Individual and Combined Effects of Medium- and Long-Chain Triacylglycerol and 2′-Fucosyllactose on Small Intestinal Morphology, Barrier Function, and Gut Microbiota in Growing C57BL/6 Mice

<b>Background/Objectives:</b> Medium- and long-chain triacylglycerol (MLCT) and 2'-fucosyllactose (2'-FL) are functional ingredients abundant in human milk; however, their effects on small intestinal development and health remain largely unknown, and no research has explored their potential combined effects. <b>Methods:</b> In this study, growing C57BL/6 mice (3 weeks old) were fed diets without or with 2.5 g/100 g of MLCT, 2'-FL, or the combination (MLCT + 2'-FL; 5:1) for 21 days. Body weight, major organ indices, small intestinal morphology-related indicators (small intestinal length, villus height, crypt depth, villus height/crypt depth (V/C) ratio, and epithelial cell proliferation), and intestinal barrier function markers (goblet cell and Paneth cell count, protein expression of ZO-1 and occludin, and levels of sIgA and LPS) were measured. <b>Results:</b> In addition to the shared promotion of epithelial cell proliferation, MLCT intervention raised villus height and crypt depth, while 2'-FL intervention elevated Paneth cell count and sIgA levels. Notably, MLCT + 2'-FL intervention offered additional advantages (increasing the V/C ratio, goblet cell count, and expression of ZO-1 and occludin) without affecting crypt depth. 16S rRNA sequencing analysis of cecal contents revealed that all three interventions mainly affected beta diversity rather than alpha diversity, and enriched differentially abundant bacterial taxa: <i>Erysipelotrichaceae</i>, <i>Faecalibaculum</i>, <i>UBA1819</i>, and <i>Faecalitalea</i> in the MLCT group; <i>Enterobacteriaceae</i>, <i>Escherichia</i>, and <i>Allobaculum</i> in the 2'-FL group; <i>Bifidobacterium</i>, <i>Romboutsia</i>, <i>Clostridia</i>, and several other bacterial taxa in the MLCT + 2'-FL group. <b>Conclusions:</b> These results indicate that MLCT and 2'-FL interventions alone appear to provide different benefits for small intestinal development, and their combination may confer more comprehensive advantages.