Abstract: Lentinan which has the biological activity of enhancing immune system,anti-cancer,antihypertensive and hypolipidemic activities,was widely used in medicine and food industry.The enzyme-assisted (papain and cellulose) pressured hot water extraction was applied to extract lentinan from fresh Lentinus edodes in the present study.The fresh Lentinus edodes was firstly prehydrolyzed with papain and cellulase,and followed by pressured hot water extraction.Afterwards,gel chromatography with Superose 6 PG was applied for the purification of lentinan.The optimal conditions of enzymatic prehydrolysis were as follows:cellulase of 1.00 g/g (calculated by dry mass of Lentinus edodes),papain of 0.024 g/g,temperature of 45℃,pH of 4.5 and enzymatic prehydrolysis time of 10 h.After that,the pressured hot water extraction was used to extract lentinan for 80 min at 115℃ with a solid/liquid ratio of 1:50(W/V).Under the optimal conditions,the extraction yield of lentinan was up to 23.6%.Then,purification of crude lentinan was carried out on Superose 6 PG with degassed purified water as eluent,and the flow rate and column temperature were 0.6 mL/min and 48℃,respectively.A homogeneous polysaccharide fraction was obtained by the two-step gel chromatography.The purified lentinan was identified by anthrone-H₂SO₄method,periodate oxidation method and infrared spectroscopy.The weight-average molecular weight of the purified lentinan was 3.75×10⁴,which was measured by gel permeation chromatography.A red shift phenomenon was observed when the Congo red reaction was used to analyse the purified lentian,which suggested the helix conformation was existed in the purified lentinan.The results of present research will supply a new approach for extraction and purification of lentian from fresh Lentinus edodes.