Study On The Preparation Of OPO By Enzymatic Transesterification Of High Oleic Peanut Oil Fatty Acid/Ethyl Ester With Palm Stearin

1,3-dioleoyl-2-palmitoylglycerol(OPO),as a new type of human milk substitute,was approved by the Ministry of Health of the People's Republic of China as a nutritional fortifier to be added to infant formula in 2008.At present,the commonly used method for the synthesis of OPO is enzymatic acidolysis.According to GB 30604-2015 "National Standard Food Nutrition Fortifier for Food Safety1,3-dioleoyl-2-palmitoylglycerol",oleic acid for the synthesis of OPO should be derived from edible vegetable oils.At present,high oleic peanut oil has been more and more widely planted.In order to increase the added value of high oleic peanut oil,high oleic peanut oil was selected as the raw material to prepare high oleic peanut oil fatty acid and high oleic peanut oil fatty acid ethyl ester,which were used as acyl donors for the synthesis of OPO,and were optimized to interact with palm stearin under the action of Novozym 40086 lipase;Then,the combination of thinlayer chromatography and gas chromatography was used to determine the changes in the components of the system during the reaction,and the in-situ infrared spectrometer was used to study the synthesis of OPO from two different acyl donors.Finally,the lowtemperature solvent crystallization method was used to enrich and reuse the oleic acid in the mixed free fatty acid,the by-product of OPO synthesis with oleic acid as the acyl donor,so as to reduce production costs and increase economic benefits.Firstly,high oleic peanut oil was saponified and acid hydrolyzed to prepare high oleic peanut oil fatty acid,and then transesterified with palm stearin under the catalysis of Novozym 40086 enzyme to synthesize OPO.The optimized process conditions were: the molar ratio of oleic acid to palm stearin 8:1(mol/mol),the reaction time 4h,the reaction temperature 60?,enzyme load 8%(based on the total mass of the substrate).Under these conditions,the content of OPO in the product obtained was(46.28±0.11)%,the percentage of sn-2 palmitic in total palmitic acid content was(66.62±1.30)%,and the content of glyceryl tripalmitate was(3.68±0.09)%,and the component indicators of the prepared product met the requirements of the national standard GB 30604-2015.Secondly,because of its simple gump three ester and acyl esters(methyl or ethyl ester)between the enzymatic transesterification could synthetic fatty acid composition and structure on the cream with people of similar target product,at the same time simple acyl ester has the characteristics of low melting point,low viscosity,thus we obtained by ethanol solution of high oleic acid peanut oil preparation high oleic acid peanut oil fatty acid ethyl ester,and then transesterified with palm stearin under the catalysis of Novozym 40086 enzyme to synthesize OPO.The optimized process conditions were: ethyl oleate: palm stearin 6:1(mol/mol),reaction time 1h,enzyme load 8%(based on total substrate mass),reaction temperature 60?.Under these conditions,the content of OPO in the product was(46.35±0.03)%,the percentage of sn-2 palmitic in total palmitic acid content was(60.73±0.23)%,and the content of glyceryl tripalmitate was(5.67±0.30)%,and the component indexes of the product met the requirements of national standard GB 30604-2015.By comparing the above two different acyl donor synthesis OPO reaction process conditions and the difference in the composition of the target product,the combination of thin-layer chromatography and gas chromatography was used to determine the components of the system(glycerol mono-,di-,tri-ester and free Fatty acid,ethyl ester),and combined with in-situ infrared spectrometer to monitor the reaction process on-line to deduce the process of the OPO synthesis of two different acyl donors: when oleic acid was used as the acyl donor,the triglyceride in the system was partially hydrolyzed into diacylglycerol and a small amount of monoglyceride under the action of lipase,and then the resulting diacylglycerol,monoglyceride and oleic acid undergo an esterification reaction under the catalysis of lipase to synthesize OPO;taking ethyl oleate as in the case of acyl donor,the triglycerides in the system are partially hydrolyzed into diacylglycerol and a small amount of monoglyceride under the action of lipase,then the produced diacylglycerol,monoglyceride and ethyl oleate undergo alcoholysis reaction to synthesize OPO under the catalysis of lipase.Finally,we analyzed the composition of the crude product of OPO synthesis with oleic acid as the acyl donor and found that: the by-product in the OPO synthesis process-mixed free fatty acids accounted for 76%(mass ratio)of the crude product,of which the oleic acid content was 68 % or so,which was unable to meet the needs of further synthesis of OPO.In order to reduce production costs and improve the utilization efficiency of oleic acid,we enriched and reused the oleic acid in the mixed acid,the by-product of OPO synthesis by low-temperature crystallization,and the optimal reaction conditions were: n-hexane as a solvent,N-hexane: fatty acid=3:1(m L /g),crystallization temperature-5°C,crystallization time 1h.The oleic acid purity of the final separated fatty acid product was 81.4%,and the yield was 61.8%.Oleic acid of this purity can meet the requirements for further production of OPO.