Breeding Of Higher γ-Aminobutyric Acid-Producing Monascus And Optimization Study On The Submerged Fermentation

γ-Aminobutyric acid (GABA) is a kind of non-protein amino acid in nature. It is an important inhibitory neurotransmitter of the mammals'central nervous system. GABA can participate in cerebral circulation, promote reproduction and activat kidney function. It also influence on depressing blood-pressure, improving growth and treatment of epilepsy. GABA has been widely used in the field of food and pharmaceutical industries.Monascus X27 was isolated from red mold rice. Through mutagenesis, medium composition optimization and optimization of fermentation conditions, GABA production of Monascus X27 has been increased, which have been used in the 300 L fermentation tank pilot.First, from the red yeast rice, red fermented bean curd, Chinese yeast and bean paste,300 strains of monascus were isolated. Through a kind of improved paper chromatography screening, the GABA production of a strain No.27 reached 2.32 g/L. No.27 was regarded as a strain of Monascus ruber by morphological means and it would be used as the starting strain for future mutagenesis experiments. By ultraviolet mutagenesis, nitrous acid mutagenesis, cobalt chloride mutagenesis and nitrous acid-UV mutagenesis, the GABA production of the mutagenic strain (No.27-A015) rose up to 3.12 g/L. After 20 generations, the mutant strain had stable GAB A production and no reverse mutation. No.27-A015 was renamed as X27.Second, results of P-B design experiments showed that the most significant factors of GABA biosynthesis were temperature, pH and ventilation rate. According to P-B design experiment results, climbing experiments were designed to determine the central point of response surface method (RSM). Based on the regression coefficients of RSM, the regression equation(Y=-209.366+3.435X,+55.945X2+0.103X3-0.059X12-5.135X22-0.0002X32-0.081X1X2-0.001X 1X3-0.019X2X3) was established. The optimized fermentation conditions of GABA production were 34℃, pH 5.5 and 120 L/h, and the other conditions were:first order seeds, tank pressure of 0.3 MPa, impeller speed of 200 r/min, inoculum concentration of 5%. Under these conditions, the production of GABA reached 9.18 g/L. Though the carbon, nitrogen source and sodium glutamate fed-batch studies, it was found that sodium glutamate had inhibition effect on the biomass of Monascus X27. The low concentration of sodium glutamate in initial fermentation was the key of Monascus biomass increasing, and can also shorten the lag phase and cycle of the fermentation, which reduced the production cost effectively. After nitrogen source optimized fed-batch study, the maximum GABA production reached 9.91 g/L.Finally, the 300L fermentation experiment of Monascus X27 was carried out by the use of the best medium components and the optimal fermenter conditions. Compared with the small fermenter, its oxygen utilization was raised, but the increasing of volumetric oxygen transfer coefficient (KLa) became slower relatively (with the increasing contents of the medium volume, KLa value growth rate subsequently slowed down), which made the commercial production base on the parameters of small-scale fermenter experiments. At last, increasing the impeller speed of early fermentation stage (within 12 hours) can improve the amount of medium's dissolved oxygen and stimulate thallis'growth. But the impeller speed couldn't exceed 250 r/min to avoid excessive shear damaging monascus X27. At the fermentation medium-term (24 hours around), adding the amount of glucose can inhibit the rapid increasing of pH, which has toxic effects on monascus X27.