Mixture-design optimization of chlorella-chitosan biocomposite films followed by juniper seed extract incorporation...

This study aims to develop biodegradable chitosan-based films containing Chlorella vulgaris biomass and tannic acid, and to further enhance the optimized matrix through the incorporation of juniper seed (Juniperus excelsa) extract (EX). The effects of EX incorporation were evaluated relative to both the baseline chitosan film and the optimized extract-free matrix film in terms of functional, antioxidant, antibacterial, thermal, and structural properties. Distinct from conventional chitosan-based films, the present work applies a mixture-design optimization strategy to a chemically complex algae-chitosan system, enabling systematic evaluation of multicomponent interactions rather than one-factor-at-a-time modifications. The film formulation was optimized using a D-optimal mixture design, with algae (ALG) (15-45%), chitosan (CH) (30-55%), glycerol (GLY) (20-45%), and tannic acid (TA) (0-15%) as independent variables and tensile strength (TS), elongation-at-break (EAB), elastic modulus (EM) and water vapor permeability (WVP) as the response parameters. The validated film (V) containing 29.7% ALG, 43.3% CH, 25% GLY, and 2% TA, showed TS of 5.72 ± 0.36 MPa, EAB of 20.07 ± 1.58%, EM of 25.96 ± 2.51 MPa, and WVP of 0.24 ± 0.02 g.mm/m².h.kPa. The optimized film was then supplemented with EX at 5%, 10%, and 15% (v/v), yielding VEX5, VEX10, and VEX15 films, respectively. Spectroscopic and thermal analyses revealed enhanced intermolecular interactions among chitosan, microalgal components, tannic acid, and juniper seed extract polyphenols, including hydrogen bonding and π-π associations, leading to a more cohesive polymer network and improved thermal stability compared to the baseline chitosan film, as evidenced by shifts in degradation-related thermal events. Scanning Electron Microscopy (SEM) observations showed a progression from smooth surfaces in the control chitosan film to increasingly compact morphologies in extract-loaded samples, with VEX15 exhibiting the most uniform structure. Antibacterial assays of film-forming solutions against Staphylococcus aureus, Escherichia coli, and Salmonella Typhimurium revealed increasing inhibition zones with higher EX content, indicating a synergistic effect with algal and polyphenolic components. Overall, the algae-chitosan-glycerol-tannic acid film system enriched with J. excelsa extract offers improved structural stability, antioxidant potential, environmentally responsive degradability, and functional performance, highlighting its potential for sustainable packaging.