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.

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.

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).

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.