Abstract: Objective: To determine the immunoenhancing activity of ternary mixtures of 2’-fucosyllactose (2’-FL), fructooligosaccharide (FOS), and galactooligosaccharide (GOS) at different ratios and to validate the immunoregulatory effects of milk formula containing prebiotic combinations. Methods: An appropriate mixing ratio of prebiotics for enhancing immune function was identified on the basis of T cell counts, macrophage number, macrophage phagocytosis, and goblet cell number in an immunosuppressed zebrafish model. This prebiotic blend was then used to develop a children’s milk formula. The effects of the milk formula on the morphology of immune organs, peripheral blood and/or spleen lymphocytes, immunoglobulins, and cytokines in cyclophosphamide-induced immunosuppressed mice were evaluated. Results: The mixtures of 2’-FL, GOS, and FOS were found to enhance both specific and non-specific immune functions in immunosuppressed zebrafish, the optimal ratio being 1:3:3. In immunosuppressed mice treated with the milk formula containing the 1:3:3 mixture, partial restoration of damaged spleen and thymus structures was observed. Additionally, the peripheral white blood cell count and the CD4+/CD8+ ratio returned to normal levels, and the concentrations of interleukin (IL)-4, IL-10, tumor necrosis factor-alpha (TNF-α), and C-reactive protein (CRP) were increased to varying degrees, with a significant increase being observed in IL-10 concentration (P < 0.05). Conclusion: The combination of 2’-FL, GOS, and FOS exhibits immunomodulatory effects depending on the mixing ratio. The children’s milk formula containing 2’-FL, GOS, and FOS at a ratio of 1:3:3 demonstrated immunomodulatory effects on immunosuppressed mice.

Key words: 2’-fucosyllactose; fructooligosaccharide; galactooligosaccharide; immune modulation; milk formula