Studies On Production Of Carotenoids By Rhodotorula Glutinis And Its Functional Properties

According to the theory of yeast fermentation and the characteristic of natural pigment, the dissertation focuses on the conditions of producing pigments with fermentation and the pigments isolation, determination, its functional properties. The main research items and results as follows:(1) Rhodotorula glutinis as a starting strain was mutated with UV and diphenylamine as a screening reagent. One carotenoid high producing strain Ruv701 was screened through UV induction of mutation. Genetic character of mutated strain was stable by fermentation test. Flask-shaking fermentation could produce carotenoid 13.25 mg/L, which was 42.16% higher than starting strain. It could produce carotenoid 37.21 mg/L in 5 L fermentation tank, which was 62.56% higher than un-mutation strain.(2) The technology for extracting pigments produced by Rhodotorula glutinis: yeast cell broken by grinding at 35℃, using petroleum ether - acetone as solvent and extracting 30min were appropriate.The reaction of dark blue between concentrated sulfuric acid and chloroform carotenoids could be qualitative analysis for the Rhodotorula glutinis pigment.HPLC could be quantitative analysis for Rhodotorula glutinis pigment. Chromatographic column RP-ODS-C₁₈, temperature of the column 35℃, mobile phase acetonitrile-tetrahydrofuram(60:40,V/V), flow speed 1mL/min, quantity of injecting 10uL.TLC , chromatographic column, spectrophotometric method, HPLC and mass spectrum could separate or analyse Rhodotorula glutinis pigments. There was β -carotene in Rhodotorula glutinis pigments, other components may be torulene, torularhoin, which were illustrated by mass spectrum or spectral analysis.The technology of supercritical CO₂ extracting Rhodotorula glutinis pigments: extracting press 18-19Mpa, extracting temperature 44.5-45.0℃, flow rate of CO₂ 4.8-10.5ml/min, extracting time 10min.(3) Through single factor experiment, the optimal carbon resource was sucrose and optimal nitrogen resource was yeast extraction powder for Rhodotorula glutinis producing carotenoid.Through uniform design, the optimal adding quantity of metal ion was K₂SO₄ 1.0%,MgSO₄ · 7H₂O 0.05%,CuSO₄ · 5H₂O 0.0045%.Through response surface test, the effect of vitamins on carotenoid fermentation wasY=12.3+0.0675X₁+0.06X₂+0.0725X₃-0.1975X₁²+0.1275X₂²-0.045X₂^*X₁-0.09X₃^*Xi+0.04X3*X2-0.1935X32. From this equality, the optimal adding quantity were VB, 0.2mg/U VB2 l.Omg/L, VC 0.7mg/L.(4) Through research of fermentation condition on high producing mutated strain Rhodotorula glutinis producing carotenoid, optimal fermentation condition on 5 L fermentation tank were initial pH value of cultural medium was 6.0, fermentation temperature 28 °C, fermentation time 76h. initial sugar concentration 20g/l, adding sugar concentration to lOOg/1, keeping residual sugar concentration 10-20g/l, dissolved oxygen 15-20%, yeast powder 30g/l. Under the fermentation condition, it could be reached at cell biomass 43.83g/l, carotenoid content 842.82 u g/g, and transformation rate of sugar was 0.372mg/g.(5) With MATLAB software, the process of fermentation was analysed and strain growing kinetic model, products forming model were built in. The fitting models could be suitable for the practical batch fermentation of carotenoid.(6) The research on property of Rhodotorula glutinis pigments showed that temperature had big effect on stability;the pigment was not stable under light, specially sunlight, also including in UV and in room light, it was stable without light, which was the notable factor (p<0.05);it was not stable in acid;the consentration for peroxide hydrogen was the notable factor (p<0.05);but antioxidatant had protection on the pigment.(7) Chemiluminescence and chemical method all showed that Rhodotorula glutinis pigments had higher antioxidant, and as increasing of concentration as improving. As the concentration was 5.57mg/mL, the antioxidant activities of the pigment to lecithin liposome system was equally 27.47% as a -tocopherol;the pigment had higher inhibiting ability on oxidation of linoleic acid liposome, at 33.33mg/mL of extraction was 4.76mg/mL as a -tocopherol in antioxidant. The pigments had very high ability of inhibiting hydroxyl radical, higher than to superoxide anion.