pH‐Shift‐Prepared Chlorella pyrenoidosa Protein/Peptide–Calcium Complexes Improve Skeletal Health by Enhanced Intestinal Calcium Utilization in Calcium‐Deficient Rats

ABSTRACT Calcium carbonate supplements often exhibit limited solubility and gastrointestinal precipitation, restricting calcium availability. To address this limitation, an enzyme‐free pH‐shift strategy was applied to prepare Chlorella pyrenoidosa protein (CPP)/peptide–calcium complexes (pH–Ca) and to evaluate their effects on calcium utilization in calcium‐deficient rats. Alkaline pH‐shift treatment promoted protein unfolding and increased the accessibility of calcium‐binding groups, resulting in an effective chelation rate of 44.04% ± 1.34% under optimized conditions (pH 13, 4 h). Spectroscopic analyses indicated the formation of predominantly amorphous protein/peptide–calcium complexes. In vivo, pH–Ca supplementation significantly improved bone mineral indices, microarchitecture, and mechanical strength compared with calcium carbonate and untreated protein–calcium controls. These skeletal benefits were accompanied by partial restoration of duodenal morphology, normalization of intestinal calcium transport–related gene expression, and increased gut microbiota diversity. Overall, pH‐shift‐prepared CPP/peptide–calcium complexes enhance intestinal calcium utilization and skeletal outcomes in calcium‐deficient rats.