The first evidence of cryo-milling improving enzymatic production of chitooligosaccharides from chitin biomass

Cryo-milling offers an energy-efficient approach to process α-chitin, the most prevalent and well-structured chitin form. In contrast to ball-milling, cryo-milling operates at low temperatures (<123 K), preventing heat-related charring and chitooligosaccharide loss. This study explored various cryo-milling conditions' impact on α-chitin, resulting in reduced crystalline plane intensity, increased surface area and smaller particle size without substrate breakdown or functional group loss. Additionally, the study investigated cryo-milled α-chitin's enzymatic hydrolysis for the first time. Using Paenibacillus sp. LS1's chitin-active-secretome, cryo-milled α-chitin exhibited a twofold increase in GlcNAc yield compared to unmilled α-chitin. Hydrolysis with recombinant Chi5 from Paenibacillus sp. LS1 resulted in twofold and fourfold increments in (GlcNAc)2 and GlcNAc yield, respectively. Overall, cryo-milling emerges as a gentle and effective alternative to ball-milling. It reduces α-chitin crystallinity and particle size, and increases the available surface area for enhanced enzymatic saccharification, thereby facilitating chitooligosaccharide production.