Stable Nano‐Casein Emulsions Prepared by a Scalable Two‐Stage High‐Pressure Homogenization Process for Oral Calcium Delivery

Abstract

ABSTRACT Nutritional emulsions are extensively used as carrier systems in foods for special medical purposes and health supplements. The rational addition and utilization of calcium are critical for realizing its functional value in supporting bone development, maintaining metabolic homeostasis, and regulating enzymatic activities. However, calcium absorption is readily affected by various physiological factors, limiting its bioavailability. In this study, a nano‐casein emulsion (NCE) was prepared using a two‐stage high‐pressure homogenization (rated at 8000 L/h throughput) on an industrial production line. Its physicochemical properties, in vitro digestion behavior, oral bioavailability, storage stability, and biocompatibility were evaluated. Compared with conventional casein emulsion (CE) prepared without high‐pressure homogenization, NCE exhibited a mean particle size of 260 ± 2.2 nm, which was 60.76% smaller than CE (662.64 ± 39.68 nm), with improved colloidal stability and viscoelasticity. Simulated gastrointestinal digestion and Caco‐2 cell transport assay demonstrated that NCE markedly increased amino acid release and intestinal absorption, achieving 2.2‐fold higher transport efficiency than CE. The calcium absorption rate was also improved (46.0% ± 3.1% vs. 40.0% ± 0.04%, * p < 0.05). Pharmacokinetic analysis further confirmed higher in vivo bioavailability, with a peak serum calcium concentration ( C max ) of 100 ± 0.426 μg/mL and an AUC increase of 67.20% compared to CE. NCE exhibited excellent physical stability, with a low instability index (0.0055 ± 0.0007) compared to CE (0.0655 ± 0.0007, *** p < 0.001), and maintained uniformity without phase separation during 15‐day storage, confirming superior colloidal and storage stability. No adverse effects or histopathological abnormalities were observed in SD rats, confirming good biocompatibility. These results indicated that industrial‐scale nano‐casein emulsions provided a stable and scalable delivery system to enhance calcium and amino acid bioavailability, offering practical support for their commercial application in functional nutrition products.