Abstract
Microalgae are promising protein sources, but improved extraction techniques are needed to ensure efficient and sustainable processing. The objective of this study was to investigate the effect of high-pressure homogenization (HPH) parameters on the extraction of soluble protein from Nannochloropsis sp. Various combinations of processing parameters, such as extraction media (water vs. phosphate buffer (0.1 M, pH 7)), biomass concentration (2% vs. 8% w/v), HPH pressure (0–1500 bar), and pass/cycle number (1–3), were used. Increasing pressure, biomass concentration, or pass number did not always improve soluble protein yield, likely due to reduced disruption efficiency at higher solids loading and post-release protein denaturation/aggregation during excessive processing. Under all pressures, protein yield at 2% (w/v) biomass in phosphate buffer decreased from two to three cycles despite greater membrane disruption, as confirmed by dual-fluorescence microscopy, suggesting that excessive processing promoted post-release protein denaturation/aggregation and interactions with other released intracellular materials, thereby reducing the soluble protein fraction. The maximum protein yield obtained at 1500 bar (after two cycles) was 80.35 ± 1.0 mg·g−1 DW, which corresponded to 25.5% recovery of the total biomass protein, and the cumulative soluble protein recovery, including the protein recovered during the washing step, reached 40.5%. The results establish an optimal HPH operating window that maximizes protein recovery while limiting the potential for excessive processing-induced protein modification, providing a practical guideline for efficient and scalable microalgae protein extraction.