Introduction: Gut health is crucial for overall well-being and can be improved through probiotics and bioactive milk-derived components like Milk Fat Globule Membrane (MFGM). This study compared MFGM from bovine, caprine and human milk for enhancing probiotic functionality and gut health using in vitro and in vivo models.

Methods: MFGM was isolated, characterized, digested and combined with three probiotic Lactobacillus strains (LGG, MTCC 5690, and MTCC 5689) to assess hydrophobicity, aggregation, co-aggregation and adhesion to HT-29 cells and interactions with pathogens. Gut barrier integrity and anti-inflammatory potential were evaluated by an FITC-dextran assay, RT-qPCR, and ELISA. The most effective treatment with probiotic LGG and MFGM from bovine, caprine and human combination was further tested in a DSS-induced colitis mouse model for body weight, organ parameters, gut permeability, and gene expression by Rt-qPCR.

Results: MFGM characterization confirmed the bioactive components. Human MFGM significantly enhanced probiotic functionality, including hydrophobicity, aggregation, co-aggregation, adhesion and pathogen interference, with the following trend: human > caprine > bovine. RT-qPCR and ELISA in HT-29 cells revealed that LGG combined with human MFGM significantly downregulated pro-inflammatory and upregulated anti-inflammatory cytokines. In vivo, DSS-induced colitis caused body weight loss (31.5 g to 28.7 g), reduced colon length (6.5 cm), increased gut permeability (~17,000 ng/mL), liver weight (1.9 g), and spleen index (0.32g), and the upregulation of pro-inflammatory genes. Probiotic or MFGM alone provided partial protection, while LGG + human MFGM offered the most effective protection. This group showed improved body weight (25.6 g), restored colon length (10 cm), reduced gut permeability to 500 ng/mL, normalized organ parameters and favorable modulation of gene expression with the downregulation of IL-1β, TNF-α, IFN-γ, IL-2 and TLR-2 and upregulation of IL-4, IL-5, IL-10, Occludin, Claudin and MUC-2.

Conclusion: The combination of human MFGM and probiotic LGG demonstrated strong anti-inflammatory and gut barrier protective effects in both in vitro and in vivo models, supporting the potential of species-specific MFGM in enhancing probiotic efficacy in gut health restoration.