Mannan interference and purification efficiency in downstream processing of precision-fermented milk proteins from Komagataella phaffii

Precision-fermented milk proteins from Komagataella phaffii are a well-established technology, but high downstream processing costs remain challenging. This study characterised extracellularly secreted recombinant β-lactoglobulin (rBLG), unphosphorylated α_s1-casein (rCSN), and lactoferrin (rLTF) based on protein and non-protein content, comparing them to their animal-derived counterparts. Three purification methods were evaluated. Two were charge-based, i.e., isoelectric point (IEP) precipitation at a pH range of 2 to 7.5 for rBLG and rCSN and 5.5 to 11 for rLTF and anion-exchange (AEX) chromatography; one was size-based membrane separation. All target proteins matched ∼95% of their animal-based counterparts in secondary structure. Irrespective of the protein, mannans (52–66% d.b., 2–242 kDa, 75–87% mannose) were the main impurity. Size-based membrane separation was ineffective due to the similar sizes of protein and mannan. Charge-based methods were more successful. AEX removed mannan effectively, increasing the protein purity from 20–41% to 64–81%, but recovered only 32–37% protein, limiting its use in the food industry. IEP precipitation worked only for rCSN, obtaining final protein purity up to 77% (in precipitated fraction) with only 7% of the casein remaining unprecipitated. Future work should focus on better mannan removal to meet purity demands for functional applications.