| In order to develop a new and natural color material, this paper reports theexperiment concerned with yellow pigment produced by Trichoderma viride.First,studies on fermentation process and dynamics of T. viride;Second, optimize culturemedium components and culture conditions in orientational culture to produced moreyellow material，then extracted, isolated and characterized the yellow metabolism toconfirm the constitutes and structures.The main results and contributions of thisresearch are as follows:The yellow metabolite of T. viride has maximal absorption peak at thewavelength of290nm and380nm, the results was close to the maximum absorptionpeaks of Monascus yellow pigment reported (370,388nm).During the processes ofbatch liquid fermentation of T. viride, the dry cell weight, residual sugarconcentration, OD value and pH degree were tested.Then the experiment data wasprocessed to obtain the kinetices changing curves.The kinetic characteristics of thefermentation implied that the type of T. viride production fermentation type is growthcoupling, and it was presumed that yellow pigment might be directly derived fromprimary metabolism.The experiment concerned with the prescription of culture medium and cultureconditions optimization of T. viride producing yellow pigment, and used responsesurface methodology(RSM) based on a three-factor Box-Behnken design ofexperiments to optimize culture conditions.The results show that the optimum culturemedium components and culture conditions are:liquid medium based on PDA(250g·L-1potato and2g·L-1Na2HPO4), culture temperature is35℃;inoculum6%;the bestcarbon source is21.7g·L-1xylose;the most suitable filled amount in250mlErlenmeyer flask was50ml, the fermentation period is152hours at120r·min-1.In theoptimal conditions, the yellow pigment color value is52.8±0.8U·mL-1.Added urea,ammonium sulfate, peptone, beef extract powder in culture medium can’t increasethe output of yellow pigment.The yellow pigments of fermentation product were separated and the mainfractions were tentatively identifed by thin-layer chromatography–Fourier transforminfrared spectroscopy (TLC–FTIR). The analysis of infrared spectrum indicates thepresence of OH,=CH CH2, C=O, COOH, NH2and ester bond, glycosidic linkagesubstructures was confrmed.Every fraction had aromatic skeleton in varyingdegrees, and the results appeared to be reinforced by the ultraviolet spectrum. In conclusion the yellow metabolite had lots of functional groups and complex structure.From the ultraviolet absoption spectrum, it is observed that three obvious absorptionpeak, indicating that the yellow fractions containing one or more six-carbon ringscharacteristic of the benzene series and related organic groups, among that fraction eand f contained polycyclic aromatic compound such like naphthalene, anthracene,phenanthrene.The compounds were processed with solvent extraction and fractionalextraction,then isolated from the fermentation extracts by repeated column chromatography (CC)on silica gel, and Sephadex LH-20, as well as by semi-preparative HPLC andrecrystallization. The structures of these compounds were elucidated by means ofspectroscopic methods including IR, UV, ESI-MS,1D-NMR (1H-NMR,13C-NMR,DEPT) and2D-NMR (1H-1H COSY, HMBC, HMQC) as well as by chemicalmethod. Totally,5compounds were isolated and structurally elucidated:E1-2, E9-1,E11-1, E11-2, E12-1.One new naturally occurring product E1-2was also isolated, andcompound E1-2ã€E11-2is isolated from T. viride for the first time. |
