Physicochemical Properties Determination of Recombinant Human Lysozyme and Its Effects on Intestinal Development in Mice.

Background/Objectives: Breast milk lysozyme is crucial for infant intestinal health. The low breastfeeding rate has driven the investigation of alternatives like hen egg white lysozyme (HEWL) for infant formula supplementation. However, HEWL differs significantly from human lysozyme. This study aimed to systematically compare the functional efficacy of recombinant human lysozyme (rhLYZ) and HEWL to assess their suitability as formula supplements. Methods: The physicochemical properties (enzymatic activity, optimal pH, thermal stability) of rhLYZ and HEWL were analyzed. Biological functions were evaluated using HT-29 intestinal cells for proliferation, differentiation, and protection against lipopolysaccharide-induced damage. In vivo effects on growth, intestinal morphology, and gene expression were assessed in a mouse pup model via transcriptomic analysis. Gut microbiota composition was also examined. Results: rhLYZ exhibited twice the enzymatic activity of HEWL, with an optimal pH of 6.0. In cellular models, rhLYZ enhanced intestinal epithelial differentiation at low concentrations. In vivo, rhLYZ supplementation significantly improved pup body weight, intestinal maturity, and villus-to-crypt ratios, outperforming HEWL. Transcriptomics revealed rhLYZ upregulated broad-spectrum antimicrobial peptides (e.g., Defa, lactoferrin) and immune-related genes, whereas HEWL induced a narrower antibacterial response and downregulated key defensins. Furthermore, rhLYZ significantly increased gut microbiota diversity and enriched beneficial butyrate-producing bacteria. Conclusions: rhLYZ more effectively mimics human milk lysozyme by promoting intestinal development, broad-spectrum immunity, and a balanced microbiota. HEWL shows a narrower functional profile. These findings provide a scientific basis for optimizing lysozyme selection in infant formula, highlighting the superior potential of rhLYZ.