Converted Ergosterol Into VD₂ In Vivo Of Pleurotus Ostreatus Under Light Conditions And Risk Assessment Of Heavy Metals

In this research,Pleurotus ostreatus was the object,the contents of ergosterol and VD₂ were used as indicators and the high performance liquid chromatography（HPLC）method was established for evaluating the quality of Pleurotus ostreatus.And then methodology of path of steepest ascend and Plackett-Burman design（PBD）were choosed for optimizing the cultivation of oyster mushroom.For the purpose of obtaining perfect cultivation method and high yield ergosterol strains and optimal media formulation,the effects of conditions of ergosterol transforming to VD₂ were studied,including different irradiation time,different parts,different species and different concentrations of ergosterol as immersion solution.At the same time,the oyster mushroom samples were collected from laboratory samples and markets in Guangzhou city.The contents of heavy metals（As,Cd,Pb）in oyster mushroom treated with microwave were determined by inductively coupled plasma-mass spectrometry（ICP-MS）.Target Hazard Quotients recommended by US EPA（Environmental Protection Agency）was used for risk evaluation.To lay the theoretical foundation for the study of ergosterol and VD₂ in Pleurotus ostreatus and provide the scientific basis for the development of natural Pleurotus ostreatus and its products,the research was established.The results were shown as below:1.High Performance Liquid Chromatography（HPLC）was established to determine the content of ergosterol and VD₂ in oyster mushroom.The C₁₈ Chromatographic（4.6 ID×250 mm,5μm）column was used for the simultaneous determination,the injection volume was 10μL,100%methanol was used for mobile phase,the flow rate was 1.0 mL/min,the column temperature was at 30℃,and detection wavelength was270 nm.The linear ranges of ergosterol and VD₂ were 101.6⁵08.6μg/mL（R2=0.999）and10.5⁵2.5μg/mL（R2=0.999）,respectively.The average recoveries rate of ergosterol in the sample was 99.06%,with an RSD of 2.93%,and the average recoveries of VD₂ was 99.77%and the RSD was 5.54%.A simple,accurate and sensitive high performance liquid chromatography method was developed to determine the content of ergosterol and vitamin D₂ in Pleurotus ostreatus.2.Selection of Pleurotus ostreatus Strains and optimization of Culture Medium for High Yield Ergosterol.The result indicated that P831 was the optimum strain among five strains,and the ergosterol content was 3.25 mg/g,which was 1.0³.0 times higher than that in other strains.The composition of oyster mushroom culture medium was optimized by response surface analysis based on Design Expert 8 software.Finally,the best medium formula was:peptone 0.35%,sucrose 3.5%,potassium dihydrogen phosphate 0.35%,and the yield of ergosterol produced by mycelium increased 12%,from 3.25 mg/g to 3.64 mg/g.It was found that the ergosterol content in fruiting bodies increased after adding urea and sucrose to the solid cultivation material.In addition,the number of lock structure in mycelial bodies increased and their size were uniform when they were treated with ultraviolet radiation and observed by Field Emission Electron Microscope（FEM）.After 20 min UV irradiation,VD₂ were detected in cap and stip.It turned out that ergosterol and VD₂ content were higher than that of the stipe.Pleurotus ostreatus’gills were covered by a large number of spores,while there were no spores on the stipe.And the metabolism and cell division ability were strong in lock structure and spores.The results showed that ergosterol content was related to the histomorphological structure and metabolic capacity of the cells.3.Optimization of cultivation media for high yield ergosterol and conversion of ergosterol to vitamin D₂ in vivoTo obtain the best medium formula of high yield ergosterol,the Pleurotus ostreatus which named P10 was used as research object and orthogonal test design was based on single factor experiment.The effects of UV irradiation on ergosterol transformation were studied,including different irradiation time,different parts,different species and different concentrations of ergosterol in immersion solution.Then the best medium formula was obtained.Its C/N ratio was at 40,the carbon source and nitrogen source were sucrose and urea respectively,and the additive was 6-BA with 0.15 mg/100mL.After optimization,the yield of ergosterol was up to 4.725 mg/g.Adding glucose in solid cultivation not only improved the growth of fruiting body,but also promoted the ergosterol content.The optimal carbon-nitrogen ratio for ergosterol synthesis is 30.Vitamin D₂ content in Pleurotus ostreatus significantly increased when UV irradiation time dose increased.Compared with 10、20、30、60、90 min UV irradiation time,90 min UV irradiation showed the best result.The result was that VD₂ content reached to 54.23±1.12μg/g.Vitamin D₂ content in the whole mushroom,such as caps and stems,all increased when UV irradiation time increased.Furthermore,the VD₂ content in the mushroom cap was the first place,the whole mushroom was the second,and the stems was the lowest.Analysis of Vitamin D₂content in different species of Pleurotus ostreatus was different.Vitamin D₂ content in different species mushroom all increased when UV irradiation time dose increased,but the rate of gaining was discrepant among different species.In different concentrations of ergosterol as immersion solution,it showed that the ergosterol content of Pleurotus ostreatus increased when it was immersed in increased dose of ergosterol solution,but the content of VD₂ did not increase significantly.When Pleurotus ostreatus was immersed in 200μg/ml concentrations of ergosterol solution,total content of VD₂ reached to 63.65±6.46μg/g.4.Determination and health risk evaluation of heavy metals in oyster mushroomsThe content of Pb,As,Cd in Pleurotus ostreatus was determined by optimized microwave digestion combined with inductive coupling and mass spectrometry（ICP-MS）.The health risk assessment of three heavy metals such as Pb,As and Cd was conducted by calculating the target hazard quotient（THQ,TTHQ）.The results showed that the best method for microwave digestion Pleurotus ostreatus samples was as follows:0.5 g mushrooms,4 mL nitric acid and 4 mL hydrogen peroxide.The standard-exceeded of Pb,As and Cd in the samples of Pleurotus ostreatus were 16.66%,5.55%and 5.55%,respectively.From the health risk to heavy metals,adults and children not exposed to the surveyed Pleurotus ostreatus have no obvious health risks.Pb,among three kinds of heavy metals had the greatest harm to the residents’health,so it should be paid more attention to.A variety of heavy metal complex risk calculations showed that,the overall TTHQ for heavy metals in adults and children was 0.818 and 1.5458.It was indicated that there was a significant and potential health risk for people who intake Pleurotus ostreatus in the market,especially for children.