Lactose-Free Fermented Milk Production Using <i>Lactococcus lactis</i> NK24 and NK34 with Immune-Enhancing Effects on RAW 264.7 Cells

This study aimed to produce lactose-free fermented milk using probiotic <i>Lactococcus lactis</i> strains and to investigate the immune-enhancing effects of fermented milk. Lactose-free milk was fermented using <i>Laticaseibacillus rhamnosus</i> GG (LGG), <i>L. lactis</i> NK24, and <i>L. lactis</i> NK34. The pH, TA, viable lactic acid bacterial counts, and physicochemical properties of lactose-free fermented milk were recorded over 28 days under refrigeration. Each water-soluble extract of lactose-free fermented milk stimulated NO production, phagocytic activity, and induced morphological changes in RAW 264.7 cells, similar to the LPS treatment group. Additionally, the mRNA levels of <i>inducible nitric oxide</i> (<i>iNOS</i>), <i>cyclooxygenase-2</i> (<i>COX-2</i>), and proinflammatory cytokines increased. Furthermore, each sample promoted protein expression of iNOS, and COX-2 and phosphorylation of p38, p65, JNK, and ERK. Therefore, lactose-free milk fermented by <i>L. lactis</i> NK24 and <i>L. lactis</i> NK34 can enhance the immune response via nuclear factor-kB (NF-kB) and mitogen-activated protein kinase (MAPK) activation and has the potential for development as a functional food.