An Adapted High-Pressure Homogenization Workflow Improves Recombinant β-Casein Recovery from Escherichia coli Inclusion Bodies

The efficient extraction and purification of recombinant β-casein from Escherichia coli is a crucial step in bioprocess engineering. This study evaluates an adapted high-pressure homogenization (HPH) workflow for recombinant β-casein recovery and compares it with osmotic shock (OS) and standard HPH based on specific protein yield, estimated relative purity, and processing time. In contrast to standard HPH, which requires separate inclusion body recovery, washing, and subsequent denaturant-mediated solubilization, the adapted-HPH workflow integrates mechanical cell disruption with inclusion body recovery during homogenization. Standard HPH was evaluated in two downstream-processing runs, whereas adapted HPH and OS were each evaluated in three downstream-processing runs using separate aliquots from the same bioreactor biomass batch. Adapted HPH demonstrated higher specific yields (4.08 mgcasein/gCDW) and required 40% less processing time compared to the OS and standard HPH methods. The specific yield obtained with adapted HPH was significantly higher than that obtained with standard HPH (p = 0.026), whereas the difference between adapted HPH and OS was not statistically significant (p = 0.086). Despite the inclusion body washing steps used for OS and standard HPH, adapted HPH achieved a slightly higher average purity than OS (63.46 ± 3.49% versus 59.38 ± 2.79%) and a purity comparable to standard HPH (68.10 ± 6.87%); however, these differences were not statistically significant. Overall, the comparative evaluation of these methods indicates that adapted HPH may provide a simplified alternative for recombinant β-casein recovery, although further validation with a larger number of independent experiments is required.