Whey Protein–Polysaccharide Nanocomplexes: Structure–Function Characterization and Bone-Related Outcomes in Ovariectomized Rats

Abstract

Background/Objectives: Estrogen deficiency is associated with impaired bone mineralization and deterioration of bone quality. Protein–polysaccharide nanostructures have attracted interest as food-grade formulations because their physicochemical properties can be modified by the nature and organization of their constituent biopolymers. This study evaluated the effects of whey protein–polysaccharide nanocomplex formulations on bone-related outcomes in ovariectomized female Wistar rats. Methods: Whey protein nanocomplexes containing either Opuntia ficus-indica mucilage or Tamarindus indica xyloglucan were produced by complex coacervation and ultrasonication. The nanocomplexes were characterized in terms of particle size, polydispersity index, ζ-potential, and morphology. Ovariectomized rats received the nanostructured formulations, their corresponding physical mixtures, the individual ingredients, or control diets for 12 weeks. Femoral bone quality was evaluated through biomechanical testing, calcium and phosphorus content, Ca/P ratio, bone mineral density, scanning electron microscopy and histomorphometry, and X-ray diffraction analysis. Results: The developed nanocomplexes exhibited particle sizes below 500 nm and polydispersity indices of approximately 0.3. Their physicochemical characteristics and morphology differed according to the polysaccharide incorporated. Ovariectomy was associated with unfavorable changes in bone mineralization, microarchitecture, crystallographic characteristics, and biomechanical competence. Among the evaluated treatments, the whey protein–O. ficus-indica mucilage nanostructured formulation (LMCC) showed the most favorable overall pattern across most bone-related outcomes, including bone mineral density, femoral calcium content, microarchitecture, hydroxyapatite crystallite size, and biomechanical properties. However, the response to nanostructuring was not uniform across the two polysaccharide systems, and the xyloglucan-based formulations exhibited parameter-dependent responses. Conclusions: The biological response to whey protein–polysaccharide formulations was associated with polysaccharide type and physicochemical organization. The mucilage-based nanostructured formulation showed a more favorable overall pattern across most evaluated bone-related outcomes in ovariectomized rats. However, calcium association within the nanostructures, calcium-release kinetics, gastrointestinal bioaccessibility, and intestinal calcium absorption were not evaluated; therefore, the mechanisms underlying these responses remain to be established. These findings should be interpreted as preclinical and should not be extrapolated directly to osteoporosis prevention in humans.