Study On The Biotransformation Of Valencene Into Nootkatone

Nootkatone is a sesquiterpene compound,which belongs to bicyclic sesquiterpene ketone of Yashilane series.Nootkatone exhibits strong grapefruit aroma at low concentrations.It also has sweet orange and woody fragrance,but it tastes bitter.It has a low odor threshold of only 1 ug/L.Nootkatone has attracted much attention due to its pleasant citrus aroma.Since its discovery,the natural distribution,crystal structure and aroma characteristics of various stereoisomers of naringin have been extensively studied.Because of its low content in natural products and stereoisomers,it can hardly be separated.Its extraction cost is very high,so the extraction and application of citrone are greatly limited.At present,there are few studies on the biotransformation of valencene to produce nootkatone at home and abroad.The purpose of this study was to screen the suitable strains for the transformation of valencene to produce nootkatone by comparing the effects of three different microbial strains.The production cost of natural flavor citrone was reduced by increasing the conversion of valencene to nootkatone and increasing its conversion rate.Then the product of nootkatone was isolated and purified,and the enzymes in the transformation process were preliminarily explored.1.The purpose of this study was to investigate the effect of Aspergillus nigerus,Mucor sp.and Yarrowia lipolytica on the conversion of valeneene to nootkatone using SPME-GC-MS.The results showed that all these three strains could transform valencene to nootkatone.Other products,such asα–terpinol and D-carvone,were also found during the biotransformation.The most amount of nootkatone was found by Yarrowia lipolytica with a yield of 252.52±15.79 mg/L and conversion rate of 27.45±1.72%.Followed by Aspergillus niger and Mucor sp.And the amounts of these two fungi were 140.94±30.26mg/L and 137.67±14.02 mg/L,and the conversion rate were 15.32±3.29%and15.45±3.36%,respectively.2.The orthogonal test was used to optimize the optimum composition of fermentation medium of Yarrowia lipolytica.The optimum combination of the culture medium was（g/L）:lactose 40,peptone 15,FeSO₄·7H₂O 0.6.After optimizing the culture medium,the yield of nootkatone reached 457.32±76.11 mg/L,and the conversion rate was 49%which increased by 21.55%.On the basis of single factor process optimization experiment,the optimum combination of transformation conditions was determined.The time of adding substrates was 36 hours when bacteria were transformed.The concentration of added bacteria was 5%,the concentration of added substrates was 920mg/L,and the transformation time was 36 h after adding substrates.Dimethyl sulfoxide（DMSO）,a water-soluble organic solvent,was used as a cosolvent with a concentration of10%.The temperature of conversion condition is 32℃,the speed is 150 rpm,and the pH is 5.30.The conversion of the product was 531.16±21.30 mg/L and the conversion was57.73±2.31%.Compared with the optimization of culture medium,the conversion rate of nootkatone was 49%,and the conversion efficiency was increased by 8.73%.The conversion was 29.92%higher than the initial conversion.3.High-speed counter-current chromatography（HSCCC）was used to isolate and purify nootkatone from valencene transformed by Yarrowia lipolytica.By using high-speed countercurrent chromatography to separate and purify the components of naringone in samples,the suitable two-phase solvent system was selected and the better solvent separation conditions were determined.High-speed counter-current chromatographic experiments were carried out by screening three non-aqueous two-phase solvent systems:n-hexane-chloroform-acetonitrile,n-hexane-dichloromethane-acetic ether-acetonitrile,n-hexane-dichloromethane-acetonitrile and water-soluble two-phase solvent system of n-hexane-ethyl acetate-methanol-water.The effects of various solvent systems on the separation were analyzed.Considering the purity and elution time of pomelone,n-hexane-methanol-water（5:4:1）was selected as the stationary phase and the lower phase was the mobile phase for elution.The elution time of pomelone was 280-340minutes,and the purity of product analyzed by GC-MS was 53.74%.The final structure was identified by mass spectrometry and infrared spectroscopy.It was identified as nootkatone.4.The main purpose is to purify the protein of Yarrowia lipolytica.in this part.Six kinds of inhibitors studied in this experiment can inhibit the biotransformation and decrease the concentration of nootkatone.Among them,1,10-phenanthroline has the strongest inhibitory effect on the transformation of valencene to nootkatone,with the inhibition rate of 12.28%.The results showed that most of the active protein components of the enzymes transforming valencene to nootkatone were in the supernatant of the crushing solution.Nine different gradients of ammonium sulfate（（NH₄）₂SO₄）were used to precipitate Yarrowia lipolytica fragments.The optimum precipitation concentration was determined to be 40%.