Hydrolysis And Interesterification Of Soy Lecithin Catalyzed By Phospholipase A1

This article studied the quantitativeanalysis methods of phosphatidylcoline(PC), lyso-phospholipids(LPC), and glycerophosphorylcholine(GPC) by Proton Nuclear Magnetic Resonance(1H-NMR)and High Performance Liquid Chromatography equipped with Evaporative Light Scattering Detector(HPLC-ELSD). Using PC(35%) as the starting material and phospholipase A1 as the catalyst, LPC was prepared in aqueous reaction system, and GPC and structural lecithin rich in linolenic acid were produced in water-in-oil emulsion system. The detailed research is as follows:(1) 1H-NMR was employed to analyzethe content of PC, LPC and GPC in the samples obtained from the hydrolysis of soybean lecithin catalyzed by phospholipase A1. The sample was dissolved in denudated methanol for1H-NMR analysis. By comparing the 1H-NMR spectra, the signals at δ = 4.14 ~ 4.16, 3.76 ~ 3.83, and 4.40 ~ 4.50 were chosen as the characteristic signals of PC, LPC and GPC, respectively. Their relative contents were calculated by the integration of these signals and their molecular weights. The results show that the recovery of PC, LPC and GPC standard samples were103.4%, 85.6% and 104.8%. The relative standard deviations(RSDs) of PC, LPC and GPC(n = 5) were 4.2%, 3.5% and 2.3%. By analysis of the hydrolyzed soybean lecithin catalyzed by phospholipase A1, the results prove that the 1H-NMR method developed in this study can be applied to determine the relative content of the PC, LPC and GPC.(2) An HPLC-ELSD analysis method of GPC was established in lecithin samples. The analysis conditions were: Alltech Silica column(4.6 mm × 250 mm,5 μm), column temperature 40 ℃, methanol moving phase, flow rate 1.0 m L/min, temperature of ELSD drift tube 65 ℃, and air flow rate 1.7 m L/min. Because of the strong polarity, the peak of GPC is seen at the last with a retention time of 4.97 min. The standard curve of GPC is Y = 2.0483 X + 7.7744(R2 = 0.9914), the limit of detection is 6.3 μg/m L(S/N = 3), and the limit of quantitationis 13.0 μg/m L(S/N = 10). The average standard recovery of GPC is 102.5% with RSD = 2.2%(n = 3), and the time of analysis is less than 6 min. This indicates that the HPLC-ESLD method can effectively separate the GPC in the spectrum and the analysis results are precise and stable. This method is suitable for the quantification of GPC derived from the enzymatic hydrolysis of lecithin.(3) The preparation of LPC was conducted in water-in-oil emulsion system catalyzed by phospholipase A1. Firstly, methanol was used as an extracting solvent to preparethe PC enrich lecithin(37%).The hydrolysis of phosphatidylcholine was performed in aqueous system fromthe hydrolysis of PC enrich lecithin catalyzed by phospholipase A1. The effects of the ratio of substrates to solvent, enzyme loading and reaction time on the yield of lysophospholipid were studied. The results show that the optimized parameters are: substrates to solvent 1:4, enzyme loading 66.7 U/m L, and2 h. The product has an acid value of 62.2 mg/g.(4) The preparation of GPC in a water-in-oil emulsion system was carried out using phospholipase A1. As the enzyme is an interfacial-active enzyme, the water-in-oil emulsion system can effectively increase the interfacial area of the reaction system and hence can enhance the enzyme activity for efficient preparation of GPC. The resultsrevealed that the optimal hydrolysis conditions were: the oil is 4 times that of the lecithin, water content is 15% that of lecithin, the enzyme loading is 10%, the hydrolysis time is 2.5 h, the GPC content in the product is 27%.(5) Phospholipase A1 was employed to catalyze the hydrolysis of PC enriched soybean lecithin and flaxseed oil under aqueous conditions to obtain GPC and linolenic acid. Then under vacuum conditions,phospholipase A1 was used again to catalyze the esterification of linolenic acid with GPC to form pinolenic-acid-rich structural lecithin. The impact of water content, hydrolysis time,vacuum time, amount of enzyme and the oil-lecithin ratio on the hydrolysis was studied.The product was methylated with boron trifluoride to determine its content of linolenic acid by GC-FID. The results show that the optimum condition are: the water content 10%, 6 h, oil to lecithin ratio 3:1. The highest linolenic oil content of the structural lecithin achieved is 27.7%.