin seed flour–based edible films and demonstrates the potential of pumpkin seed by-products as value-added biopolymer matrices for stable probiotic delivery in food bioprocessing and functional food development.