β-lactoglobulin from precision fermentation and milk exhibit similar protein structure but distinct aggregation behaviour: a multi-length-scale perspective.

Precision fermentation is a well-established biotechnology which has recently emerged as a platform for manufacturing nature-equivalent dairy proteins, including β-lactoglobulin (BLG). Here, we compare fermentation-derived BLG (f-BLG) with milk-derived BLG (m-BLG) from primary to quaternary structure and relate these findings to aggregation under UHT-like conditions. LC-MS and LC-MS/MS revealed that f-BLG consists of several proteoforms, predominantly a mature sequence containing an N-terminal "EAEA" extension. Despite this modification, 2D methyl NMR and SEC-UV-MALS confirmed equivalent tertiary and quaternary structures for f-BLG and m-BLG. However, the altered N-terminus markedly affected thermal stability. At 140 °C, m-BLG became opaque after approximately 1200 s, whereas f-BLG remained translucent throughout 1800 s. UPLC-UV showed that 80-90% of m-BLG aggregated within 30 s at 140 °C, while no detectable aggregation occurred for f-BLG. Consistent with these findings, m-BLG exhibited a steeper concentration-dependent viscosity increase. Krieger-Dougherty modeling estimated that heated m-BLG approached close packing (Φm = 0.68) at 6 wt% protein after 60 s, whereas f-BLG remained below Φm = 0.60. f-BLG's increased thermal stability is attributed to the additional glutamic acid residues in its N-terminal extension, which results in a more negative surface charge and increased electrostatic repulsion. Molecular dynamics simulations further indicate that these negatively charged N-termini remain outward facing upon protein association, potentially maintaining electrostatic repulsion at the aggregate surface and thereby limiting further aggregate growth. Together, these results demonstrate that a subtle N-terminal modification in precision fermentation-derived BLG markedly reduces aggregation and enhances stability under UHT-like conditions.