Synthesis Of Functional Lipids By Microalgae Oil And The Mechanism Of Enzymatic Reaction

Functional fats and oils are riched in n-3 polyunsaturated fatty acid(n-3 PUFAs),preventing and/or reducing the risk of cancer,cardiovascular disease,podagra,and stroke and so on,has been a hot research in modification of fats and oils.N-3 PUFAs,particularly EPA and DHA,are rooted in human mind as "gold oil".In recent years,stearidonic acid(SDA)as an essential fatty acid can be metabolized into EPA and DHA,showing that SDA is a new source to replace with EPA and DHA.Moreover,many studies have domanstrated that SDA not only has some similar biological characteristics,but also shows some different biological functions(e.g.SDA is an important source to synthesize prostaglandin E2).At present,SDA is mainly existed in fish,plant and marine microalgae.Naturally,fish oil have a low content of SDA.The concentration of SDA in plant oils like echium oil is largely associated with temperature,light intensity.Compared with fish oil and plant oil,marine microalgae like Isochrysis galbana have many advantages of fast growth rate,high lipid productivity and high content of SDA.Therefore,a microalgae strain named by Isochrysis galbana(Lgalbana)to produce a high content of SDA is selected in this project.A flat-plate photobioreactor is applied to cultivate I.galbana to build some kinetic models about the cell growth,lipid formation and consumption of sodium nitrate and then find the correlation between lipid formation and initial concentration of sodium nitrate.After collected enough algae powder,microalgae oil is extracted to analyze the lipid classes and regiodistribution of fatty acid in each lipid to produce functional lipids.Triacylglycerols(TAGs)rich in SDA from I.galbana is used to produce monoacylglycrols(MAGs)and structured triacylglycerols(STAGs)rich in n-3 PUFAs by green and environmental-friendly enzymatic reactions.Moreover,n-3 fatty acids from I.galbana are used to prepare the human milk fats by lipase-catalzed reaction of microalgae oil from Nannochloropsis oculata.During the enzymatic reactions,some novelty characteristics of selected lipases and their fatty acid selectivity and regiospecificity are addressed,providing a theoretical and practical knowledge in modifying fats and oils.1.A quantitative description of microalga growth and the "trade-off" between biomass production and lipid accumulation will provide a kinetic insight of the dependency of biomass and components on growth conditions and the interplays between lipid/protein production and N-limitation.The examination of the dependency of Isochrysis galbana growth on initial concentration of sodium nitrate(NaNO3)in a flat-plate photobioreactor(FPPBR,25 L)revealed that the maximal lipid 106 mg L-1 is produced at 25 mg L-1 NaNO3 and whereas the accumulation of biomass,protein and starch increases as NaNO3 increases to 100 mg·L-1.The analysis and model fitting results indicate that I.galbana growth and production of lipids in a FPPBR can be quantitatively described by Baranyi-Roberts,Logistic equation and Luedeking-Piret model in a satisfactory manner,respectively.25-75 mg L-1 was found to be proper level of NaNO3 to achieve a good trade-off balance to produce both high quantities of biomass and lipids.Based on the predictions of growth model,after culturing middle stationary phase under the high nitrogen medium,switching of culture for algal cells to a nitrogen-free medium and/or high illumination intensity might be preferable strategy to achieve high level accumulation of lipids.2.Accelerated solvent extraction(ASE)was applied to extract the oil from I.galbana powder.The performance of three methods for extracting(traditional extraction,Soxhlet extraction and ASE)was investigated using a green and environment-friendly solvent.The results showed that ASE method had the advantages of shorter extraction time,less solvent,lower labor intensity and easier operability in comparison to other extraction methods.95.24%of microalgae oil could be extracted the microalgae oil by ASE using the 3:1 of volume ratio for 90%ethanol to n-hexane.Triacylglycerols(TAGs)and free fatty acids(FFA)from microalgae oil(I.galbana)were separated by silica gel column.The fatty acid composition of TAGs and FFA were determined by GC-FID;moreover,GC-FID and TLC technologies were applied to determine the regiodistribution of fatty acid in TAGs for developing the functional lipids from TAGs and FFA in I.galbana.3.Using anchovy oil and TAGs from microalgae oil with low content of n-3 PUFAs as substrates,CAL-A was the best candicate lipase to produce the monoacylglycerols(MAGs)enriched in n-3 PUFAs by ethanolysis;furthermore,the fatty acid selectivity and regiospecificity of selected lipases for producing the MAGs rich in n-3 PUFAs by ethanolysis of oils were inverstigated to address some novelty characteristics and/or application of lipases.The results showed:(1)CAL-A displayed the most strongest discrimination agaist SDA,ETA,EPA,DPA and DHA in comparsion to Novozym 435 and Lipozyme TL IM.(2)The reaction parameters of CAL-A were optimized as follows:the weight ratio of ethanol to oil was 3:1,reaction temperature was 35?,water content was 5%-6%,lipase loading was 10%.Under these conditions,after 48 h of ethanolysis,the content of n-3 PUFAs in MAGs could reach up to 88-92%,which were higher than that of other lipases recorded in available literature.(3)In the excessive present of ethanol,the order for cleaving the fatty acid in TAGs by CAL-A was saturated fatty acid,monosaturated fatty acid,n-6 fatty acid,?-linolenic acid(ALA)and n-3 fatty acid.(4)During the ethanolysis,the results from 13C nuclear magnetic resonance showed that CAL-A displayed the non-regiospecificity.(5)The kinetic equation of CAL-A for producing MAGs rich in n-3 PUFAs by ethanolysis of oils was built.(6)Liquid lipase can be an alternative source to replace with immobilized lipase for functional lipids production.4.Using Lipozyme TL IM with cheaper price as biocatalyst,a two-step enzymatic process was proposed to synthesize the structure TAGs(medium chain(C8)-n-3PUFAs and palmatic acid-medium chain(C8))by enzymatic reaction of sardine oil and microalgae oil from I.galbana.Moreover,the difference about property of enzymatic reaction of fish oil and microalgae oil was elaborated to illustrate a new approach for producing the structured lipid.The results were listed as follows:1)First-step enzymatic process for MAGs production by ethanolysis of oils.(I)the optimal conditions for producing MAGs by Lipozyme TL IM were:reaction temperature was room temperature,water content was 5-9%,the molar ratio of ethanol to oils was 32:1,reaction time was 6 h.Under these conditions,the yield of MAGs could be in the range from 30%to 33%.The content of PA,ALA,SDA,EPA,DHA and total n-3 PUFAs in 2-MAGs by fish oil and microalgae oil was 13.71%,1.44%,2.72%,9.35%,22.42%and 39.01%;24.26%,6.92%,25.16%,1.17%,6.91%and 41.11%,respectively.The content of PA+n-3 PUFAs in 2-MAGs from microalgae oil was 65.37%,which was higher than that in 2-MAGs from fish oil.(?)The results of 13CNMR showed that the MAGs production by Lipozyme TL IM was mainly sn-2 MAGs(around 90%).(?)the specificity of Lipozyme TL IM for ethanolysis of oils showed the sn-1,3 specific selectivity.2)Second-step enzymatic process for synthesizing the structured lipid by esterification of 2-MAGs and caprylic acid(C8).The optimal conditions for second-step enzymatic process were:n-hexane was used as media,reaction temperature was 40,the molar ratio of 2-MAGs to caprylic?acid was 1:3,reaction time was 24 h.Under these conditions,the maxmium rate of esterification for structured lipid by Llipozyme TL IM was 91.84%.The content of PA+n-3 PUFAs in STAGs from microalgae oil and fish oil was 23.26%(ALA,2.67%;SDA,8.93%;EPA,0.28%;DHA,2.17%)and 18.06%(ALA,0.41%;SDA,0.81%;EPA,3.02%;DHA,7.55%),resepectively.3)The crystallization profiles and melting profiles of substrate(fish oil and microalgae oil),2-MAGs and structured TAGs was studied and found that the temperatures of crystallization and melting for structured lipid were lower than those of substrate and MAGs.5.Free fatty acids rich in ALA,SDA and DHA from I.galbana and TAGs rich in palmatic acid at sn-2 position from Nannochloropsis oculata were selected to synthesize human milk fats(HMFs)rich SDA by enzymatic interesterification or acidolysis using 3 kinds of commercial lipases.Enzymatic method for HMFs production can improve the content of n-3 PUFAs and nutritional value.The optimal conditions and fatty acid selectivity of biocatalysts were investigated to propose a new approach for producing HMFs using microalgae oil as substrate.The results were concluded as follows:(1)The interesterification or acidolysis of CAL-A did not happen under the anhydrous condition using n-hexane as media.(2)The reaction parameters of Novozym 435 and Lipozyme TL IM were optimized:the molar ratio of microalgae oil to free fatty acid rich in n-3 PUFAs was 1:3,reaction temperature was 60 ? lipase loading was 10%,reaction time was 24 h.Under these conditions,the content of ALA,SDA,total n-3 PUFAs in HMFs by Novozym 435 and Lipozyme TL IM could be 3.63%,5.45%and 12.67%;7.32%,3.22%and 13.91%,respectively.(3)The incorporation order(from high to low)for n-3 fatty acid selectivity of Novozym 435 and Lipozyme TL IM was ALA>SDA>DHA.(4)During the enzymatic reaction,the acyl migration for Novozym 435 and Lipozyme TL IM was observed;moreover,the rate of acyl migration for Lipozyme TL IM was lower than that Novozym 435.(5)After the modification of microalgae oil from Nannochloris oculata,HMFs had a higher content of n-3 PUFAs and over 60%of palmatic acid at sn-2 position.(6)The crystallization profiles and melting profiles of HMFs by enzymatic reaction were determined as alternative substrates to produce dairy product,bread,ice-cream and so on.