Study On Production Of Perylenequinones By Submerged Fermentation And Separation And Purification

Hypocrellins and other perylenequinones were some good potential photosensitizer. They also were used as a new generation of light dynamic antitumor drugs. The stomata of Shiraia bambusicola were the main sources of hypocrellins, However, It was limited as a kind of wild resource. Therefore, producing perylenequinone pigments by fermentation was one of main trends. Shiraia sp. super-H168 was studied to fermentation perylenequinones in this paper. The analysis method, composition of liquid fermentation media culture conditions and extraction and purification technology of pigments were developed. The conclusion was as follows:1) A method to analyze single pigment in the extraction was established. Phecda C18 purchased from Hanbon Sci.&Tech. was used, the ratio of acetonitrile and water was 6:4, the flow-rate was 1.0 mL/min, the column temperature was 25℃. Peak area and sample concentration is a significant positive correlation for EC in 37.72～754.40 mg/L range. For hypocrellins A (HA), peak area and sample concentration is a significant positive correlation in 14～219 mg/L range Ultraviolet and visible spectrophotometer was adopted to determine the total amount of pigments, the wavelength was 460 nm, OD value and sample concentration is a significant positive correlation in this situation.2) The composition of culture for submerged fermentation ofShiraia sp. super-H168 was gained through single factor test and response surface. The optimum fermentation culture was: Potato 200 g/L Glucose 60.95 g/L, Peptone 12 g/L, MgSO4 0.80 g/L, and NaCl 5.0 g/L, the production of pigments was 2.07±0.20 g/L in this situation.The culture conditions: pH 8.0, media volume 50 mL/250 mL, inoculation 7%, temperature 30℃, rotate speed 200 r/min, seed culture time 42 h, fermentation period was 3 days. After optimization of conditions, the total production of pigments was 2.795 g/L by shake flask culture, which was three times more than basic medium.3) The extraction and purification process of perylenequinone pigments from fermentation broth was as follows:(a) Extraction method: 90% ethanol, the ratio of fermentation broth and ethanol was 1:7, extraction temperature was ambient, and extraction was repeated two times, the time of extraction was 2.0 h.(b) Concentration: the extraction solvent was CH2Cl2, the ratio of concentration and CH2Cl2 was 5:2, extraction temperature was ambient, extraction was repeated two times, the time of extraction was 24 h.(c) Partial purfiy by Low pressure column chromatography: packing material was polyamide with 100～200 mesh, The mobile phase was methanol–water 45:55 (v/v) with a flow-rate of 3.5 mL/min. the column temperature was 20℃,the column size was 1.5 cm×20 cm. (d) Purify by Semi-preparation HPLC : Packing material was Phecda C18 from Hanbon Sci.&Tech., the ratio of acetonitrile and water was 6:4, the flow-rate was 35 mL/min, the column temperature was 25℃.After above purification steps, purity of elsinochrome C and hypocrellin A are more than 98% and 90%, respectively. The total recovery rate of pigments is 76.6%.