| n-3 polyunsaturated fatty acids, such as ecosapeatanolic acid (EPA) and docosahexaenoic acid (DHA), play an important role in human nutrition, health and development. As the natural form of EPA and DHA, the type of triglycerides is the best form to consume, which contains a small quantity of EPA and DHA. This paper aims at preparing triglycerides containing 45% EPA and DHA through transesterification catalyzed by lipases in solvent-free system, which mainly includes:1 The water content and hydrolysis activity of 10 kinds of lipases including lipase J, U, P, T, B, L, M, N, Lipozyme TL IM and Lipozyme RM IM are estimated, and the method of estimating the transesterification activity of lipases in solvent-free system is established, on whose basis the transesterification activity of 10 lipases are determined. The lipase M from Candida Rugosa has the highest hydrolysis activity of 10 lipases mentioned, which is 4702.7 U/g; lipase L from Candida Tropicalis has the second highest hydrolysis activity. Lipozyme TL IM and Lipozyme RM IM have hydrolysis activity of 356.7 U/g and 407.2 U/g, respectively. The reaction of measurement with ethyl caprylate and glycerol trioleate as the substrates is under the conditions as follows:the weight ratio of ethyl caprylate and glycerol trioleate is 1:1, enzyme dosage is 1%, the temperature is 60℃, and the time of reaction is 12h. The lipase M, whose hydrolysis activity is highest, also has the highest transesterification activity, which is about twice of Lipozyme RM IM (usually used as the catalyst of transesterification). So the lipase M is chosen as the catalyst of transesterification to prepare triglycerides containing 45% EPA and DHA. It is also found that the hydrolysis activity and the transesterification activity of lipases are absent of general correlation by comparison.2 A method for the determination of mono-, di-, and triglycerides by a high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) is established. Chromatographic analysis is carried out on a Rx-SIL column (250mm X 4.6mm id,5μm) with hexane- isopropyl alcohol-acetic acid (90:10:0.2, V/V) as the mobile phase. The flow rate is 0.8 mL/min. The drift tube temperature of ELSD and flow rate of carrier gas(nitrogen) are set at 60℃and 1.4 L/min respectively. It takes only 12 min to finish the analysis of a sample. The peak area and quality of sample injected have a linear relationship, and the C.V. is less than 1.5%. The detection limit is from 0.02μg to 0.05μg and the average recovery is 98.25%-103.08%. The method can be used for rapid and accurate determination of mono-, di-, and triglycerides, despite of the composition of fatty acids.3 The chosen lipase M is used as the catalyst of transesterification to prepare triglycerides which contains 45% DHA and EPA totally. The advantages and disadvantages of acidolysis and interesterfication are compared and interesterfication is chosen although acidolysis has a little faster reaction speed. Transesterification is carried out with fish oil ethyl esters and triglycerides (containing 28.87% EPA and DHA) as substrates. The effect on transesterification of temperature (40℃-70℃), reaction time (6-60h), the enzyme dosage(40-200U), substrate weight ratio (2:1-1:3), water dosage (0.5%-8%) and the content of EPA and DHA in ethyl esters are investigated, and it can be conclude through orthogonal experiment that reaction time has the greatest effect on transesterification, which is followed by enzyme dosage, the substrates weight ratio has the least effect. The favorable conditions obtained are: reaction temperature 60℃, reaction time 24h, the substrate weight ratio 5:4, enzyme dosage 80U, without adding water to the reaction system. Under these conditions, the triglycerides obtained contain 32.40% EPA and 13.10% DHA, and the immobilized enzyme can be used seven times. It is found that lipase M favors EPA by comparison between the content of EPA and DHA in products and substrates.
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