Whey Protein Phospholipid Concentrate and Its Fractions as a Diet Intervention Enhance Bone Health and Alter the Gut Microbiome in Weanling Mice

Early post-weaning growth is a critical window for bone development, and diet plays a central role in establishing peak bone mass in early life. Whey protein phospholipid concentrate (WPPC), a co-product of whey protein isolate manufacturing, is enriched in bioactive lipids and proteins that may support bone development and calcium homeostasis. This study aimed to investigate the effects of WPPC and its protein- and lipid-enriched fractions on bone development, gene expression, and gut microbiota in weanling mice. WPPC was fractionated using temperature-dependent centrifugation, microfiltration, and ultrafiltration, yielding lipid- and protein-rich components that were used to supplement isocaloric diets. Mice were fed either lipid fraction (Fat), protein fraction (Protein), or whole WPPC (WPPC) (10% by kcal; ~8.7% w/w) for 12 weeks. Compared to controls, the Protein and WPPC groups exhibited significantly increased femur length (4.44% and 4.01%, respectively), while the Fat group showed significantly higher bone mineral density (6.15%). Quantitative PCR of jejunal tissues revealed upregulation of calcium transporter genes (Cldn2, Cldn12, and Pmca1) in WPPC-fed mice with no changes in vertebral bone markers of osteocyte differentiation. Circulating iFGF23 levels also remained unchanged, suggesting limited endocrine involvement. Gut microbiota analysis via 16S rRNA sequencing showed diet-specific shifts, including increased Akkermansia and Streptococcus in the WPPC group and elevated Lactobacillaceae in the Protein and Fat groups. These findings demonstrate that WPPC and its enriched macronutrient fractions promote skeletal development and modulate calcium uptake and gut microbial composition, supporting their potential as functional ingredients for bone health applications in early life.