Abstract: Human milk free oligosaccharides (HMOs) play a critical role in the early development of the infant immune system. Goat milk free oligosaccharides (GMOs) exhibit greater structural and quantitative similarity to HMOs compared with bovine milk free oligosaccharides (BMOs). In this study, neutral and acidic free oligosaccharide fractions were efficiently prepared from human, goat, and bovine milk using DEAE-52 weak anion-exchange cellulose chromatography combined with graphitized carbon solid-phase extraction. The immunomodulatory activities of these fractions and their underlying mechanisms were comparatively investigated in RAW264.7 macrophages. The results showed that all oligosaccharide fractions exhibited immunomodulatory activity. Notably, human milk sialylated free oligosaccharides (SHMOs) and goat milk sialylated free oligosaccharides (SGMOs) demonstrated stronger activity than bovine milk sialylated free oligosaccharides (SBMOs), potentially mediated through upregulation of the mitogen-activated protein kinases signaling pathway. Finally, online liquid chromatography-mass spectrometry was employed for qualitative and quantitative comparative profiling of SHMOs, SGMOs, and SBMOs. The results revealed that SGMOs shared greater structural homology with SHMOs, which may account for their enhanced immunomodulatory potency.

Key words: human milk; goat milk; bovine milk; free oligosaccharides; immunomodulation; mechanism

摘要： 母乳游离寡糖（human milk free oligosaccharides，HMOs）对婴幼儿早期免疫系统构建至关重要。羊乳游离 寡糖（goat milk free oligosaccharides，GMOs）在含量及结构上较牛乳游离寡糖（bovine milk free oligosaccharides， BMOs）更接近HMOs。采用DEAE-52弱阴离子纤维素柱结合石墨碳固相萃取柱高效制备母乳、羊乳和牛乳中性 及酸性游离寡糖组，对比其对RAW264.7细胞的免疫调节活性，并探讨潜在机制。结果发现，所有寡糖组均具有 免疫调节活性，其中母乳酸性游离寡糖（human milk sialylated free oligosaccharides，SHMOs）和羊乳酸性游离寡 糖（goat milk sialylated free oligosaccharides，SGMOs）的活性强于牛乳酸性游离寡糖（bovine milk sialylated free oligosaccharides，SBMOs），其可能通过上调丝裂原活化蛋白激酶信号通路发挥免疫调节作用，最后结合液相色 谱-串联质谱技术对SHMOs、SGMOs及SBMOs进行定性及定量比较分析，结果显示，SGMOs结构更接近SHMOs， 这可能与其更强的免疫活性相关。

关键词: 母乳；羊乳；牛乳；游离寡糖；免疫调节；机制