Separation Of α-Linolenic Acid From Silkworm Pupa Oil By Simulated Moving Bed Chromatography And Microfluidic Enzymatic Preparation Of Monodispersed Lipid Microcapsules

Silkworm chrysalis as a resource for medicine and food is rich inα-linolenic acid and other unsaturated fatty acids,which is an ideal raw material for the preparation of new structural triglycerides.In this paper,α-linolenic acid in silkworm oil was isolated by simulated moving bed（SMB）chromatography.Two kinds of structural triglycerides were prepared by enzymatic method using microreactor,and a high performance liquid chromatography-evaporative light scattering detector（HPLC-ELSD）coupled method was established to analyze the triglyceride composition.In addition,microfluidic droplet method was constructed to prepare the monodisperse structure lipid microcapsules by the caffeic acid-grafted chitosan aqueous solution.The results of the study are as follows:For the first time,a method for the separation ofα-linolenic acid with high purity from silkworm pupa oil was developed using SMB chromatography.The initial conditions were investigated using a C₁₈8 single column:the mobile phase was 95%ethanol solution,the flow rate was 1.5 mL/min,and the material concentration was 68 mg/mL.At this time,the purity of ethylα-linolenate was 100±0%with the yield with 21.4±0.8%.On this basis,continuous separation was performed using 4-zone SMB chromatography.When the feed flow rate was0.48 mL/min,the mobile phase flow rate was 1.53 mL/min,and the running time was 147 s,the purity of ethylα-linolenate was 97.86%±0.7%,and the yield was 2.51±0.12 mg/mL.The simulation mathematical model of the SMB separation process was established using MATLAB software,and the purity of the ethylα-linolenate reached 98.12%when the feed flow rate was 0.51 mL/min and the mobile phase flow rate was 1.49 mL/min.A continuous flow microreactor immobilized packed with enzyme was designed.α-linolenic acid of high purity obtained with SMB chromatography was used to modify palmitate triglyceride（PPP）and mulberry seed oil triglyceride respectively and APA（A:alpha-linolenic acid,P:palmitic acid）type breast milk fat substitute and n-6:n-3 matching type structural triglycerides were successfully prepared.The results showed that the content ofα-linolenic acid in the APA-type human milk fat substitute reached 69.56%when Lipozyme RMIM was used as the catalyst and the substrate ratio was 1:7,the flow rate was2μL/min,and the temperature was 60°C.Compared with conventional reactors,microreactors can make the required ALA reduce by 41.6%,and the reaction time reduce abruptly to 1/15.When the substrate ratio was 1:9,the flow rate was 2μL/min,and the temperature was 50°C,the ratio of the n-6:n-3 ratio of the structural triglycerides was 1.25.Compared with conventional reactors,the microreactors can make the reaction temperature dropp by 28.6%,and the reaction time drop sharply to 1/8.These results demonstrated the high-efficiency,time-saving and process green energy-saving features of immobilized enzyme microreactors for the synthesis of structural triglycerides.The triglyceride composition of structural triglycerides and the mechanism of microfluidic reaction enhancement were analyzed using a HPLC-ELSD assay and a kinetic model.By DSC,TGA and HPLC-ELSD characterization,the melting point and crystallization point of the structural triglycerides prepared by the microreactor reduced by3.26°C and 0.25°C,the thermal reduction stage increased 33%,and the structural change was as high as 95%compared with the conventional reactor.These changes showed that microreactors have an accelerating effect on the enzymatic synthesis of structural triglycerides.Compared with conventional reactors,the kinetic constants of enzymatic reactions in the microreactors were reduced to 1/7,and the internal and external mass transfer coefficients were increased by more than 9 times.This confirms that the process enhancement of the microreactor originates from its unique mass transfer,heat transfer and efficient technical features.The microfluidic antioxidation wall material embedding technique was constructed to prepare monodisperse lipid microcapsules.Caffeic acid-grafted chitosan（CA-g-Ch）was prepared using caffeic acid and chitosan as raw materials.Its aqueous solution was used to prepare monodisperse droplets of structural triglycerides by microfluidic droplet technology.Monodispersed lipid microcapsules were obtained after drying.The results showed that0.1%Tween 20 solution can contribute to a high encapsulation efficiency of 67%,uniform size,stable morphology,good rheological properties,high dispersion,and structural lipid microcapsules possessing excellent antioxidant properties.In summary,the high-purityα-linolenic acid was successfully isolated from silkworm pupa oil by SMB chromatography,and two novel structural triglycerides were prepared using a continuous flow enzymatic microfluidic technique.Droplet preparation technology has prepared monodisperse lipid microcapsules with CA-g-Ch as a wall material,providing technical support for the development of new structural lipid products with high nutritional value,strong oxidation resistance and good bioavailability.