Construction Of The PksCT Gene Disruptant In Monascus Ruber And Study On Producting Monascus Pigment In Submerged Fermentation & Solid-state Fermentation By The Disruptant

The filamentous fungus Monascus is one of the important microorganism resources of China.The physiologically active substances that Monascus producted had been researched widely.Mnascus pigments is a natural substance,wnich is playing more and more important poles in modern food industry.However,due to citrinin contamination during the fermentation process,it is called in question about food safety evaluation of Monascus fermentation.In this pater,a pksCT-replacement vecter was transformed into M.ruber11(Mr11) to disrupt its pksCT gene,and a pksCT-disrupted strain was gained,whose capacity of producing citrinin was sharply decreased.Then,the process of production of Monascus pigments in submerged fermentation and solid-state fermentation by a pksCT-disrupted strain was studied. The main contents are illustrated as following:1.The pksCT-replacement vector pHD116 was transformed into protoplast of M.ruber 11 to disrupt its pksCT gene.Then the methods of hygromycin resistance and genomic-PCR were used to screen disruptants.A pksCT-disrupted strain Mr11k was gained,whose capacity of producing citrinin was decreased by about 99%compared with the original strain.At same time,its capacity of producing Monascus pigments was increased by 16.6%.2.Response surface methodology(RSM) based on a three-factor Box-Behnken design of experiments was used to increase the Monascus pigment content of Monascus-fermented rice(MFR) by Mr11k in solid fermentation.The optimum cultivating conditions were as follows:moisture content 32.5%;cultivation time 16d and temperature 30℃.In these conditions,the color value of MFR produced by Mr11k reached to the maximam,which was 5120 U/g,and citrinin wasn't detected in the MFR.3.An optimum medium was obtained through single factor test and Response surface methodology(RSM) experiments.The optimal medium for flask-liquid-state fermentation were rice power 5%,NaNO₃ 0.2%,KH₂PO₄ 0.05%,K₂HPO₄ 0.1%, MgSO₄ 0.1%.Using this optimal medium,the culture conditions for producing Monasus pigment were investigated.The optimum cultivating conditions were as follows:fermentation time 144hr,temperature 28℃,seed age 72 hr,seeding volume 6%,filled volume 40mL/250mL flask,initial pH6.7.In these conditions,the color value of fermentation broth was improved to 170.6 U/mL,and the content of citrinin was 2.8μg/mL.4.Based on the optimal medium and condition for flask-liquid-state fermentation, the fed-batch fermentation was performed with strain Mr11k in 5L fermentor.When the fermentation was conducted with agitation at 150 r/min and aeration at 2 sL/min for 120 hr,the final Mnascus pigments yield by Mr11k reach to158 U/mL,and the content of citrinin was 2.2μg/mL.