Biologically induced spontaneous alginate gelation mediated by extracellular products of Lactiplantibacillus plantarum

Abstract

Biological regulation of polysaccharide assembly represents an emerging concept in biofabrication. However, spontaneous gelation of natural polymers driven by bacterial extracellular metabolism remains largely unexplored. This study aimed to characterize a previously unrecognized alginate gelation phenomenon induced by Lactiplantibacillus plantarum without the intentional addition of conventional crosslinking agents. The effects of alginate concentration, bacterial cultivation time, and incubation temperature on gel formation were systematically investigated. The contributions of bacterial cells and cell-free supernatants were evaluated to identify the active fraction responsible for gelation. The role of acidification was examined using defined organic and inorganic acids, and the resulting gel microstructures, secondary Ca 2+ crosslinking capability, processability, and bacterial viability during refrigerated storage were assessed. Reproducible gelation was achieved using 1% (w/v) sodium alginate following 24 h of bacterial cultivation and subsequent incubation at 4 °C. Gel formation depended on bacterial growth, alginate concentration, and low-temperature treatment. Cell-free supernatants retained gelation activity, whereas bacterial cells alone failed to induce gel formation, indicating that extracellular bacterial products mediated the process. Although acidification alone was sufficient to initiate visible gelation, gels formed using defined acids exhibited microstructures distinct from those induced by bacterial extracellular products. Furthermore, the resulting gels remained processable, supported secondary Ca 2+ crosslinking, and maintained high bacterial viability during refrigerated storage. These findings demonstrate that extracellular products of L. plantarum can induce alginate gelation under mild conditions without intentionally added conventional crosslinkers. The distinct gel microstructures suggest that bacterial extracellular metabolism may influence gel architecture beyond acidification alone. This biologically induced gelation process provides a foundation for developing bacteria-directed biofabrication strategies, living biomaterials, and biologically engineered hydrogel systems.