Research Of Process Optimization For Produce β-carotene And Metabolic Regulation By Blakeslea Trispora From Corn Steep Liquor

β-carotene is one of the carotenoids which is widely used in the field of medicine and food as antioxidant. Corn steep liquor is one of the most important by-product of production of corn starch, in the industrial production of fermentation is used as nitrogen source. In this paper, pretreatment method of corn steep liquor was studied, then used pretreated corn steep liquor as the sole nitrogen source by using Blakeslea trispora to fermentation product β-carotene, the fermentation medium were optimized through mathematical statistics, the purpose was to enhance the ability of these strain to produce β-carotene by using corn starch as raw material. The details are as follows:（1）Using single-factor test to determine the optimal seed medium and cultivation condition, there were the optimum seed medium: corn flour 3%, soybean flour 3%, Mg SO₄ 0.02%, KH₂PO4 0.1%, VB₁ 0.001%. The optimum culture conditions as follow: temperature 28℃, shaking speed 200 r/min, initial p H 6.5, seed age of strain（+） 20 h and strain（-） 25 h. Based on the optimization seed medium and culture conditions, medium composition was selected by single-factor, Plackett-Burman design and response surface methodology, optimal culture medium as follow: glucose 7%, soybean flour 0.7%, wheat bran extract 3%, Mg SO₄ 0.05%, KH₂PO4 0.3%, VB₁ 0.001%, soybean oil 1%. According to the determined optimum conditions, the maximal β-carotene production was 47.20mg/L, which agreed with the predicted values well.（2）Using single-factor test to determine the effect of some metal ions which contained in corn steep liquor on β-carotene and biomass,the result showed that when 0.006% Zn²⁺ or 0.007% Mn²⁺ added into the medium, the production of β-carotene increased 17.9% or 44.06% compare with blank, this demonstrates that β-carotene biosynthesis was stimulated by Zn²⁺ and Mn²⁺ in Blakeslea trispora, different concentrations of Fe³⁺, Cu²⁺ and Ca²⁺ would inhibit the synthesis of β-carotene.（3）Using single-factor test to determine pretreatment method of corn steep liquor, alkaline treatment and Na₂ S sediment treatment was used, study found that the alkaline treatment could remove the metal ions in corn steep liquor, but this method could not improve β-carotene production. The method of Na₂ S as follow: add Na₂ S into corn steep liquor and then filtration the precipitation with p H6.5, Na₂ S optimum adding amount was 0.58g/100 m L corn steep liquor. Then use of treated corn steep liquor fermentation, the maximal β-carotene production was 46mg/L which 1.8 times than untreated.（4）Use the pretreatment corn steep liquor as nitrogen source to produce β-carotene, optimal culture medium obtained by single-factor, Plackett-Burman design and response surface methodology, the optimization culture conditions as following: glucose 9%, corn steep liquor 4%, Mg SO₄ 0.04%, KH₂PO4 0.3%, VB₁ 0.005%, soybean oil 2.34%, p H 6.5. According to the determined optimum conditions, the maximal β-carotene production was 60.36mg/L, which agreed with the predicted values well.（5）Using single-factor test to study metabolic stimulators on β-carotene production. The optimum conditions as following: 1.5% n-dodecane, 1% n-hexane, 0.001% ketoconazole and 0.15% Tween-80 were added before inoculate, 0.1% β-ionone was added after 36 hours of age inoculation, 3% liquid paraffin was added into the medium after 24 h older inoculation, 30μmol/L H₂O₂ was added into the medium after 36 h older inoculation, metabolic stimulators of study could enhanced β-carotene production after optimized.