Carotenoid Analysis and Evaluation of Phycocyanin Recovery and Biological Activity Using Different Methods of Extraction from Arthrospira fusiformis (Oscillatoriales, Cyanobacteria) Originated from Lake Chitu, Oromia, Ethiopia

Abstract

Spirulina grows naturally in some alkaline lakes in warm tropical regions. Spirulina flourishes in saline-alkaline lake of the East African Great Rift Valley lakes, including Lake Chitu. Spirulina is famous and important source of phycocyanin pigment and various carotenoids with numerous applications. However, there are no investigations and clear scientific reports showing analysis of the total carotenoids and phycocyanin content of Spirulina isolated from Lake Chitu. Scientific reports have proven that various health benefits and applications of Spirulina in food, pharmaceutical, cosmetic and natural colouring industry does emerge from its high-quality protein, carotenoids, and particularly phycocyanin content. This study has aimed to conduct spectrophotometric evaluation of total carotenoid content of Spirulina culture identified and isolated from Lake Chitu and to extract and analyze phycocyanin pigment extracted using different extraction techniques from these cultures and then to evaluate the biological activity of partially purified lyophilized extracts at different concentrations. For this study, Arthrospira fusiformis containing samples were collected, identified and isolated, mass produced at laboratory scale, and then harvested and dried. Total carotenoid content was estimated using spectrophotometric analysis after extraction using 80% Acetone. Solvent type used for the study was optimized and crude phycocyanin extracts were obtained using classical mortar and pestle, freeze-thaw, homogenization with beads, ultrasound assisted extraction and microwave assisted extraction methods under same conditions of biomass to volume ratio of 1:50 w/v and solvent parameters. Extraction efficiency of each extraction methods employed and phycocyanin concentration, yield and purity were calculated using standard equations for analysis and comparison of the extracts. The presence of phycocyanin pigment was validated using sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Partial purification of extracts was done using the ammonium sulfate precipitation and dialysis method. Antimicrobial activity of the partially purified lyophilized phycocyanin extracts at different concentrations was conducted against four different gram-positive and gram-negative bacterial strains. Antioxidant activity of these samples was also evaluated using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay method. The total carotenoid content of 3.912 mg.g -1 was present in the dry weight of Spirulina. 0.1M sodium phosphate buffer was found to have the highest extraction efficiency at 0.415 mg.ml-1 phycocyanin concentration. The ultrasound assisted extraction method was found to give the highest yield of phycocyanin than the other methods at 36.35 mg.g -1 with an extraction efficiency of 84.5 %. SDS-PAGE has validated the presence of α and β subunits of phycocyanin pigment in the samples. After purification, a food grade phycocyanin purity was achieved with the highest value of 0.98. All extracts have shown effective antibacterial activity against all pathogens tested with demonstrated dose dependency. The DPPH assay has shown effective antioxidant activity of samples ranging between 48.9 to 74.3 % of radical scavenging activity across the different samples and concentration. This study has revealed the potential of Lake Chitu’s Spirulina as a good source of highly useful phycocyanin pigment compounds and carotenoids which have various applications in different sectors of industry and health.