| Ergothioneine(EGT),2-mercapto-histidine-trimethyl inner salt,is a rare natural amino acid and can be used as a good natural antioxidant with strong anti-oxidation and anti-inflammatory properties.It is effective and safe.It has been used as a good natural antioxidant in food,cosmetics,medicine and other fields.At present,there is no ergothioneine reference material in China.The price of ergothioneine reference substance is more expensive and the quality is uneven,which brings inconvenience to the detection of related laboratories.Therefore,the development of ergothioneine reference material is necessary.Some edible fungi are rich in ergothioneine.In this paper,the establishment of ergothioneine detection method and the selection of high-content ergothioneine edible fungi,the preparation of high-purity ergothioneine and the stability of high-purity ergothioneine in aqueous solution are studied in order to provide a reference material for the development of ergothioneine.The main content and research results are as follows:The determination method of ergothioneine in edible fungi was established by UPLC,and the contents of ergothioneine in 24 kinds of edible fungi from different producing areas were analysed.A high-content ergothioneine edible fungus was selected to provide a basis for the selection of raw materials for the preparation of ergothioneine standard materials.The determination was performed by BEH HILIC(2.1 mm×100 mm×1.7 μm)with mobile phase A:acetonitrile 0.1%formic acid,and mobile phase B:water with 0.1%formic acid.The flow rate was 0.4 mL/min,detection wavelength was 262 nm,and sample volume was 3 μL.The concentration of ergothioneine showed a good linear relationship in the range of 1 to 50 mg/L.The regression equation was Y=32529X+6006.6,R2=0.9999.The method had good repeatability and reproducibility in the range of 80.0%～120.0%.It was a reliable method for the determination of ergothioneine in edible fungi.Using this method,the ergothioneine content in different edible fungi varieties was determined and compared with the reported literature.The results showed that the experimental determination of ergothioneine content in edible fungi(Boletus,Shiitake)was more consistent with reported literature data;There is a big difference between the dry samples and fresh samples of the same type of edible fungi;the higher content of ergothioneine in the dry samples of edible fungi fruiting bodies is Boletus edulis(1.020%)>Pleurotus ostreatus(0.285%)>Morel(0.110%).The content of ergothionine in edible mycelium is higher.The content of ergothioneine in edible mycelium is higher.The high ones are Shiitake(0.345%)>Pleurotus ostreattus(0.083%)>Cordyceps militaris(0.079%).The scraps of Pleurotus ostreatus(0.530%)are also rich in ergothioneine.Taking comprehensive considerations into consideration,the mycelium of mushrooms,Cordyceps militaris,Boletus edulis,Pleurotus ostreatus,Morel,and Pleurotus ostreatus mycelium and leftovers are used as raw materials for preparing ergothioneine standard substances,which can not only reduce the cost of raw materials.Taking comprehensive considerations into consideration,the mycelium of mushrooms,Cordyceps militaris,Boletus edulis,Pleurotus ostreatus,Morel,and Pleurotus ostreatus mycelium and leftovers are used as raw materials for preparing ergothioneine standard substances,which can not only reduce the cost of raw materials.Using commercially available crude ergothioneine with a nominal purity of 98% as raw material,qualitative analysis of the impurities was carried out by UPLC-Q-TOF-MS,and the three impurities in the crude ergothioneine,using the response signal values of the main components and impurities as indicators to optimize the analytical liquid purity determination conditions to quantitatively analyze the three impurities,and use the semi-prepared solution to purify the crude product to prepare pure ergothioneine.Using ergothioneine purity,yield,peak time,single preparation time,and single preparation mobile phase consumption as indicators,the semi-preparative high performance liquid chromatography conditions were optimized.The optimized analytical liquid purity determination conditions are:BEH HILIC(2.1 mm×100 mm×1.7 μm)column,wavelength 292 nm,concentration 1000 mg/L,mobile phase A:acetonitrile,B:0.1%formic acid water;gradient elution program:0～2 min,5%B,2～15 min,5%～60%B,15～17 min,60%B,17～17.1,60%～5%B,17.1～20 min,5%B.The optimized semi-preparative high performance liquid chromatography separation and preparation conditions for ergothioneine are:Xbridge Prep HILIC column(19 mm×250 mm×5 μm)column,75%acetonitrile-water solution flowing isocratic elution,flow rate 10 mL/min,The injection volume is 3.5 mL,the detection wavelength is 262 nm,and the needle washing program is set between each needle sample.The elution method is:gradient elution,phase A is water,phase B is acetonitrile,0～1 min,25%～60%A,1～11 min,60%A,11～11.1 min,60%～25%A,11.1～16 min,25%A,running time 16 min,under this preparation conditions,the purity of ergothioneine increased from 97.63%to 99.10%.Using the 99.10%purity of ergothioneine as raw material,we studied the influence of temperature,pH,reducing agent DTT concentration,ergothioneine concentration,and light external physical and chemical factors on the stability of ergothioneine aqueous solution,and explored the state standard substance feasibility of preparation.The results showed that the influence of five single factors on the stability of ergothioneine aqueous solution was light,ergothioneine concentration,pH,concentration of reducing agent,and temperature in order.(1)Light has a significant effect on the stability of ergothioneine aqueous solution.When the ergothioneine aqueous solution is placed under ultraviolet light,visible light,full light,and dark conditions for 14 hours,The content of ergothioneine decreased by 20.4%,16.3%,40.4%,and 3.94%respectively compared with the initial value.The stability of ergothioneine under the four light sources is in order of protection from light,visible,ultraviolet,and full light,indicating that storage in the absence of light is beneficial to the stability of the aqueous solution of ergothioneine.(2)At three concentrations of 100 mg/L,500 mg/L,and 1000 mg/L,the ergothioneine aqueous solution is more stable at low concentrations.The storage time of 100 mg/L,500 mg/L,and 1000 mg/L ergothioneine aqueous solutions was 8 days,and the minimum storage rates of ergothioneine were 87.12%,85.4%,and 63.9%,respectively.(3)The pH has an effect on the stability of the ergothioneine aqueous solution.Within 8 days,the stability of the high-purity ergothioneine solution in the two solutions with pH 6 and 7 is better than that of the solution with pH 8.(4)When the storage time is≤3 days,the reducing agent DTT can stabilize the thioneine solution.When stored,the ratio of C(DTT):C(EGT)is 0.5:1,1:1,2:1.Below,the DTT ratio has a similar effect on the stability of ergothioneine.(5)Under the conditions of-20℃,4℃and 20℃,temperature has no significant effect on the stability of the ergothioneine aqueous solution,which is unstable when stored at-20℃,4℃and 20℃for 8 days.Comprehensive consideration,ergothione should not be made into a solution standard substance. |
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