Studies On Genetic Transformation System, Color Value And Citrinin Of Monascus Purpureus

The application of Monascus has a long history in china. Presently, the Monascus products are widely used in rice wine fermentation, food additives, heath care and medicines. In recent years, researches on the lattermost one have become the hotspot, both in China and abroad. But the identification of Citrinin in the metabolites of Monascus in 1995, however, has arose close attention among the researchers around the world. This mycotoxin is tightly controlled in the food and feed in western countries.In order to disrupt pks gene and make non-citrinin producing strains of Monascus purpureus.We studied the method of protoplast preparation and REMI transformation. To establish protoplast-mediated genetic transformation system of Monascus purpureus9903A, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 32℃. Mycelia obtained through cellophane-mediated culture for 50-60 h were adequate to for protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase at the concentration of 0.4% lysing enzyme,0.8% cellulose and 0.6% snailase respectively was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentration on the protoplasts preparation, 1 mol/L sorbitol was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 1.5-h. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of M. purpureus, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth. According to the study of protoplasts prepatation, we can adcance the efficiency of protoplasts regeneration.Fristly, the conserved sequence including partial ITS1, ITS2 and entire 5.8S rDNA was amplified from the chromosomal DNA of 9903A, and sequenced. The strain was confermed as Monascus purpureus by the BLAST analysis of network service. The result showed that the method of transformation was able to transform other gene into M. purpureus.Five hundred and nity eight transformants were obtained on the selective plates containing hygromycin B. Six of the transformants were subjected to characterize extensively the pigment production in solid state fermentations. After several times of screening, transformant ZM0303 was selected.At the same time, aiming to increase the yield of Monascus pigments and to decrease the citrinin content, we studied the culture medium and the fermentation conditions of solid fermentation of Monascus purpureus 9903A.An optimum medium was obtained through single factor tests and optimization tests.During the research, we found that the addition of octanoic acid or MgSO4?7H2O could improve the color value while decrease the citrinin content.The studies on the process conditions of solid fermentation also focused on the effects of temperature. When the temperature increases within the rang of 30-36℃, the color calue of red rice was fund to improve, too. However, only at lower temperature(30℃)could a remarkably lower citrinin content be obtained.