Abstract

Provided an integrated expression-to-encapsulation strategy for rBLF delivery. • NaAlg complexation protected rBLF from gastric degradation. • MTGase-crosslinked rBLF hydrogel enabled sustained anthocyanin release. 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 tran