Extraction、Isolation And Identification Of Pigment Secondary Metabolites From Trichoderma Cyanodichotomus TW21990-1

Trichoderma cyanodichotomus TW21990-1 was isolated from the lake wetland of Dongting Lake in Hunan Province.The stability and molecular structure of blue pigment obtained from T.cyanodichotomus TW21990-1 were identified based on the optimization of its culture conditionand extraction method.Specific research results are as follows:（1）T.cyanodichotomus TW21990-1 was inoculated in potato dextrose agar medium and cultured for 7 days.The colonies were uniform,the aerogenic mycelia were dense and velvety,the conidia were gray green and covered the whole plate with no obvious odor,and the surface produced obvious diffused blue pigment.The optimal culture temperature for producing blue pigment was 25℃.（2）Optimization of medium conditions for production of blue pigment from T.cyanodichotomus TW21990-1.Through single factor experiment and Box-Behnken central combination design experiment,the optimal culture conditions for blue pigment production of T.cyanodichotomus TW21990-1 were determined as follows:glucose was selected as carbon source,0 g/L agar,the initial p H was 4.8,culture temperature was 25℃,and incubated for 7 days.Under the above culture conditions,the extraction rate of blue pigment was the highest.（3）The average inhibition rate of T.cyanodichotomus TW21990-1 against plant pathogens was 20%～68.57%on PDA plate.Among them,the inhibition rates of TW21990-1 against Rhizoctonia solani,Botrytis cinerea,Botryosphaeria parva,Gaeumannomyces graminis var.tritici,Fusarium oxysporum f.sp.cucumerinum Owen were 68.57%,65.20%,56.25%,56.12%and 50.06%,respectively.The inhibition rate of TW21990-1 against Fusarium moniliforme was as low as 20%,while against other pathogenic fungi was intermediate,and blue pigment was produced around the inhibition zone.（4）Optimization of extraction method of T.cyanodichotomus TW21990-1 blue pigment:Through single factor experiment and orthogonal test,the extraction conditions of the highest extraction rate of blue pigment crude extract were as follows:extraction solvent was anhydrous ethanol,extraction temperature was 55℃,solid-liquid ratio was 1:20 g/m L,extraction time was 80 min,ultrasonic power was 150 W.The blue pigment ethanol solution was scanned at 200-750 nm by enzyme labeling instrument.It was found that the blue pigment crude extract had a maximum absorption peak at 335 nm and 580 nm respectively.（5）The stability of blue pigment was tested.The results show that blue pigment has the following characteristics in terms of physical and chemical properties:blue pigment can withstand ultraviolet light and sunlight,but is not suitable for exposure to direct sunlight.Blue pigment is more sensitive to temperature.In addition,blue pigment has a certain tolerance to acidic environment,but not to alkaline environment.The effects of various metal ions on the stability of blue pigment were different,among which Fe³⁺and Al³⁺had the greatest effects.H₂O₂,Vc and EDTA-Na₂showed strong achromatic effect on the blue pigment.Na HCO₃and sodium citrate also had a certain effect on the stability of blue pigment,while sodium benzoate had a significant effect on color protection.Glucose,Na Cl,KCl,citric acid and other organic acids had no significant effects.Maleic acid has a certain color protection effect.Therefore,the blue pigment is suitable to avoid direct sunlight and weak acid preservation.（6）Isolation and identification of monomer compounds.The crude extract of blue pigment was isolated and purified by repeated column chromatography on silica gel and Sephadex LH-20 as well as high performance liquid chromatography（HPLC）.The structures of these compounds were elucidated by means of spectroscopic methods including IR,UV,ESI-MS,1D-NMR(¹H-NMR,¹³C-NMR)and 2D-NMR（¹H-¹H COSY,HMBC,NOESY）as well as by chemical method.Totally,3 compounds were isolated and structurally elucidated:（4R,5R）-4-（hydroxymethyl）-5-methyl-ethylene sulfite,compound 2 and euonymol,which all isolated from T.cyanodichotomus for the first time.Among them,（4R,5R）-4-（hydroxymethyl）-5-methyl-ethylene sulfite and compound 2 were new compounds that have never been reported.