Extracellular expression of recombinant bovine lactoferrin in Pichia pastoris with enhanced activity and a thermostability analysis for food application.

Cost-effective oral delivery of bovine lactoferrin remains a challenge. It is currently the most expensive dairy ingredient in the industry, due to its low native yields (0.5 g/L) and lengthy purification process. To overcome these challenges, recombinant bovine lactoferrin (rbLF) was produced extracellularly from a Pichia pastoris (KM71-H) host with the goal of creating a simple, yet effective production stream. To validate the successful expression, protein activity was investigated in terms of antibacterial analysis and iron-binding ability. To further validate its application in the food processing field, we measured the thermal stability of rbLF up to 90°C for 5 min to represent boiling conditions. When compared with native lactoferrin (cbLF), the rbLF showed similar antibacterial activity against Gram-negative bacteria, Escherichia coli (E. coli), enhanced antibacterial activity against Gram-positive bacteria, Staphylococcus aureus (S. aureus), and enhanced iron(III)-binding (p ≤ 0.0001). Whereas both cbLF and rbLF remained effective against E. coli until 60°C and effective against S. aureus until 70°C, they both completely lost their activity when exposed to 90°C against either bacterial strain (n = 3). High Performance Liquid Chromatography (HPLC) (n = 3) and Differential Scanning Calorimetry (DSC) (n = 2) revealed that rbLF and cbLF showed a two-fold decrease in structure at 70°C and a complete loss at 90°C. In summary, this study developed an extracellular expression platform for rbLF in a Pichia pastoris system that behaves similarly to cbLF. The produced rbLF shows promise as a valuable material for new potential downstream applications in the food industries.