Bioengineering of probiotic-loaded hydrogel films with high antimicrobial activity

Abstract

The rise of antimicrobial resistance complicates the management of infected wounds, necessitating novel therapeutic strategies. Probiotic-based therapies offer a promising alternative, but their efficacy depends on delivering a high concentration of viable, active microorganisms to the wound site. Alginate hydrogels are excellent carriers, but freshly prepared probiotic-loaded films often lack immediate therapeutic activity. This study aimed to investigate the effect of a post-immobilization cultivation period on the viability and antagonistic activity of Bifidobacterium bifidum LVA-3 and Lactobacillus bulgaricus 1Z 03501 immobilized in calcium alginate films. The central hypothesis was that this cultivation step would function as an in-situ bio-activation process, enhancing the films' therapeutic potential.

Methods. B. bifidum LVA-3 and L. bulgaricus 1Z 03501 were immobilized in calcium alginate films. The films were then cultivated in a nutrient medium for 2, 4, or 6 days at 37 °C. Viable cell counts were determined by plate counting after film dissolution. Antagonistic activity was assessed using an agar overlay diffusion method against pathogenic test strains (Staphylococcus aureus 209, Pseudomonas aeruginosa 9027, and Escherichia coli B), measuring the diameter of inhibition zones. The experimental data revealed that uncultivated films (Day 0) showed no antagonistic activity. Post-immobilization cultivation led to a significant increase in viable cell counts for both strains, with populations rising by over 100- to 500-fold within 2 days to therapeutically relevant levels (>10¹⁰ CFU/mL). B. bifidum LVA-3 showed rapid growth peaking at day 2, while L. bulgaricus 1Z 03501 maintained a high, stable population through day 6. This increased cell density directly correlated with the emergence of potent antagonistic activity against all three pathogenic strains. In summary, it can be concluded that a post-immobilization cultivation step is a critical bio-activation process that transforms probiotic-loaded alginate films from passive carriers into functionally potent biomaterials. This strategy effectively increases probiotic viability to therapeutic concentrations and enables the in-situ production of antimicrobial compounds. This two-step approach of immobilization followed by cultivation presents a novel method for developing high-efficacy probiotic formulations for applications such as bioactive wound dressings.