Ultrasound assisted methods for enhanced extraction of phycobiliproteins from marine macro-algae, Gelidium pusillum (Rhodophyta)

Extraction of phycobiliproteins (R-phycoerythrin, R-PE and R-phycocyanin, R-PC) from macro-algae is difficult due to the presence of large polysaccharides (agar, cellulose etc.) present in the cell wall which offer major hindrance for cell disruption. The present study is aimed at developing most suitable methodology for the primary extraction of R-PE and R-PC from marine macro-algae, Gelidium pusillum (Stackhouse) Le Jolis. Such extraction of phycobiliproteins by using ultrasonication and other conventional methods such as maceration, maceration in presence of liquid nitrogen, homogenization, and freezing and thawing (alone and in combinations) is reported for the first time. Standardization of ultrasonication for different parameters such as ultrasonication amplitude (60, 90 and 120 µm) and ultrasonication time (1, 2, 4, 6, 8 and 10 mins) at different temperatures (30, 35 and 40 °C) was carried out. Kinetic parameters were estimated for extraction of phycobiliproteins by ultrasonication based on second order mass transfer kinetics. Based on calorimetric measurements, power, ultrasound intensity and acoustic power density were estimated to be 41.97 W, 14.81 W/cm2 and 0.419 W/cm3, respectively. Synergistic effect of ultrasonication was observed when employed in combination with other conventional primary extraction methods. Homogenization in combination with ultrasonication resulted in an enhancement in efficiency by 9.3% over homogenization alone. Similarly, maceration in combination with ultrasonication resulted in an enhancement in efficiency by 31% over maceration alone. Among all the methods employed, maceration in combination with ultrasonication resulted in the highest extraction efficiency of 77 and 93% for R-PE and R-PC, respectively followed by homogenization in combination with ultrasonication (69.6% for R-PE and 74.1% for R-PC). HPLC analysis was carried out in order to ensure that R-PE was present in the extract and remained intact even after processing. Microscopic studies indicated a clear relation between the extraction efficiency of phycobiliproteins and degree of cell disruption in a given primary extraction method. These combination methods were found to be effective for extraction of phycobiliproteins from rigid biomass of Gelidium pusillum macro-algae and can be employed for downstream processing of biomolecules also from other macro-algae.