Functional Characterization of Breast Milk-Derived Lactobacillus paragasseri HUH02 Postbiotic Preparations with...

Breast milk-derived lactic acid bacteria (LAB) are promising sources of functional postbiotics. In this study, we evaluated the immunomodulatory, epithelial barrier-enhancing, and anti-melanogenic properties of nonviable HUH02 preparations, including heat-killed cells (HUH02-HK) and microfluidized preparations generated at 20,000 psi (HUH02-2psi) and 40,000 psi (HUH02-4psi), derived from the breast milk-derived Lactobacillus paragasseri HUH02 (L. paragasseri HUH02). HUH02 derivatives were evaluated in RAW264.7 macrophages, HaCaT keratinocytes, and B16F1 melanoma cells. All exhibited low cytotoxicity at functional concentrations. In Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, HUH02 derivatives markedly reduced nitric oxide production and suppressed the expression of inflammatory mediators, restored macrophage morphology toward a non-activated phenotype, and reduced iNOS protein expression. In HaCaT keratinocytes, HUH02 derivatives enhanced wound closure and the expression of epithelial barrier-associated genes, including ZO-1 and Occludin. Moreover, microfluidized derivatives, particularly HUH02-2psi and HUH02-4psi, significantly enhanced transepithelial electrical resistance, indicating improved epithelial barrier integrity. In B16F1 melanoma cells, HUH02-derived preparations exhibited antimelanogenic effects by reducing intracellular melanin accumulation and suppressing the expression of melanogenesis-related genes and proteins, including Tyr, TRP-1, TRP-2, and MITF. HUH02 was identified as L. paragasseri based on 16S rRNA, average nucleotide identity, and whole-genome phylogenetic analyses. Genome-based safety assessment revealed the absence of antibiotic resistance genes, virulence factors, plasmid replicons, and pathogenicity-associated markers. These findings demonstrated that breast milk-derived L. paragasseri HUH02 postbiotics exhibit multifunctional biological activities, including immunomodulatory, epithelial barrier-enhancing, and antimelanogenic effects.