Effects of sequential ball milling and loosenin-like protein treatment on pulp fibers

Loosenins are non-hydrolytic proteins that enhance cellulose accessibility by disrupting hydrogen bonding. This study investigates the effect of a novel fungal loosenin-like protein, PcaLOOL12, on never-dried hardwood kraft pulp fibers pre-treated by mechanical ball milling. Ball milling at varying energy levels (50–150 kWh/t) was used to open the fiber structure and improve protein accessibility. A treatment energy of 50 kWh/t was selected for subsequent experiments. Fiber morphology, water retention, and dissolution behavior were analyzed as a function of PcaLOOL12 dosage and incubation time. Mechanical treatment increased fiber width, fibrillation, and water retention, with minimal impact on fiber length or nanoscale porosity. PcaLOOL12 further enhanced fibrillation and water retention, particularly at higher dosages (5–10 wt%) and longer treatment times (24–72 h), suggesting surface-level delamination. No significant increase in nanoscale porosity was observed, indicating surface-specific action. CED-solubility decreased following protein treatment, possibly due to fibril aggregation or altered fiber–solvent interactions. These results demonstrate that combining mechanical and protein treatments can modulate fiber morphology and accessibility, supporting their use in bio-based product development.