Microalgae proteins are increasingly recognized in the food and nutraceutical industries for their functional versatility and high nutritional value. Mild alkaline treatment is commonly used for cell wall degradation and intracellular protein solubilization, consequently enhancing the protein extraction yield. The findings of this study reveal that alkaline treatment alone, even at higher NaOH concentration (up to 0.3 M) and treatment time (up to 90 min), was ineffective (max. 2.4% yield) for the extraction of protein from Auxenochlorella protothecoides biomass. This challenge was significantly reduced through synergistic application of mechanical cell disruption using high-pressure homogenization (HPH) and alkaline solubilization. Single-pass HPH (35 k psi) alone without alkaline treatment led to 52.3% protein solubilization from wet biomass directly harvested from culture broth, while it was only 18.5% for spray-dried biomass. The combined effect of HPH and alkaline (0.1 M NaOH) treatment significantly increased protein extraction yield to 68.0% for a spray-dried biomass loading of 50 g L⁻¹. Through replacing spray-dried biomass with wet biomass, the requirement of NaOH was reduced by 5-fold to 0.02 M to achieve a similar yield of 68.1%. The process integration of HPH with the mild alkaline solubilization and utilization of wet biomass from culture broth showed high potential for industrialization of microalgae protein extraction. This method achieves high extraction yield while reducing alkaline waste and eliminating the need for energy-consuming drying of biomass, thereby minimizing the environmental impact.

Chlorella vulgaris (CV) is a promising sustainable protein source; however, its rigid cell wall limits protein extraction efficiency. To address this challenge, we previously developed a two-step process involving dry bead milling (BM) followed by alkaline extraction, which achieved a protein solubilization yield of 47.3% using 0.1 M sodium hydroxide (NaOH). To further improve protein recovery, reduce alkaline consumption, and simplify processing, the present study developed single-step extraction strategies that integrate cell disruption and protein solubilization in a single operation. Among the evaluated approaches, a single-step high-pressure homogenization (HPH) process coupled with 0.1 M NaOH achieved a protein solubilization yield of 80.2% after two passes (~5 min, 35,000 psi), representing a 1.4-fold and 1.7-fold increase compared with the single-step BM process (wet BM, 1 h) and the previously reported two-step process (2 h), respectively. Protein functional properties were influenced by the extraction method, with the single-step BM extract showing superior water absorption capacity, foaming capacity, and emulsifying performance. Collectively, the single-step HPH process maximized protein extraction yield and process efficiency, whereas the single-step BM process enhanced protein functionalities. These findings advance the efficient utilization of CV as a sustainable alternative protein source.