Abstract
The development of sustainable plant protection strategies requires stable and environmentally compatible delivery systems for beneficial microorganisms. In this study, Bacillus subtilis was encapsulated within chitosan-based cast films to evaluate bacterial viability, sustained biological activity, and antifungal efficacy. Films prepared from chitooligosaccharide (COS) and chitosans of low, medium, and high molecular weight (CS-LMW, CS-MMW, CS-HMW) were characterized in terms of morphology, mechanical performance, and pH-dependent swelling behavior. The viscosity of the chitosan solutions increased markedly with molecular weight from 73 cP (COS) to 614 cP (CS-HMW), while film thickness ranged from 34 ± 1.5 to 57 ± 2.3 µm. Mechanical performance improved significantly with increasing molecular weight, with maximum tensile stress exceeding 200 MPa for CS-HMW films, while swelling studies confirmed pronounced pH-dependent behavior consistent with the polyelectrolyte nature of chitosan. Encapsulation effectively preserved bacterial viability and metabolic activity over time. The intrinsic antifungal activity of chitosan synergized with the biocontrol activity of B. subtilis against Fusarium avenaceum and Alternaria solani. The highest antifungal performance was observed for CS-HMW films, which produced inhibition zones up to 84.6 ± 5.0 mm against A. solani. These findings demonstrate that chitosan-based cast films serve as effective carriers for beneficial microorganisms, providing environmental protection and regulated biological activity. The combination of a bioactive polymer matrix with a potent biocontrol agent represents a promising eco-friendly approach to sustainable plant protection.