Characteristics Of Penicillium Citrinum Mutant F-33 With The Ability Of Producing Red Pigments

Penicillium citrinum F-33 was a mutant which could produce red pigments. It was obtained by plasmogamy after ⁶⁰Co mutagenesis from Penicillium citrinum 4011 strain.The main research aspects were focused on the growth characteristics compared with other mutants with different pigments,red pigment diversities affected by nutrients in medium,and the purification of red pigments.Morphology study of 4 strains was made under different medium including PDA,MEA,YES and CYA,and RAPD-PCR was used to analyze the genetic diversity between 4 strains.The mutant strain's ability of pigment production changed much.Strain F-33 can produce abundant of red souble pigments in PDA medium and MEA medium,but produce a little pigments in YES medium and CYA medium.There would be a positive relationship between the color production with the nuclease P1 in the mutants,i.e.,colony with dark,deep green color suggested a low enzyme activity,but grey-green colony gave out a high output of nuclease P1 in a higher possibility.Colony of this strain is dark green in face,the ability of production of nuclear P1 is low.Penicillus of Penicillium citrinum F-33 is not strong but highly concentrated,conidiophore is rich.Six primers with rich polymorphisms were chosen from 24 random primers by RAPD-PRC in order to analyze the difference between the four strains genome.The results showed that there were some genetic differences between strain F33 and other strains.In detail,strain F-33 is similar to Penicillium citrinum 4011 while Penicillium citrinum J1Y6 is similar to Penicillium citrinum F5-5 by homology comparison.Colorimeter and CIELAB system was adopted to analyze production of red pigment under different conditions.Combined with TLC,parameters such as Chroma(bigger was better) and Hue angle(smaller was better) were used to measure the pigments in the natural status.In PDA medium,the parameters(Chroma,Hue angle) cultivated 48hours,72hours and 96hours at 28℃were(51,39),(47,26),(39,19) respectively,they are stable,the parameters(Chroma,Hue angle) under same condition at 25℃were (36,27),(52,29),(19,72).Combined with TLC,the stripe at 28℃was much more obvious than 25℃;the stripe at 25℃even showed yellow,further more, Hue angle at 25℃made a great accretion because red medium became yellow. Strain produced No red pigment at 33℃,while it stopped growing at 37℃,their Chroma almost less than 20.So 28℃was the optimal temperature,for strain to produce red pigment.Acetic acid buffer,phosphorice acid buffer and citric acid buffer well showed inhibition on the production of pigments.The Chroma of pigment in 3 mentioned medium were all less than 20,and the stripe of pigments were obscure in TLC.Carbon source had inhibition on red pigment' synthesis,short chain carbon source affected more than long chain carbon source.Glucose was the worst one while the starch was the best one.Polyphenols in PDA may be the crucial reason for pigments' synthesis.Nitrogen source will promote the pigrnent's synthesis,especially the poly peptone and tyrptone,the parameters(Chroma,Hue angle) cultivated 96 hours reached(59,35) and(60,33) respectively.Compared with PDA(39,19),the color of medium with poly peptone and tyrptone was more saturated but less pure.From the TLC,all stripes of the red pigrnents were tailing except the pigment in PDA,the TLC method in this paper was no longer fit to analyze those pigment.It suggested pigments would become complex with the culture time increased.Centrifugation,extraction,column chromatography and HPLC was used to purify the pigment from broth.Ethyl acetate was the best extract solvent for red pigment.Acetone was the best eluant to in column stuffed with Kisselgel 60G, it could elute a red pigment crudeâ… .Methanol will wash-out another red pigment crudeâ…¡after acetone' elution.2 finals including red pigment and yellow pigmentâ… -b were obtained by HPLC,max UV-vis absorption wavelength ofâ… -a is 518nm,max UV-vis absorption wavelength ofâ… -b is 390nm.