Development of a Layer-by-Layer Zein/CMCS Microcapsule Platform for Bacteriophage Delivery: A Proof-of-Concept Study Using a Model Phage in Sea Bass

Bacteriophages (phages) offer a targeted biocontrol solution, but their direct application is hampered by environmental instability. To address this, we developed a novel, food-grade microcapsule system for phage delivery using layer-by-layer (LbL) self-assembly of zein and carboxymethyl chitosan (CMCS). Lytic phages targeting specific spoilage bacteria were successfully encapsulated via electrostatic interactions. Characterization confirmed the formation of a multilayer structure, driven primarily by hydrogen bonding and electrostatic forces between the wall materials. The microencapsulation markedly enhanced phage stability against thermal (60 °C and 70 °C) and extreme pH (2.0, 12.0) stresses and provided a controlled release profile in a simulated fish exudate. When applied to fresh-cut sea bass (Lateolabrax japonicus), the phage-loaded microcapsules (CMCS3), constructed via a three-layer zein/CMCS LbL assembly, significantly delayed the pH rise during refrigerated storage, maintaining a final pH of 6.28 compared to 7.28 in the control group after 5 days. The microcapsules also effectively suppressed microbial growth (total viable count (TVC) was maintained below 6 log CFU/g) and controlled lipid oxidation (thiobarbituric acid reactive substances (TBARS) values were kept at 0.62 mg malondialdehyde/kg) while better preserving texture and color stability compared to free phages. This zein/CMCS-based LbL system presents a promising strategy for advancing phage-based biopreservation in aquatic products through enhanced physical protection, sustained release, and improved stress tolerance.