| Perennial herb valeriana officinalis L.belonging to the family of Valerianace and genus of Valerian L.was a traditional Chinese medicine with its roots as a medicine.Valeriana officinalis oil extracted from valeriana officinalis L.has good pharmacological activities:used for treating about coke,insomnia,depression,etc;having certain protective effect on the central nervous system,cardiovascular system,liver,bravery,and kidney;as well as having strongly effects of anti tumor and anti dementia.With the abundant valeriana resources and its high economic value,the study of valeriana resources development and utilization has great significance.The article using the root of valeriana officinalis L.from guizhou as a raw material to study valeriana officinalis oil extraction technology and its chemical composition,to separate the endophytic fungi which can produce the valtrate and to optimize its shaking flask fermentation conditions,aimed to provide the reference for development and utilization of valeriana resources.The main results were as follows:1.Valeriana officinalis oil extraction technology researchBy using steam distillation,supercritical CO2 fluid extraction and subcritical fluid extraction to extract valeriana officinalis oil respectively,this chapter aims to optimize the extracting conditions as index of valeriana officinalis oil yield.(1)Steam distillation to extract valeriana officinalis oilThe solid-liquid ratio,the extraction time and the material granularity of steam distillation were optimized by orthogonal design to determine the best conditions:when the solid-liquid ratio was 10 mL/g,the material granularity was 50 mu,extract time was 2.5 h,the valeriana officinalis oil yield was(0.835±0.002)%.(2)Supercritical CO2 fluid extraction to extract valeriana officinalis oilOn the basis of single factor experiment,the Box-Benhnken experiment design was used to optimize extraction pressure,extraction temperature,extraction time and CO2 flow rate of supercritical CO2 fluid extraction.By doing quadratic regression analysis for the experiment result with SAS8.1 statistical software,a better fitting regression model was established,its correlation coefficient R2 = 0.9376.The best conditions were determined through the model:when extraction pressure was 21 MPa,CO2 flow was 20 L/h,extraction temperature was 45 ?,extraction time was 90 min,the valeriana officinalis oil yield was(4.250±0.003)%.(3)The subcritical fluid extraction to extract valeriana officinalis oilThe single factor experiments and uniform design experiment were adopted to optimize extraction pressure,extraction temperature,material liquid ratio,extraction time of subcritical fluid extraction.By doing quadratic polynomial stepwise regression analysis for the experiment result with DPS7.05 statistical software,a significantly regression equation was established,its correlation coefficient R=0.9998,the value F=533.9032,P-value=0.00 1 9,the residual standard deviation S=0.0080,Durbin-Watson statistics d=2.2500.The best conditions were determined through the equation:when extraction pressure was 0.47 MPa,solid-liquid ratio was 11.0 mL/g,extraction temperature was 47 ?,extraction time was 25 min,the valeriana officinalis oil yield was(3.383±0.004)%.2.Valeriana officinalis oil chemical composition analysisChemical composition analysis of valeriana officinalis oil extracted by the above three methods through GC-MS technology showed that the alkene,the ketone,the esters,the alcohols,the acids were mainly compounds in valeriana officinalis oil,and bornyl acetate was the highest relative content of components in valeriana officinalis oil of three methods to extract.52 components,relative content in more than 0.1%,were identified from valeriana officinalis oil extracted by steam distillation,and the relative contents of its peak areas were 95.21%in total valeriana officinalis oil,including bornyl acetate(29.75%).79 components,relative content in more than 0.1%,were identified from valeriana officinalis oil extracted by supercritical CO2 fluid extraction,and the relative contents of its peak areas were 95.13%in total valeriana officinalis oil,including bornyl acetate(28.53%).57 components,relative content in more than 0.1%,were identified from valeriana officinalis oil extracted by subcritical fluid extraction,and the relative contents of its peak areas were 96.41%in total valeriana officinalis oil,including bornyl acetate(29.63%).3.The separation and identification of endophytes for producing the valtrate12 strains of endophytic fungi were separated and purified from the roots of valerian officinalis L.by the streak plate method.Using the HPLC to test the fermentation broths of 12 strains of endophytic fungi,it found that XCS-6 strains can produce valtrate.Morphological features show that XCS-6 strains colonies were pale,radiating growth faster hyphae,28? culture after 4 day colonies diameter(5.7±0.1)cm on PDA plate.The microstructure of the XCS-6 strains by using insert method to observe showed that hyphae have septa,conidiophores and spores colorless,unit cell,ovoid,smooth surface,and the dendritic structure by single conidiophores from hyphae.There was 100%homology between the ITS sequence of XCS-6 strains and the GeneBank HQ270170.1 by combining with molecular biology identification,so the XCS-6 strains belonged to Basidiomycota,Agaricomycetes,Cantharellales,Ceratobasidiaceae,Ceratobasidium(Ceratobasidium sp.AG-A).4.The fermentation conditions of endophytes for producing the valtrateOn the basis of single factor experiment,valtrate production as index,the Box-Benhnken experiment design was used to optimize shaking flask fermentation time,fermentation temperature and shaking speed of XCS-6 strains.By doing quadratic regression analysis for the experiment result with SAS8.1 statistical software,a better fitting regression model was established,its correlation coefficient R2=0.9929.The best conditions were determined through the model:when fermentation time was 7.5 day,fermentation temperature was 27?,haking speed was 170 r/min,the valtrate production was(2.265±0.003)?g/mL. |
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