Study On Production Of β-carotene By Rhodotorula Sp. And Its Conversion To Vitamin A Palmitate In Vitro

Rhodotorula RM which can produce β-carotene was isolated from yellow wine lees of Fujian, then the strain was improved by protoplast mutagenesis, the mutation breeding and optimization of media ingredients were studied, and the culture conditions of the mutant were optimizated too, the amplification tests were implemented at the sametime. The β-carotene was transformed into vitamin A and then into vitamin A palmitat in vitro in the futher studies.The main results were as follows:1. In order to obtain a high-yield strain of carotenoid, the protoplasts of Rhodotorula RM were prepared at first, and then was mutated by N+ ion mutagenesis and 60Coy-ray radiation. The results showed that the optimum conditions of the formation and regeneration of protoplast were as follows:the cell age at 14h, pretreatment with 0.05mol/L EDTA+0.5mol/L β-mercaptoethanol 30min,17% sucrose as osmotic stabilizer for dilution and medium of regeneration culture, catalyzed the reaction with 1.0% snailase 45min at 30 ℃. Then the protoplast was mutated with dose of 1.5×1014ion/cm2 by 10 keV N+ implantation and mutant RMA25 was obtained, which the production of carotenoid increased by 104.96% compared with Rhodotorula RM.And then mutant RMB91 was obtained by 1kGy 60Coy-ray radiation mutagenesis of RMA25. Compared with the original strain RMA25, the quantity of carotenoids of the mutant RMB91 come up to 1078.00μg/L, increased with 16.58%. after ten times of subculture, its hereditary property was stable, which suggested a bright prospect of application.2. To study the optimization of mutant RMB91 growth as well as the carotenoid production, The single-factor method and orthogonal test were employed.The results showed that the optimum seed medium were:2%glucose,2% peptone,1% yeast extract, with 30 mL pH6.0 fermentation liquid added into 250 mL Erlenmeyer flasks, agitation rate of 240r/min at 28℃,24h as optimum seed conditions. And the optimum fermentation medium were:175g/L molasses,30g/L corn steep liquor,20g/L NH4NO3,20mg/L A12(SO4)3·18H2O,5mg/LFeSO4·7H2O,40mg/L ZnSO4·7H2O,0.7g/L MgSO4·7H2O,0.2g/L K2HPO4, lmg/L VB2, with 50 mL pH6.0 fermentation liquid added into the 250 mL Erlenmeyer flasks,10.0% inoculum size with 180r/min agitation rate as optimum fermentation conditions. During the incubating, the temperature was varied, the first 48h at 30℃, then kept 24h at 25℃. And feeding 5g/L ethanol at 12h.10L fermenter fermentation tests showed that the biomass of the cell, the carotenoid content and carotenoid yield of the fermentaion were respectively up to 17.21 g/L,292.27μg/g and 5030.00μg/L, Increased by 5.32%,24.02% and 30.62% over than in Erlenmeyer flasks. Its optimum fermentation condition was 5L fermentation liquid,10.0% inoculum size,1.5vvm ventilatory capacity, pH6.0 (ammonia water as regulator), dissolved oxygen exceed to 20%, feeding 25g ethanol with 32mL/h at 24h, During the incubating, the temperature was varied, the first 48h at 30℃, then kept 24h at 25℃.3. In vitro, studies were conducted for conversing β-carotene, which was produced by Rhodotorula into retinol. Results showed that β-carotene 15,15’-monooxygenase,which come from chicken intestinal mucosa, can cleave β-carotene into retinal, and then be deoxidated to retinol. The optimum conditions for conversion were as follows:β-carotene 123 mg/L, pH 8.0 PBS O.lmol/L, sodium deoxycholate 3.5mmol/L,1.5 mmol/L oleic acid, 0.5mmol/L d-a-tocopherol,3.0g/L Tween 40, respectively. Under the optimum conditions, The enzyme reaction was conducted at 37℃ for 7h, its retinol content come up to 42mg/L, which showed a conversion efficiency of 63.96%(mol/mol), with 2.81 nmol mg-1 h-1 β-carotene 15,15’-monooxygenase activity.4. The conversion of retinol above-mentioned in non-aqueous phase was conducted, the results showed that vitamin A palmitate can be synthesised by retinol and palmic acid through Lipozyme. The optimum reaction conditions for conversion were n-hexane as solvent, Lipozyme as catalyst, the concentration ratio of substrate10:30(retinol:palmic acid),45℃ 6h with agitation rate of 100r/min, the conversion efficiency come up to 62.38%.