Study On Ergosterol And Vitamin D₂ In Lentinus Edodes Fruit Body And Saccharomyces Cerevisiae

The content of ergosterol and vitamin D₂ in lentinus edodes fruit body had studied,the extraction method of ergosterol was optimized,the method for simultaneous determination of ergosterol and vitamin D₂ was established in lentinus edodes fruit body,the effects of ergosterol and vitamin D₂ in saccharomyces cerevisiae cells was explored,the stability of ergosterol and vitamin D₂ in saccharomyces cerevisiae cells was studied.It provided theoretical support and technical reference for studying ergosterol and vitamin D₂ in lentinus edodes fruit body and saccharomyces cerevisiae.The results of the study are as follows:Different alcohols,alkaloids and extractants in the extraction process of ergosterol from letinous edodes fruit body were compared based on the extraction rate of ergosterol by ultrasonic extraction,isopropanol,KOH as saponification liquid and petroleum as extractant were selected for this investtigation.The effects of liquid to solid ratio,alcohol alkali solution volume ratio,alkali concentration and ultrasonic time on extraction rate of ergosterol were discussed through single factor test and response surface analysis.The extraction conditions were optimized as follows:54.64mL/g of the ratio of liquid to material,4.83 of the volume ratio of alcohol and alkali solution,the concentration of KOH as 4.37mol/L,22.14min of the ultrasonic time.Under this condition,the extraction rate of ergosterol in lentinus edodes had reached 3.372 mg/g.The method for simultaneous determination of ergosterol and vitamin D₂ in lentinus edodes fruit body by high performance liquid chromatography was established.The detection wavelength,flow phase,flow phase velocity and column temperature were explored and optimized.It was finally determined that the detection conditions were C₁₈?Agilent ZORBAR SB,4.6mm×250mm,5?m?color column;the mobile phase:methanol;the detection wavelength:270nm;the flow velocity:1.5mL/min;the column temperature:30?;the sampling size:10?L.Established liquid chromatography coupled with tandem mass spectrometry?LC-MS/MS?method for simultaneous determination of ergosterol and vitamin D₂content in Lentinus edodes.The sample solution was prepared by ultrasonic extraction combined with saponification,ergosterol and vitamin D₂ were separated by using C₈?Agilent ZORBAR SB,15 mm×2.1 mm,3.5?m?as chromatographic column and purified at 25? of column temperature,270 nm of detection wavelength and the flow rate of methanol aqueous solution at 1.5mL/min in the gradient elution methods.The purified samples were tested by mass spectrometry with electrospray ionization positive ion?ESI⁺?mode and multi reaction?MRM?monitoring mode,and ion source injection voltage?IS?at 5500 V,ionic source temperature?TEM? at 250? and ion source spray velocity at 5 L/min were applied as mass spectrometer conditions when the set pressure of air curtain gas?CUR?,atomization gas?GS₁?and auxiliary gas?GS₂?were controlled at 20 psi,45 psi,60 psi,respectively.Results showed that the retention time of ergosterol and vitamin D₂ chromatographic peaks were at 4.49 and4.26 min,respectively,the content of ergosterol?0.40?3.60 mg/L?and vitamin D₂?0.22?2.20 mg/L?had a good linear relationship with the peak area?R²=0.9995?within a certain content range.The average recovery rate of ergosterol and vitamin D₂ were within 86.7% to 102.4%,and the relative standard deviation were within 1.02% to 4.13%,respectively according to methodological validation.The collect samples were detected by LC-MS/MS established in this experiment and HPLC recommended by the national standard.The detection results suggested that no significant difference?p>0.05?was found by two methods utilized for samples analysis,however detecting time for sample by LC-MS/MS was shortened by half hour compared with HPLC approach and indicated the established LC-MS/MS method build in this study was quickly,accurately and sensitively for simultaneous determination of ergosterol and vitamin D₂ content in Lentinus edodes,It will provides technical support for the quality detection of edible mushrooms related products and lay the foundation for simultaneous determination of other isomers in the futures.The saccharomyces cerevisiae strain GIM 2.198 with high vitamin D₂ content was selected as the research object from four strains of saccharomyces cerevisiae.The culture medium for improving the bacterial concentration,the content of ergosterol and vitamin D₂ was optimized On the basis of the basic culture medium,and the similarities and differences between the three kinds of optimized medium were compared,it was found that the culture medium formula for improving the content of ergosterol and vitamin D₂ was the same.Under this culture medium,the bacterial concentration was 12.4073 mg/mL,the ergosterol content was 11.0249 mg/g,the vitamin D₂ content was 0.4497 mg/g.It showed that ergosterol production is related to the production of vitamin D₂.The content of vitamin D₂ under white,ultraviolet,red,red-blue light and sunlight irradiated by GIM2.198 saccharomyces cerevisiae was compared.It was found that five kinds of lights could increase the content of vitamin D₂ in saccharomyces cerevisiae,among which ultraviolet light was most significant to increase the content of vitamin D₂,so ultraviolet light was chosen as light source to treat zymotic fluid to improve vitamin D₂ content in GIM 2.198 saccharomyces cerevisiae.Effects of ultraviolet irradiation time,irradiation height and culture time on bacteria,ergosterol and vitamin D₂ content in saccharomyces cerevisiae were compared by the single factor analysis.On the basis of the single factor analysis,the optimal portfolio of ultraviolet irradiation time,irradiation height and culture time was optimized by orthogonal analysis.It was found that the order of the effects of three treatment conditions on vitamin D₂ content was culture time>ultraviolet irradiation time>irradiation height.The best treatment method was 1.0 h of the ultraviolet irradiation time,20 cm of tne irradiation height,and 72 h of the culture time.Under the conditions,the content of the bacterial concentration was 5.0239mg/mL,the content of ergosterol was 10.6733 mg/g and vitamin D₂ was 1.2867 mg/g.The effects of the standard solution of ergosterol in different concentrations on the reduction of ergosterol and vitamin D₂ in saccharomyces cerevisiae was explored.It was found that soaking in 300mg/L standard solution of ergosterol was the best way to reduce the attenuation of the content of ergosterol and vitamin D₂.Under this concentration,ergosterol can be restored to 89% of the content before being soaked,and vitamin D₂ can be restored to 80% of the content before being soaked.The difference of the content of ergosterol and vitamin D₂ was compared between saccharomyces cerevisiae and lentinus edodes.It was found that the content of ergosterol and vitamin D₂ was significantly higher than that of lentinus edodes in saccharomyces cerevisiae GIM2.198.The difference of ergosterol content was not significant before and after ultraviolet irradiation?p>0.05?.The difference of vitamin D₂ content was significant before ultraviolet irradiation?0.05>p>0.01?,and the difference of vitamin D₂ content was very significant after UV irradiation?p<0.01?.