Breeding On Mutagenesis And Fusion Of Monacolink-high-yielding Monascus Protoplasts And Optimizing Of The Fermentation Conditions

The MonacolinK of Monascus，which have a good effect on the patiens withhigh cholesterol．The original strain Q-11，which had higher yield of MonacolinK was adoptedthe dual technology of UV mutagenesis and fusion of protoplast．Through the firstscreening by bacteria form，the repeat screening of detection by HPLC and geneticstability experiment， we selected three stable and high-MonacolinK-yieldingMonascus strains．(1) In the study，the formation，regeneration，UV mutagenesisand the fusion of protoplasts were researched． The result showed that theprotoplasts with higher yield and quality were obtained by the Mycelium age of theQ-11was36h，time of enzymolysis was2.5-3h，and enzymatic system(0.3%lysozyme+0.5%snailase+0.5%cellulase)． The optimal condition for UVmutagenesis of Monascus protoplast was the protoplast in the25W UV lamp at an20cm distance for80-100s，and the death rate reached88.7%-91%．Then achieveda stable and high-MonacolinK-yielding Monascus strain X-60，which yield was30%higher than Q-11． The protoplast fusion rate of X-60and Q-11was3.56%．And by fusing X-60and Q-11，we finally got five mutants which havinghigher yield of MonacolinK than Q-11，and they were R-22，R-101，R-39，R-51，R-58．Among them，R-39increased most．Comparing to Q-11and X-60，the rateof R-39increased120%and69.2%．R-58was another higher yield of Monacolinkand it increased86%and43.1%than Q-11and X-60．(2) Then the high-yieldstrains of R-22， R-101， R-39， R-51， R-58were tested by genetic stabilityexperiment．The results showed that the Coefficient of Variation of most high-yieldfusant reached18.54%，and its genetic stability was weak．However，the Coefficientof Variation R-22，R-51and R-58less than1%，they were proved to be the stablegenitic strains and no apparent reverse mutation．And the MonacolinK content ofR-58was highest，the mean reached2.1396mg/g．Compare with the original producing strain，choicing R-58has a higher production value．The mutant R-58had emerged more different phenomenon under the different cultures， whichcombined with original producing strain Q-11．The MonacolinK was extracted from the Red yeast powder by ultrasonicmethod， and the content of MonacolinK was quantitatively determined byHPLC． On the basis of single-factor experiments， the optimum extractionconditions of MonacolinK was obtained through the Box-Benhnken design andresponse surface analysis．The result show that the optimum extraction conditionsof MonacolinK were as follows． Ultrasonic power68.02%， ultrasonic time28.67min，and material to liquid ratio was1:5.45(m/v)．Under this conditions，thepredicted value and the validated value of MonacolinK were300.56ug/g and296.40ug/g．The medium components and fermentation time of solid-state fermentation forMonacolinK production by the strain R-58was optimized． The single-factorexperiments showed that the best initial water content of stromal was35%，the bestcarbon source is glucose，the optimal nitrogen source was soy protein powder，andthe optimum fermentation time was15d．But ZnSO4was found not the necessarycomponent in the fermentation of R-58，so do not add Zn．Through orthogonaldesign，the best solid-state fermentation medium of R-58as follows．Glucose5%，soy protein powder4%， MgSO40.5%， KH2PO40.3%． Under the optimumconditions，the validated value of MonacolinK was3.206mg/g，it is1.5times morebefore optimization．Adding Yeast inducer can promote MonacolinK formation，the single-factortests showed that the product of MonacolinK would be the highest when the addedtime of yeast was the second day of Monascus fermentation，the addition amount ofyeast was1ml and the age of yeast was60hours．Ethanol，penicillin and ultrasound can improve cell permeability，and they hadgreat impact on the product of MonacolinK content．The result of test showed that MonacolinK would be the highest when the ethanol concentration in2.5%．Thepenicillin had optimum effect on Monascus cell permeability on the condition ofthe addition amount of penicillin was4thousand units and the added time was thesecond day of Monascus fermentation．Besides，when ultrasonic time was10minand ultrasonic power40%，ultrasound would regulate the metabolic capacity ofMonascus and improve the production of MonacolinK．...