Enzymatic Synthesis Of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol And Preparation Of High Similarity Human Milk Fat Substitutes

1-Oleoyl-2-palmitoyl-3-linoleoylglycerol?OPL?is the most abundant triacylglycerol?TAG?in Chinese breast milk,which accounts for about 25%.Infant formulas?IF?are the most ideal substitute food for babies,and the fat of which is named human milk fat substitutes?HMFS?.At present,the HMFS on the market are maily developed based on the composition of foreign human milk fat,no attention has been paid to the ingredients of Chinese human milk fat.OPL has not been added to infant formulas,and OPL didn't get widespread attention in IF,resulting the significantly difference between HMFS and Chinese human milk fat on the Chinese market.Therefore,in order to make the TAG composition of HMFS more similar to Chinese human milk fat,HMFS rich in OPL were prepared by combination of enzymatic acidolysis and physical blend.The main results in this study were as follows:Firstly,58? palm stearin was fractionated in a solvent system to obtain high purity of tripalmitin?PPP?.After fractionation,the contents of PPP and palmitic acid were 90.69%and92.03%,respectively.The feasibility of preparing OPL was evaluated by comparing the two-step acidolysis and one-step acidolysis reaction routes of palm stearin and fatty acid mixture?oleic acid+linoleic acid?.The result shows that the OPL content was 55.91%after the two-step acidolysis,which was higher than the one-step acidolysis?42.07%?.The result also shows that two-step acidolysis reaction was obviously better than one-step acidolysis reaction.Therefore,two-step acidolysis reaction route was selected to synthesize OPL.Subsequently,the reaction conditions for the two-step acidolysis of OPL were optimized,including the effects of molar ratio of palm stearin to fatty acid acyl donor,molar ratio of oleic acid to linoleic acid,enzyme types,enzyme load,reaction temperature,and reaction time on the OPL content and the acyl migration was controlled by measuring the content of sn-2palmitic acid.The optimal acidolysis condition results were found as follows:molar ratio of palm stearin to fatty acid acyl donor,1:14;molar ratio of oleic acid to linoleic acid,1:1;Lipozyme NS40086,enzyme load,10%;reaction temperature,60°C;reaction time,4 h.The acidolysis reaction was magnified 50 times under the optimal conditions with OPL content and sn-2 palmitic acid content of 57.79%and 87.57%,respectively.The pilot experiment products were purified by molecular distillation.Under the optimal conditions?condenser temperature of 30?;preheating temperature of 60?;evaporation surface temperature of 200?;feed rate of 2.5 mL/min;scraper speed of 120 r/min;pressure of 1Pa?,the content of TAG increased from25.39%to 96.49%,the content of diacylglycerol was 3.36%,the content of free fatty acid was0.15%,the content of OPL was 57.65%,the content of sn-2 palmitic acid and the relative content of sn-2 palmitic acid were 87.90%and 82.93%,respectively.This indicated that the OPL in the product mainly existed in the form of sn-OPL.Next,physical blending model of HMFS was established.Under the model,three kinds of HMFS were prepared of vegetable oils,vegetable oils/basa catfish oil,vegetable oils/basa catfish oil/OPL based on the composition of regular fatty acid,regular sn-2 fatty acid and regular TAG in Chinese human milk fat,including HMFS?A?SO:PNO:CO:PO:HOSO:CNO=24.20%:6.39%:0.63%:39.58%:1.11%:0.59%:27.49%?,HMFS?B?SO:CO:PO:CNO:BCO=13.06%:6.97%:8.72%:16.65%:54.59%?and HMFS?C?SO:CO:PO:CNO:BCO:OPL=6.42%:5.55%:0.85%:14.41%:33.58%:39.21%?.Finally,three HMFS prepared by physical blending were assessed,results show the G_FA?Goal of fatty acid??G_{sn-2 FA}?Goal of sn-2 fatty acid?and G_TAG?Goal of triacylglycerol?of HMFS?A were 82.35,19.07,and 66.51.The G_FA?G_{sn-2 FA}n-2 FA and G_TAG of HMFS?B were 95.65,58.20,and 73.85.The G_FA?G_{sn-2 FA}n-2 FA and G_TAG of HMFS?C were 91.33,79.53,and 82.35.The G of HMFS?A,HMFS?B,and HMFS?C was 55.98,75.90,and 84.40,of which HMFS?C rich in OPL prepared by combing enzymatic acidolysis synthesis and physical blending were considered highest similarity to Chinese human milk fat.The content of OPL and relative content of sn-2 palmitic acid in HMFS?C was 29.09%and 61.60%,which was similar to Chinese human milk fat.In conclusion,OPL was synthesized through enzymatic acidolysis and purified by molecular distillation.HMFS rich in OPL were successfully prepared by enzymatic acidolysis synthesis and physical blending which were similar with Chinese human milk fat.This research is of great significance for improving the quality of IF in the Chinese market and for the health of Chinese babies.