Enzyme-assisted extraction of anticoagulant polysaccharide from Liparis tessellatus eggs

This study aimed to recover a heparin-like anticoagulant polysaccharide from Liparis tessellatus eggs (PLE) by using enzyme-assisted extraction technique. Extraction experiments were carried out using three different enzymes (Alcalase®2.4 L, Flavourzyme®500 MG, and Protamex®) under different conditions of temperature (45, 50, and 55°C), pH (6.5, 7.0, and 7.5), incubation time (24, 36, and 48 h), and enzyme to substrate ratio (E/S=0.5, 1.0, and 1.5%, w/w), which were combined according to a D-optimal design. Statistical analysis of extraction results allowed identifying the variables with greater influence on the extraction yield, and selecting the conditions that maximize the PLE extraction. The best extraction results were achieved when using the Protamex® enzyme in an E/S ratio of 1.34% (w/w), pH 6.60, 47.40°C, during 26.50 h. Under these conditions, a polysaccharide yield of 2.10% (w/w) was obtained. Clotting time measurements, activated partial thromboplastin time, and prothrombin time for evaluation of the anticoagulant properties of PLE were determined and showed increasing activities in correlation with the concentrations used. In the final step, the heparin-like nature of PLE was confirmed by digestion with heparinases I, II, and III, which showed ΔDiHS-0S, ΔDiHS-6S, ΔDiHS-diS1, and ΔDiHS-diS2 at compositions of 0.04, 0.03, 0.35, and 0.24 mol/g, respectively.