| With the development of the economy and the advancement of science and technology,people’s requirements for quality of life are increasing,and the global environment,especially the marine environment,is becoming more and more prominent.As a kind of dominant bacteria with strong resistance to stress in marine environment,marine red yeast is rich in nutrients and can metabolize various carotenoids including astaxanthin,and has various kinds of degradable petroleum compounds and purified water bodies.Physiological functions have become a research hotspot in recent years.Astaxanthin has broad application prospects in food,medicine,cosmetics and other industries because of its strong anti-oxidation,aging ability and coloring function.Carotenoids such as astaxanthin cannot be self-generated in animals.Microorganisms are currently a good source of carotenoids.Marine red yeast can metabolize and produce a large number of carotenoids,and has the characteristics of simple fermentation conditions and short growth cycle.Therefore,this study aims to optimize the HPLC elution conditions of Rhodotorula sp.The fermentation culture method,separation and purification of its metabolites and the biological activity of metabolites were studied.The first is the establishment of high performance liquid chromatography elution conditions.The metabolites of Rhodotorula sp.were analyzed by high performance liquid chromatography.The two systems of ammonium acetate methanol and formic acid acetonitrile were selected and selected.Through repeated debugging analysis of different time and mobile phase ratios,the optimal gradient separation conditions were finally established: 50 m M ammonium acetate and methanol system(0-15 min: 1 % methanol,99% 50 m M ammonium acetate;15-60 min: 60% methanol,40% 50 m M ammonium acetate),detection wavelength 265 nm,flow rate 1 m L/min,column temperature 35 ° C,Ampere C18 column(25 cm × 4.6 mm,5 um),injection volume 10 ul.The preparation method of the fermentation broth was studied.The high-performance liquid chromatography analysis of the original fermentation broth,the supernatant fermentation broth and the fermentation broth of the bacterium was finally carried out by adding an appropriate amount of methanol to the original fermentation broth,using rotation.The evaporator was concentrated to prepare a sample to be tested.Followed by the determination of the main metabolites of Rhodotorula sp.,and the optimization of its fermentation conditions.The astaxanthin standard solution was analyzed by high performance liquid chromatography,and it was preliminarily concluded that the main metabolite,the metabolite in the 19.4 min peak,was astaxanthin.Further,by mass spectrometry and nuclear magnetic resonance analysis,it was determined that the main metabolite of Rhodotorula sp.was astaxanthin with a yield of 18.6 mg/m L.The fermentation conditions of astaxanthin-producing were optimized.The single factor experiment showed that the optimal amount of acetylsalicylic acid for the fermentation of astaxanthin by Rhodotorula sp.was 100.00 mg/L.The addition amount is 100.00 mg/L,and the optimal fermentation time is 5 days.The response surface method was used to optimize the fermentation conditions of astaxanthin produced by Rhodotorula sp.,and the regression model of Rhodotorula sp.fermentation culture conditions was established.The regression equation was solved and the astaxanthin production reached the maximum value of 27.00.At mg/m L,the fermentation time was 6.64 days,the zinc sulfate addition was 113.50 mg/L,and the acetyl salicylic acid addition was 111.00 mg/L.In order to facilitate the actual operation,the fermentation conditions optimized by the model are: fermentation time is 6 days,zinc sulfate addition is 113.00 mg/L,and acetyl salicylic acid addition is 111.00 mg/L.Under these conditions,the astaxanthin production was 28.40 mg/m L,which was similar to the model prediction,which further verified the reliability of the model.The optimized astaxanthin production increased by 52.7%.The optimal conditions for the production of astaxanthin from Rhodotorula sp.are: activation medium: glucose(20 g/L),beef extract peptone(10 g/L),yeast extract(5 g/L),Sodium chloride(10 g/L),magnesium sulfate(3 g/L),dipotassium hydrogen phosphate(7 g/L),activated for 1 day.Fermentation medium: glucose(20 g/L),beef extract peptone(10 g/L),yeast extract(15 g/L),sodium chloride(10 g/L),magnesium sulfate(0.15 g/L)Dipotassium hydrogen phosphate(0.25 g / L),Tween(1 g / L),zinc sulfate(113 mg / L),acetyl salicylic acid(111 mg / L),fermentation for 6 days.Finally,the metabolites of Rhodotorula sp.were isolated and purified,and the biological activities of metabolites were studied.Five kinds of metabolites were isolated and purified by preparative high performance liquid chromatography.High performance liquid chromatography,mass spectrometry and nuclear magnetic resonance analysis were used to confirm that these five compounds are different configurations of astaxanthin.Astaxanthin,the main metabolite of marine red yeast Rhodotorula sp.,was evaluated for fibrinolytic activity,DPPH free radical scavenging ability,infrared spectrum analysis and ultraviolet wavelength absorbance analysis.Rhodotorula sp.metabolically produced and isolated and purified astaxanthin has no fibrinolytic activity;it has strong scavenging ability to DPPH free radicals,and its scavenging ability is slightly lower than vitamin C at low concentrations;high concentration When the scavenging ability is similar to that of vitamin C,astaxanthin has a strong antioxidant capacity;in the infrared spectrum,due to the C=O bond in the astaxanthin structure,the C=C bond in the aromatic,and the CC conjugate The stretching vibration of CH bond in the system formed three distinct absorption peaks;the absorbance of astaxanthin was scanned by ultraviolet spectrophotometer at 200-600 nm,and the most characteristic absorption of astaxanthin at 480 nm was obtained. |
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