Fermentation-Dependent Cell Wall-Associated Immunostimulation in a Red Pepper-Based Lactiplantibacillus plantarum CJLP243 Fermentate

Lactic acid bacteria-fermented food materials are promising sources of functional ingredients, but the active components underlying their bioactivity often remain unclear. We localized the macrophage-stimulatory activity of a red pepper-based fermentate prepared with Lactiplantibacillus plantarum CJLP243. Compared with the unfermented matrix and a non-fermented matrix supplemented with freeze-dried CJLP243, the fermentate induced greater IL-6 and TNF-α production in J774A.1 macrophages. Activity was reproducibly concentrated in the pellet-derived lysate-insoluble fraction (PDIF) and remained in residual debris after solvent extraction. LTA-directed fractionation and analytical screening did not support lipoteichoic acid as the principal determinant. Lysozyme markedly reduced activity, whereas proteinase K had a partial effect, a pattern consistent with a lysozyme-sensitive, cell wall-associated structure or cell wall–matrix complex, within which peptidoglycan-associated material remains a plausible candidate; contributions from co-associated components cannot be excluded. Targeted metabolomics revealed fermentation-associated changes in pellet-associated amino acid pools, consistent with broader adaptation of amino acid and nitrogen metabolism during fermentation; their biochemical linkage to cell-wall precursor pathways provides metabolic support rather than direct structural evidence. These findings identify a reproducible, process-linked insoluble fraction with macrophage-stimulatory activity and provide a framework for developing and standardizing fermentation-derived functional food ingredients.