A composite protein enriched with threonine, lysine, and tryptophan improves osteoporosis by modulating the composition and metabolism of the gut microbiota

Abstract

Osteoporosis is a metabolic bone disorder characterised by impaired bone metabolism and increased fracture risk. Protein constitutes a vital source of skeletal nutrition, and the appropriate supplementation of high-quality protein plays a significant role in improving osteoporosis. The global dietary trend is shifting towards sustainable, non-animal protein sources. However, individual non-animal proteins often exhibit low digestibility and imbalanced essential amino acid profiles. Furthermore, the essential amino acids threonine, lysine, and tryptophan have been demonstrated to participate in bone metabolism regulation, exerting positive effects on bone health. Therefore, this study designed a multi-source protein blend and evaluated its digestibility characteristics alongside its potential role in alleviating osteoporosis. Based on FAO/WHO essential amino acid reference patterns, soy, whey, and yeast proteins were selected and combined using grey relational analysis to formulate a composite protein (MPH) with a balanced amino acid composition enriched with threonine, lysine, and tryptophan. In addition to nutritional considerations, the present study was motivated by the limited domestic production of whey protein in certain regions, including China, and the increasing interest in diverse and sustainable protein strategies. Partial substitution of whey protein with plant- and microbial-derived proteins could be a feasible approach for reducing reliance on animal-derived protein sources while maintaining balanced amino acid delivery. Following in vitro digestion simulation, MPH demonstrated the highest rate of digestion and decomposition within the same timeframe. We evaluated its efficacy in a retinoic acid-induced osteoporosis rat model and explored the underlying mechanisms via gut microbiota 16S rRNA sequencing, PICRUSt-based functional prediction, and metabolomic analysis. Results demonstrated that MPH increased body weight and serum calcium/phosphorus levels, upregulated type I procollagen N-terminal propeptide (PINP) and osteocalcin (OC), downregulated type I collagen carboxy-terminal cross-linked telopeptide (CTX-1), reduced abnormally elevated alkaline phosphatase (ALP), and improved femoral and tibial length, bone mass, trabecular architecture, and bone mineral density, thereby effectively alleviating osteoporotic symptoms. Additionally, MPH modulated the gut microbiota—enhancing the relative abundance of Lactobacillus, Akkermansia, and Ruminococcus—and promoted the production of key metabolites such as β-hydroxybutyrate (β-HB), lipoxin A4 (LXA4), indole-3-propionic acid (IPA), and butyrate. These findings indicate that the developed composite protein exerts significant ameliorative effects on osteoporosis.