Synthesis And Characterization Of Low Saturated Transesterified Fat Using Free And Indigenously Immobilized Lipase In Solvent-free Medium

The demand and sales scale of food special oil base oil in China are growing constantly.Enzymatic transesterification is an important technology for preparing high-quality food special oil base oil.However,lipase used for transesterification has been monopolized by foreign countries and has become a bottleneck in the production of high-quality food special oil base oil,which severely restricts the processing technology level of special food oil in China.At the same time,traditional base oils contain trans fatty acids（TFA）or a large amount of saturated fatty acids（SFA）,which poses a risk of cardiovascular disease.Based on this,this project uses high oleic sunflower seed oil（HOSO）and palm stearin（PS）as raw materials,selects domestically produced lipases with low cost and high catalytic activity,and develops immobilization techniques for the enzymatic preparation of food specific oil-based oils.The preparation process of enzymatic preparation of low saturation（SFC<40%）base oil is established,and the physicochemical properties of the products are investigated.To provide theoretical support and technical guidance for promoting the efficient catalytic preparation of food specific oil-based oils using domestic lipases.The main research findings are as follows:（1）A domestically produced liquid Pichia pastoris lipase（PPL）with low price and high catalytic activity was screened,which can replace expensive imported lipases for the development and preparation of specialized food oil base materials.Based on the exploration of the influencing factors,the optimal preparation process was obtained using the response surface methodology（RSM）:reaction time of 25 minutes,enzyme addition amount of 17%,and reaction temperature of 48℃.Under these conditions,a base oil with SFC₅ of 22%and SFC₂₅ of 11%could be obtained,which was also confirmed in a 10-fold amplification experiment(SFC₅ was 22.35±0.87%,SFC₂₅ was11.67±0.84%),demonstrating a good model was established.Three base oils with different characteristics were prepared using the model,and their triglyceride（TAG）composition,thermal properties,and crystal forms were examined,confirming that triunsaturated triglycerides（U₃）and trisaturated triglycerides（S₃）were converted to dibasic monounsaturated triglycerides（S₂U）and monosaturated dibasic triglycerides（SU₂）after electrification exchange,with an electrification rate of 76.40%.Someβ-crystalline was transformed into smaller needle-shapedβ’-crystals required for specialized food oil base materials,forming a more compact crystal aggregate and network structure.（2）In order to overcome the high sliding melting point（SMP）of the product prepared by PPL,a domestically produced liquid Aspergillus niger lipase（ANL）was further screened.Based on the exploration of the influencing factors,the best reaction conditions were optimized using RSM:reaction time of 44 minutes,enzyme addition of 13%,and reaction temperature of 48℃.Under these conditions,the esterification exchange product had an SFC₅ of 23.03±0.46%,SFC₂₅ of 7.01±1.25%,and SMP of30.25℃.The analysis of the electrification exchange product showed that ANL had better catalytic effect than PPL,with more conversion of U₃ and S₃-type TAG in the base oil to S₂U and SU₂,with an electrification rate of up to 94.07%.Most of theβ-crystals were transformed into smaller needle-shapedβ’-crystals,and the resulting crystal aggregate and network structure were more compact.（3）In order to improve the catalytic performance and stability of the free PPL,a low-cost domestically produced epoxy-based resin ES-108B was used to immobilize the liquid enzyme PPL by covalent binding,preparing immobilized Pichia pastoris lipase（ES-PPL）.The optimal reaction conditions were explored using single-factor experiments:reaction time of 60 minutes,enzyme addition of 16%,and reaction temperature of 70℃.Under these conditions,the SFC₅ was 32.56±1.09%,SFC₂₅ was8.01±0.99%,SFC₃₅ was 1.89±0.58%,and SMP was 35.75±0.49℃.Compared with free PPL,immobilized enzyme ES-PPL has better thermal stability,retains high catalytic activity at 70℃,and still shows good catalytic activity after being reused six times.Additionally,the SMP of the base oil was significantly reduced（<37℃）,with lower content of S₃ and U₃-type TAG,and higher electrification rate of 99.67 The content of S₂U and SU₂-type TAG increased,andβ’-crystal form is more easily formed.（4）In order to improve the catalytic performance and stability of free ANL,an epoxy-based resin carrier LX 1000EP was selected to effectively immobilize the liquid enzyme ANL,and the effect of immobilized Aspergillus niger lipase（EP-ANL）in catalyzing the production of zero trans fat and low saturated specialized food oil base materials was explored.The optimal experimental conditions were obtained by RSM optimization:reaction time of 63 minutes,enzyme addition of 8%,and reaction temperature of 48℃.Under these conditions,the SFC₅was 25.35±0.87%,SFC₂₅ was6.17±0.8%,SMP was 30.25±0.5℃,and electrification rate was as high as 99.76%.The EP-ANL still showed good catalytic activity after being reused five times.The content of S₃ and U₃-type TAG decreased more,and the content of S₂U and SU₂-type TAG increased,with a smallerβ’-crystal form forming more compact clustered spherical crystals.Additionally,compared with free ANL,the optimal enzyme addition of immobilized EP-ANL decreased by 38.5%,indicating that immobilization improved the catalytic performance of the enzyme.