| Silkworm pupae oil is rich inα-linolenic acid,which is an ideal source for the human body to supplementω-3 series essential fatty acids.However,the palmitic acid at the sn-1,3position of silkworm pupae oil triglyceride accounts for a large proportion,thereby reducing its digestion rate and increasing the risk of cardiovascular disease.Therefore,it is necessary to rearrange the fatty acid composition and distribution of silkworm pupae oil triglycerides,such as replacing sn-1,3 palmitic acid withα-linolenic acid.At present,immobilized enzymatic modification of lipids has problems such as low reaction efficiency,poor specificity of lipase enrichment of unsaturated fatty acids,and low purity of triglyceride monomers.Therefore,in this thesis,a new process of silkworm pupae oil modification with magnetic response micro-flow field immobilized new lipase was constructed to improve the reaction efficiency.A new lipase with the ability to enrich unsaturated fatty acids was rationally designed to enhance the specificity of enzymatic reactions.Sequential simulated moving bed chromatography was used to separate easily absorbedα-linolenic acid triglyceride monomers,and microfluidic technology was used to embed structural lipids with protein-polyphenol complexes as wall materials to improve their storage stability.The main results are as follows:(1)A new solvent-free catalytic system for the preparation of structured lipids by immobilized enzyme-modified silkworm pupae oil in a magnetic response micro-flow field was constructed.The magnetic nanoparticle immobilized enzyme was loaded on the inner wall of the microchannel by the magnetic field,and the silkworm pupae oil and freeα-linolenic acid were introduced into the microchannel to continuously prepare structural lipids rich inα-linolenic acid.The results showed that Aspergillus niger Lipase was screened from the four lipases as a catalyst.Under optimum conditions,the ratio ofα-linolenic acid at the sn-1,3 position of the product was 62.14±0.31%,and the ratio at the sn-2 position was 53.77±0.56%.It was significantly higher than that of silkworm pupae oil(α-linolenic acid accounted for 25.66±0.07%at sn-1,3 and 48.78±0.19%at sn-2,p<0.05).Compared with the batch reactor and the packed microreactor,the reaction time was shortened from 2.5～48 h to 2 min,the optimum reaction temperature was decreased from60～70°C to 50°C,the reuse of immobilized enzyme was increased from 6 times to 13 times(2 h each time),and the remaining enzyme activity was more than 90%.Therefore,the magnetic response micro-flow field immobilized enzyme catalytic system achieved green,efficient,rapid,and continuous preparation of silkworm pupae oil structural lipids rich inα-linolenic acid.(2)Based on rational design,lipase mutants with the ability to enrich unsaturated fatty acids and high catalytic activity were constructed.Lipase 6A12,4GXN,and 7BUK were screened with the enrichment ability of polyunsaturated fatty acids as the index.The lipase6A12 with a stronger affinity to C18 fatty acid substrate was molecularly modified to construct lipase mutants with high catalytic activity for the preparation of structured lipids.The results showed that the mutant K100F was obtained by molecular modification with lipase 6A12 as wild type,and its catalytic activity for C18 fatty acid substrate was increased to 5369.2±63 U/g,which was 5 times and 2 times that of lipase 6A12(1366 U/g)and ANL(2538.6±19 U/g),respectively.In the magnetic response micro-flow field immobilized enzyme catalytic system,the immobilized lipase K100F was used as the catalyst to prepare structured lipids,and the sn-1,3-α-linolenic acid of the product was obtained.The proportion of 85%,compared with the commercial lipase ANL(α-linolenic acid at sn-1,3 accounted for62%),the enrichment ability ofα-linolenic acid was increased by 37.1%.Therefore,the catalytic activity andα-linolenic acid enrichment ability of lipase mutant K100F were significantly improved in the process of enzymatic preparation of structured lipids.(3)The structural lipid monomer 1,2,3-Tri-α-linolenic acid triglyceride(Ln Ln Ln)rich inα-linolenic acid was isolated from silkworm pupae oil by sequential simulated moving bed chromatography.Based on the separation conditions of Ln Ln Ln by preparative chromatography on a single column,the sequential simulated moving bed chromatography was used for the continuous separation of Ln Ln Ln,and the mass transfer diffusion model was used to analyze the process mechanism.The results showed that when the feed flow rate was 0.4 m L/min,the mobile phase flow rate was 4.5 m L/min,the sub-step running time ratio was 1:1:3(S1:S2:S3),and the running period was 150 s,the purity of the obtained structured lipid monomer Ln Ln Ln was as high as 95.15±2.06%.The preparation efficiency of sequential simulated moving bed chromatography(347.1 mg/h)was significantly higher than that of a single column(13 mg/h),and the solvent consumption of sequential simulated moving bed chromatography(0.36 BV/h)was significantly lower than that of a single column(2.39 BV/h).The mass transfer diffusion mathematical model of the Ln Ln Ln monomer was constructed by sequential simulated moving bed chromatography.The theoretical value of the model is in good agreement with the experimental value(R~2=0.9688),indicating that the constructed mass transfer diffusion model can be used to analyze the separation process mechanism.Therefore,the constructed sequential simulated moving bed chromatography process can efficiently separate high-purity Ln Ln Ln monomer.Compared with traditional single-column preparative chromatography,it has a better separation effect and lower separation cost.(4)Microcapsules were prepared by embedding structured lipids in protein-polyphenol complexes based on microfluidic technology to improve storage stability.The silkworm pupae glutelin was used as the raw material,and the problem of the low solubility of silkworm pupae glutelin was solved by“p H drift”(p H 12～7)technology.The protein-polyphenol complex was prepared as the embedding wall material,and the oxidation resistance,emulsification,emulsion stability,encapsulation efficiency,and storage stability of the embedded structural lipids were investigated.The results showed that the emulsifying properties of silkworm pupae glutelin-caffeic acid and silkworm pupae glutelin-rutin were80.15±0.88%and 86.78±0.98%,respectively,which were higher than that of silkworm pupae glutelin(51.16±0.58%).The emulsion stability of the prepared structural lipid microcapsule droplets reached 92.36±2.55%and 95.88±2.36%,respectively,and the antioxidant activity was significantly higher than that of glutelin(p<0.05).The acid value of the lipid in the microcapsules placed at room temperature for 30 days was measured,and it was found that the change was not significant(p<0.05),while the acid value of the unembedded structural lipid increased significantly(p<0.05),and rancidity occurred.Compared with traditional wall materials such as chitosan and whey protein,the microcapsules prepared by the new wall material silkworm pupae gluten-polyphenol have a considerable or even longer shelf life(more than 30 d).Because extracting silkworm pupae oil and processing silkworm pupa protein can improve the resource utilization rate and product-added value of silkworm pupae,the cost of preparing microcapsule wall material by combining silkworm pupae gluten with polyphenols is significantly lower than that of other source proteins.Therefore,the prepared structured lipid microcapsules have good storage stability and can supplement protein and polyphenols while supplementingα-linolenic acid.In summary,this thesis constructed a new process for the preparation of structured lipids by immobilized lipase-modified silkworm pupae oil in a magnetic response micro-flow field.The specific lipase was rationally designed to enrich polyunsaturated fatty acids,and the Ln Ln Ln monomer rich inα-linolenic acid was isolated by sequential simulated moving bed chromatography.Furthermore,the silkworm pupae glutelin-polyphenol complex was used to develop structured lipid microcapsules with good storage stability,to provide theoretical and technical support for the extension of the sericulture industry chain and the industrialization of structured lipids. |
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