Research Of Breeding And Fermentation Conditions Of High-lovastatin Monascus Strains

In recent years,with the improvement of living conditions,more and more people suffer from cardiovascular diseases.As the world recognized lovastatin as an ideal agent for the treatment of cardiovascular diseases,the demand of this drug is increasing rapidly.But some of the problems,low yield,long cycle and high cost in the production process limit the practical application of Monascus-lovastatin.In this paper,Monascus product lovastatin were isolated from red yeast rice in Fujian,traditional mutation and genome shuffling were used to achieve high-lovastatin strains.Then study on the solid and liquid fermentation conditions,and to explore the method of lovastatin separate.The main results are as follows:1、By means of dilution plate count,Monascus strain named SH2-7 was isolated and purified from red yeast rice,with high producing lovastatin,was identified as Monascus purpureus based on morphological and ITS sequence.Through spore suspension UV mutation and microwave mutation,five mutant strains with high lovastatin production were screened as parental genomic library of genome shuffling:4-4,5-5,W2,W42,W44.2.The optimum conditions of preparation of Monascus protoplast were as follows:lywallzyme concentration,1.0%;mycelium age,66h;enzymolysis time,3h;enzymolysis temperature,30℃;the stablizer buffer system was pH 6.0 hypertonic phosphate buffer,regeneration medium was hypertonic screening medium contaning 0.6 mol/L NaCl.Under the optimum conditions,the protoplast yields and regeneration rate were up to 2.47×107/mL and 1.45%.The protoplast fusion conditions were as follows:the protoplasts were mixed together after two inactivation methods of UV irradiation for 120s and microwave irradiation for 300s.The protoplast fusion rate reached 1.95%under 30 ℃ for 12min in 30%PEG containing 0.03mol/L Ca2+.3.A fusant R"-30 with higher production of lovastatin and genetic stability was obtained after three rounds of genome shuffling from parent strains 4-4,5-5,W2,W42 andW44.Optimum conditions for liquid fermentation were as follows:initial medium pH,5.0;medium volume,50mL/250mL;rotation speed,150r/min;temperature,30℃;seed age was 66h and the inoculum volume was 8%.Fermentation culture medium for lovastatin production was composed of glycerol,3%;soybean powder,1.5%;ZnSO4·7H2O,0.66%;KH2PO4,0.017%;MgSO4·7H2O,0.15%.The production of lovastatin by Monascus R"-30 reached to 615.3mg/mL after fermentated 12d in all these conditions.Optimum conditions for solid state fermentation were:initial water content of substrate,35%;rice 400g/1.1cm medium thickness;inoculum volume,8%;rice as basic medium,put glycerol 5%,soybean powder 1.0%,FeSO4·7H2O 0.2%,MgSO·-7H2O 0.1%in seed liquid.The production of lovastatin by Monascus R"-30 reached to 10.05g/kg after fermentated 16d in optimum conditions.4、Separation and purification of lovastatin in liquid and solid state fermentation by successively through silica gel column and LH20 column,gradient elution of organic solvents with different polarity.Liquid fermentation:the purity of lovastatin reach 93%,and 45%fermented lovastatin were obtained after separation;solid state fermentation:the purity of lovastatin reach 70%,with the recovery yield of 55%.