Study On The Preparation Of L-?-glycerophosphocholine From Egg Yolk Powder

L-?-glycerophosphocholine(L-?-GPC,GPC)is a natural choline compound,which attracts in different industries includes food and pharmaceutical.The egg yolk phosphatidyl choline(PC)is a cheap raw material for the production of the L-?-GPC.However,the existing techniques to production and purification is inadequate to get high-quality L-?-GPC.Therefore,the development of high value-added L-a-GPC production from readily available raw material is of great signi:ficance to large-scale production.In this study we aimed to produce high-quality of the raw material for L-a-GPC from egg yolk powder using hydrolysis and evaporative light scattering detection(ELSD)based high-performance liquid chromatography(HPLC)techniques.The initial separation was carried out by natural settlement instead ofsolvent extraction,and the process parameters were optimized.At first,GPC is identified by using HPLC-ELSD technique.The specific chromatographic conditions include are:alltima silica column(4.6 mm×250 mm,5?m);the mobile phase is methanol:water:acetic acid:triethylamine-(90:10:0.18:0.02,v:v:v:v),and the flow rate is 1.5 mL/min isocratic elution;column temperature is 45?;gain of 5;carrier gas pressure is 40 Psi;the mode of sprayer is heating,power level is 50%;the temperature drift of tube is 65?.The retention time of L-a-GPC is about 6.6 min,which is below the previous method.Then,the methodological verification is performed on the optimal detection conditions.The results showed that the repeat ability,stability,and precision are good.The detection limit of L-a-GPC is 0.05 mg/mL and the limit of quantification is 0.08 mg/mL.Therefore,it can be used as a rapid and scientific method to the testing of L-a-GPCThen,the egg yolk powder is washed with acetone to remove oil and fat and to obtain an acetone-insoluble matter.Then,PC in acetone insoluble matter is further extracted with ethanol.The results of single factor and orthogonal test showed that in the acetone extraction stage,the temperature is 20?,the solid to liquid ratio is 1:7,and extracted 96.9%of PC in 70 min.In addition,98.8%of PC extracted using ethanol extraction stage with in the optimum temperature of extraction is 40? and,in the ratio of solid to liquid is 1:8 in 50 min.The L-a-GPC is prepared by catalytic hydrolysis from the PC extracted in last step.After the catalytic hydrolysis using sodium methoxide,sodium ethoxide and phospholipase as catalysts is compared.As a result,it is found that the enzymatic catalysis reaction in the aqueous system yields 76.3%of L-a-GPC,with poor reproducibility and long reaction time.When the sodium methoxide is the catalyst,the yield of L-a-GPC can reach 88.1%after 2h of reaction;whlie using sodium ethoxide as catalyst,yields about 60%of L-a-GPC after 2h.Moreover,the catalytic effect of methanol as a solvent is better than that of ethanol.In summary,the methanol-sodium methoxide system has a short reaction time and high yield of L-?-GPC.This optimized conditions will be followed in further experiments.Through the single factor and response surface optimization experiments,the model equation is established,R-Squared=0.9956,Adj R-Squared=0.9906,indicating that the equation has a higher degree of fitness.The contribution of each factor can be obtained by F test:reaction temperature>solid-liquid ratio>sodium methoxide addition amount>reaction time.The optimal reaction conditions are as follows:time 97.82 min,temperature 36.67?,catalyst dosage 4.99%,solid to liquid ratio 1:7.58.Under these conditions,the average L-a-GPC yield is 97.82%,which is in good agreement with the predicted value of 99.31%.Model equations can be used as a prediction of theoretical values.Finally,L-?-GPC production liquid is purified to prepare high-purity GPC.After the rotary evaporation of reaction solution,it is naturally divided into two distinct layers after subsiding at room temperature for 1 h,and the upper layer is discarded.Through silica gel column(3cm × 30cm),some fatty acids and other phospholipid components are removed.The optimal conditions are selected as follows:wet installing,eluent:mixed solvent of methanol:water 9:1(v:v),flow rate is 2 ml/min,mass ratio of sample to silica gel is 1:20.Removing Na+ and other cations and adjust the PH through the cation and anion exchange resin with the mass ratio 1:2.The ratio of 001 X 7 resin to eluent is 1:25(m:v),and Na+ are removed completely at 70min.Finally obtain a colorless L-?-GPC with a purity of 99.80%by decolorizing with the activated carbon and a recovery rate of 86.5%.The final purified L-a-GPC is characterized using HPLC-ELSD,infrared spectroscopy(JR),nuclear magnetic resonance(NMR)and mass spectrum(MS).The results are in good agreement with the standards.