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

• Published in: Future foods : a dedicated journal for sustainability in food science Vol. 12; p. 100735
• Main Authors: Nugroho, Aryo D., Ji, Rensong, Chin, Yi Ling, Heck, Albert J.R., Reiding, Karli R., Boom, Remko M., Keppler, Julia K.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2025
• Subjects: Cellular agriculture; Exopolysaccharide; Heterologous proteins; Mannoprotein; Pichia pastoris; Recombinant casein; Recombinant lactoferrin; Recombinant β-lactoglobulin; Recombinant lactoferrin; Cellular agriculture; Heterologous proteins; Pichia pastoris; Mannoprotein; Exopolysaccharide; Recombinant casein; Recombinant β-lactoglobulin
• ISSN: 2666-8335; 2666-8335
• DOI: 10.1016/j.fufo.2025.100735

•Mannans, the main impurity of yeast-based milk proteins, make up 52–66% dry matter.•Recombinant milk proteins are 95% similar in secondary structure to bovine ones.•The size of mannans overlaps with proteins, complicating membrane separation.•Anion exchange gives high purity but limits recovery and its use in food industry.•Recombinant αs1-casein can be precipitated at its isoelectric point, at pH ∼4.5. 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 αs1-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. [Display omitted]