Comparative study on the optimization of phycobiliprotein isolation from Arthrospira platensis (spirulina) using mechanical, freeze-thaw, and enzymatic methods

Abstract

Arthrospira platensis (spirulina) is a prokaryotic microalga, rich in protein with a favorable amino acid profile. Most of its proteins are phycobiliproteins – light blue, fluorescent and water-soluble molecules, utilized in the food industry as colorants and nutrients. Despite the existence of numerous methods for separating and purifying these proteins from spirulina, they are often tedious and time-consuming. The study aimed to select the optimal method for phycobiliprotein extraction from a 5% Arthrospira platensis solution in succinate buffer (0.05 M, pH 6.5). Three approaches were evaluated: mechanical grinding (planetary ball mill, 48.2 rcf, samples after 10-40 min), freeze-thaw cycles (up to 8 cycles, samples collected after 2-8 cycles), and enzymatic lysis using lysozyme (1 mg/ml, incubation for 10-80 min, 30°C, 2 Hz). After extraction, samples were centrifuged (2652 rcf) to remove cell debris. Soluble proteins were then precipitated using ethyl alcohol at concentrations of 70%, 75%, 80% and 85%. The precipitated protein was centrifuged (9300 rcf), dissolved in deionized water and analyzed for total protein content using the Lowry method. Phycobiliprotein content was determined by measuring absorbance at 615 and 652 nm. The most effective phycobiliprotein isolation was achieved using the cyclic freezing and thawing method (4-6 cycles). Enzymatic lysis proved least effective, while ball milling yielded good results, though inferior to freeze-thawing. Increased processing time (grinding, lysozyme incubation, freeze-thaw cycles) enhanced the isolation efficiency of both total protein and phycocyanin. Regardless of the extraction method, 85% ethanol was most effective in precipitating phycobiliproteins.