Ultrasound-assisted protein extraction for deep proteome analysis of Spirulina and Chlorella microalgae

Microalgae Spirulina ( Arthrospira platensis ) and Chlorella ( Chlorella vulgaris ) are gaining attention as sustainable alternative protein sources to address the needs of a growing global population. However, their tough cell walls present a significant challenge for efficient and more quantitative protein extraction. This study introduces a simple, cost-effective, and scalable "one-pot" protocol for extracting microalgal proteins by ultrasound-assisted methods in an aqueous buffer. The combination of ultrasonication and sodium deoxycholate successfully breaks down the resilient cell walls, improves protein solubility in water, and achieves high extraction yields of 81–86%. This performance significantly surpasses traditional methods, such as Tris-HCl buffer (0.7–1.2% yields) and chloroform/methanol solvent (4.5–14% yields). Using this method, 1,354 proteins were identified in Spirulina and 771 in Chlorella, each with over 10% sequence coverage. This enabled the recovery of numerous bioactive peptides, including 161 candidates for Spirulina and 60 for Chlorella from phycobiliproteins, all comprising at least three amino acid residues. These peptides hold the potential for creating bioactive hydrolysates and functional ingredients for food and pharmaceutical applications, following detergent removal via appropriate purification. This optimized protocol offers a promising solution for large-scale, safe microalgae processing and facilitates the production of biopeptides from microalgae species. • Effective cell lysis using ultrasound assisted mechanical method • One pot extraction and digestion of microalgae proteins • Improved extraction yields with one pot protocol • Detailed proteome characterization of Spirulina and Chlorella • Recovery of almost 200 bioactive peptides from microalgae