| Tuna oil is abundant in omega-3 series polyunsaturated fatty acids(PUFAs),especially docosahexaenoic acid(DHA).DHA,also known as "brain gold",has the function of promoting the development of the nervous system,improving brain function and enhancing cognitive ability.Therefore,high purity DHA monomers are of great clinical research value.In this paper,the crude tuna oil was used as the raw material to systematically study the process from the refining of tuna raw oil to the preparation of high-purity DHA ethyl esters(EEs),aiming to provide the theoretical basis and experimental basis for the further development and utilization of DHA.The main research contents and conclusions were as follows:(1)Using tuna crude oil as raw material,the traditional fish oil refining process and molecular distillation technology were combined to compare the effects of the modified refining process and the traditional refining process on fish oil decolorization,and to further investigated the physical and chemical properties of fish oil and the change pattern of volatile components during the refining process.The results were as follows,both refining processes could effectively improve the color of tuna oil,especially the modified refining process had more obvious decolorization effect on the fish oil,and the fish oil did not show recoloration during the refining process;After both refining processes,tuna oil reached the industry standard of first grade refined fish oil,but the modified refining process had greater ability to improve each chemical index of fish oil.n terms of fatty acids,both refining processes had a slight effect on the fatty acid composition of tuna oil,and neither resulted in an increase in trans fatty acid content(P(29)0.05).In terms of volatile substances,both refining processes achieved significant(P(27)0.05)deodorization.Compared with the two processes,tuna oil processed by the modified refining process had the least volatile components(38 species),and the overall flavor of fish oil was softer,plumper and more natural.In addition,this study established a multivariate statistical analysis method to distinguish four fish oil refining process characteristics from the flavor perspective,which can be used for the identification of the refining process,where the differential components help to characterize the flavoring qualities of fish oil.(2)Using refined ethyl ester type tuna oil as raw material,the DHA content of more than70% was enriched and separated through multi-stage molecular distillation process;The results were as follows,the optimized process conditions of one-stage molecular distillation,at a distillation temperature of 90°C,a feed rate of 2 m L/min and a scraper speed of 250 r/min,resulted in(13.32±0.19)% and(44.51±1.70)% of EPA and DHA in the heavy phase fraction,respectively,and the recovery of the heavy phase fraction was(46.42±1.65)%;The changes of EPA and DHA contents in heavy and light phases were investigated at different stages of molecular distillation,and it was determined that two-stage molecular distillation is more helpful for the enrichment of DHA;On this basis,a response surface-artificial neural network model was established to optimize second stage of molecular distillation process,and the optimal process conditions were determined as follows: distillation temperature of 110°C,feed flow rate of 1.38 m L/min,and scraper speed of 296.65 r/min,at which the DHA content reached 72.31%.(3)The tuna oil treated by thin film distillation and molecular distillation was used as raw material to compare the ability of silver nitrate-silica gel column chromatography,cuprous chloride-silica gel column chromatography,high speed counter current chromatography and preparative high performance liquid chromatography to separate and purify DHA.The results were as follows,using hexane-acetone as the mobile phase,the flow rate of the chromatographic column was controlled at 1.2～1.5 m L/min,and the loading volume was 1000 mg.After the separation and purification by silver nitrate-silica column chromatography and cuprous chloride-silica column chromatography,the purity and recovery of DHA were(98.99±0.76)% and12.67% and(84.38±0.12)% and(2.24±0.15)%,respectively;using n-heptane-ethanol-acetonitrile(10:5:5,v/v/v)as the solvent system,with the upper phase as the stationary phase and the lower phase as the mobile phase,the flow rate was adjusted to 5 m L/min,the host speed was set to 800 r/min,the injection amount was 200 mg,and the purity and recovery of DHA reached(85.76±1.19)% and(54.23±1.02)%,respectively,after 150 min of high-speed counter-current chromatography;using methanol-water(90:10,v/v)as the mobile phase,the flow rate was adjusted to 520 m L/min,the injection amount of 1200 mg,and the purity and recovery of DHA reached(95.74±0.53)% and(60.65±2.53)%,respectively,after 75 min of preparative high performance liquid chromatography.(4)The preparative liquid chromatography had low toxicity solvent system,high injection amount,shortest elution time and high purity of DHA,but it also has the problems of excessive solvent consumption,so the conditions of the preparative liquid chromatography continued to be optimized.Exploration of the best process for the separation of DHA from analytical liquid chromatography and scaling up to preparative liquid chromatography.Taking the purity and recovery of DHA as indicators,the mobile phase ratio and injection amount were adjusted again to determine the optimal process for the purification of DHA by preparative liquid chromatography.The results were as follows,under the conditions of methanol-water(92:8,v/v),flow rate of 530 m L/min and injection volume of 4500 mg,the purity and recovery of DHA reached at(95.30±0.04)% and(81.23±1.16)%,respectively. |
