Biosynthesis and functionalization of recombinant bovine lactoferrin: Alginate-mediated stabilization and MTGase-induced gelation for anthocyanin delivery.
Bovine lactoferrin (BLF) is a milk-derived glycoprotein with diverse biological activities, including antimicrobial, antioxidant, and immunomodulatory functions, with potential for applications in functional foods and nutraceuticals. However, its practical utilization remains limited by key challenges: low efficiency of traditional extraction methods, poor gastrointestinal stability, and a narrow range of application forms. To address these issues, this study developed an integrated strategy spanning from efficient production to functional application. First, the Pichia pastoris expression system was optimized through signal peptide engineering and chaperone co-expression, achieving high-yield secretory production of recombinant BLF (rBLF) at 232.6 mg/L in shake-flask fermentation. Subsequently, rBLF was complexed with sodium alginate (NaAlg) via electrostatic self-assembly to form nanocomposites for gastrointestinal protection. The rBLF-NaAlg complex exhibited remarkable resistance to simulated gastric digestion, with only a 23.7% reduction in particle size, significantly lower than the 71.1% reduction observed for free rBLF. Furthermore, a three-dimensional hydrogel delivery system was constructed through microbial transglutaminase (MTGase)-catalyzed cross-linking. This system demonstrated encapsulation and sustained release of anthocyanins as a model bioactive compound. The gel structure and performance showed a clear enzyme concentration dependence: moderate cross-linking (<80 U/g) resulted in a uniform and dense network, enhancing encapsulation efficiency and providing controlled release. This work establishes a complete technological pathway from high-yield production and stabilization to functional expansion, demonstrating that rBLF can be transformed from a labile bioactive protein into a dual-functional material with both nutritional and delivery capabilities. This integrated strategy provides a viable solution for developing advanced BLF-based functional foods and nutraceutical delivery systems.