Enzymatic synthesis method of UPU type glyceride

Abstract

The invention belongs to the technical field of UPU type glyceride synthesis, and particularly relates to an enzymatic synthesis method of UPU type glyceride. The UPU type glyceride (OPO, OPL and LPL) is prepared by taking fatty acids OA, LA and PPP or glycerol as reaction substrates and taking an immobilized enzyme ANL and MARE as a catalyst. And removing the fatty acid, the monoglyceride and the diglyceride in the reaction product to obtain the triglyceride. According to different solubility of each component in a reaction product in ethanol and water, an ethanol / water system is adopted to perform liquid-liquid extraction on the reaction product, after the reaction product is added into an ethanol water solution, layering occurs, an upper layer is mainly triglyceride with relatively low density, and an ethanol solution (the polarity of the triglyceride is minimum and the solubility of the triglyceride in ethanol is highest) is used as a lower layer; and the lower layer is mainly an ethanol-water-fatty acid, monoglyceride and diglyceride solution with higher density. After liquid separation and solvent removal, triglyceride with a higher melting point is further removed by utilizing a solvent crystallization method, and the UPU type structural grease with higher purity is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of UPU-type glyceride synthesis, and particularly relates to an enzymatic synthesis method of UPU-type glyceride. Background Art

[0002] As the most ideal nutritional source for infants, the unique fat composition and structure of breast milk play an irreplaceable role in the growth and development of infants. Approximately 70% of the triglycerides in breast milk fat have palmitic acid (C16:0) as the main fatty acid, and approximately 70% of its palmitic acid is specifically esterified at the sn-2 position of the glycerol backbone, which is also the UPU-type structured lipid, while palmitic acid in common vegetable oils is mainly distributed at the sn-1,3 positions. This structural difference results in the lipids in traditional formula milk being prone to binding with calcium ions in the intestine to form insoluble calcium soaps, which not only reduces the absorption rates of calcium and fat but may also cause problems such as infant constipation and intestinal inflammation. Therefore, the development of human milk substitute fat with UPU structured lipid as the core to simulate the composition and function of breast milk fat has become a research hotspot in the field of infant formula foods.

[0003] In recent years, with the rapid development of enzyme catalysis technology, function evaluation models, and synthetic biology, the research on human milk substitute fat has advanced from single-component bionics to functional collaborative design and has achieved remarkable progress in industrial applications. The enzymatic interesterification technology has become the mainstream process due to its high selectivity and mild reaction conditions. Among them, the use of immobilized lipase to catalyze the directional synthesis of palmitic acid oleic acid triglyceride can increase the palmitic acid content at the sn-2 position to more than 50%. Most of the enzymatic preparations of OPO- and OPL-rich structured lipids at home and abroad use oils and fats rich in PA at the sn-2 position (sn-2PA), such as tripalmitonin (PPP), palm stearin, lard, etc., and OA, LA, or plant oils rich in two fatty acids as raw materials and are synthesized under the catalytic action of sn-1,3 specific enzymes. Among them, lard is a natural source of fat, and its lipid composition and molecular structure are similar to those of human milk fat. Using palm stearin as the raw material, first, the TAG rich in PA is converted into 2-palmitic acid monoglyceride (sn-2Monoglyceride Palmitate, 2-MP) under the enzymatic hydrolysis of lipase, and then 2-MP is esterified with OA and LA using lipase, which is the most commonly used method in current production research.

[0004] When preparing structured lipids by enzymatic methods, it is necessary to add an excessive amount of fatty acids in the substrate to make the reaction proceed in the forward direction. Therefore, there are relatively large amounts of fatty acids in the reaction products. There are also reaction by-products such as monoglycerides and diglycerides, and these by-products in the products need to be effectively separated.

[0005] Currently, the commonly used separation and purification methods include: (1) Molecular distillation method: Molecular distillation, also known as short-path distillation, is a method for liquid-liquid separation by utilizing the difference in the average free path of molecular motion of different substances at a certain temperature and vacuum degree. It is suitable for separating and purifying natural products with high boiling points, thermal sensitivity, and easy oxidation. Compared with traditional distillation methods, it improves the problem of high-temperature damage to thermosensitive and easily oxidized components. It does not require the use of organic solvents, has a large processing capacity and high efficiency, and is currently the most commonly used separation method in industry. However, during the reaction process, it requires high temperatures (150 - 300 °C), which may still cause oxidation and conformational changes of fats and oils, resulting in a darker color of the fats and oils and a decrease in purity and quality. (2) Alkali refining and deacidification method: That is, adding an alkali solution to neutralize free fatty acids to form soapstock, and then removing it by centrifugation or water washing. The disadvantage is that when the fatty acid content is relatively high, a large amount of alkali is consumed, and it may cause damage to the synthetic product. It is often used in the last step of deacidification to remove a small amount of residual FFA. (3) Supercritical CO2 fluid extraction method: It has the advantages of simple process, no residual organic solvents, mild operating conditions, and no environmental pollution. However, because it needs to be carried out under the condition of providing the critical pressure of carbon dioxide (7.38 MPa), the requirements for equipment are relatively high, and the current popularization degree in China is relatively low. (4) Solvent extraction method; The solvent extraction method, also known as liquid-liquid extraction method, is a method for separating each component of a sample by utilizing the difference in solubility in a solvent. It requires the use of a large amount of organic solvents and corresponding equipment for removing organic solvents. Although the yield is not as high as that of the molecular distillation method, it can be carried out at a low temperature to ensure the maximum protection of the reaction product.

[0006] Based on this, we propose an enzymatic synthesis method of UPU-type glycerides, hoping to solve the deficiencies in the existing technology. Summary of the Invention

[0007] The purpose of the present invention is to provide an enzymatic synthesis method of UPU-type glycerides for the existing problems.

[0008] The present invention is realized through the following technical solutions:

[0009] An enzymatic synthesis method of UPU-type glycerides includes the following steps:

[0010] S1. After dissolving the substrate in a rotary flask, cool it to 45 - 55 °C, then add immobilized lipase accounting for 5 - 15% of the total mass of the substrate, seal the rotary evaporation flask with plastic wrap, and rotate the reaction on a rotary evaporator;

[0011] S2. Immediately after the reaction ends, filter to separate the lipase and the reaction product;

[0012] S3. Use an ethanol / water system to perform liquid-liquid extraction on the reaction product, and then reduce the pressure to remove the solvent from the supernatant after extraction;

[0013] S4. Purify by solvent crystallization method to obtain high-purity UPU-type glycerides.

[0014] Further preferably, the substrate described in step S1 is a mixture of fatty acids and PPP.

[0015] Further preferably, the fatty acid is one or more of oleic acid and linoleic acid.

[0016] Further preferably, the immobilized lipase described in step S1 is immobilized lipase ANL@MARE.

[0017] Further preferably, the rotation speed during the rotation reaction in step S1 is 80 - 150 r / min, and the reaction time is 2 - 8 h.

[0018] Further preferably, both step S1 and step S2 are completed under nitrogen protection and explosion-proof environment. The reaction product needs to be stored under nitrogen and protected from light, and 0.02% natural vitamin E antioxidant is added during storage.

[0019] Further preferably, the specific operation of liquid-liquid extraction in step S3 is as follows: Add the reaction product to an ethanol aqueous solution of 85 - 88%. The volume ratio of the ethanol aqueous solution to the product is 6 - 10:1. Heat in a water bath at 60°C to make the upper and lower layer solutions clear. Let it stand for 1 h, separate the upper layer, repeat the above operation more than once, and remove the solvent from the supernatant under reduced pressure.

[0020] Further preferably, the specific operation of the crystallization method in step S4 is as follows: Add the triglyceride product after liquid-liquid extraction to petroleum ether with a boiling range of 30 - 60°C at 6 - 15 times the volume, place it at -20°C, and let it stand for 24 h to obtain high-purity UPU-type glycerides.

[0021] The present invention has the following advantages compared with the prior art:

[0022] 1. The present invention uses fatty acids OA, LA and PPP or glycerol as reaction substrates and immobilized enzyme ANL@MARE as a catalyst to prepare UPU-type glycerides (OPO, OPL, LPL). Remove fatty acids, monoglycerides and diglycerides from the reaction product to obtain triglycerides. Utilize the different solubilities of each component in the reaction product in ethanol and water, and use an ethanol / water system to perform liquid-liquid extraction on the reaction product. After adding the reaction product to the ethanol aqueous solution, stratification will occur. The upper layer is mainly a glyceride and ethanol solution with a lower density (the triglyceride has the lowest polarity and the highest solubility in ethanol), and the lower layer is mainly a solution of ethanol-water-fatty acid, monoglyceride and diglyceride with a higher density. After separating and removing the solvent, further remove the triglyceride with a higher melting point by solvent crystallization method to obtain a UPU-type structured lipid with a higher purity.

[0023] 2. The present invention adopts the liquid-liquid extraction method, which can be operated at room temperature, avoiding the deterioration of the quality of oils and fats caused by heating. Moreover, it does not require expensive molecular distillation equipment. The whole process is simple to operate, with low cost and good effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a flow chart of the enzymatic synthesis of UPU-type glycerides of the present invention;

[0025] Figure 2 It is an HPLC-RID diagram for the detection of synthesis products of different oils and fats;

[0026] Figure 3 It is an HPLC-RID diagram for the detection of synthesis products of OPO, OPL, and LPL;

[0027] Figure 4 It is a high-performance liquid chromatography diagram of OPO, OPL, and LPL before and after low-temperature solvent crystallization separation;

[0028] Figure 5 It is a liquid chromatography diagram of purified OPO;

[0029] Figure 6 It is a liquid chromatography diagram of purified OPL;

[0030] Figure 7 It is a liquid chromatography diagram of purified LPL. DETAILED DESCRIPTION OF THE INVENTION

[0031] In order to further explain the present invention, the following specific embodiments are described below.

[0032] Example 1

[0033] The enzymatic preparation of OPO (1,3-dioleoyl-2-palmitoyl glycerol), comprising the following steps:

[0034] (1) Mix oleic acid and PPP in a molar ratio of (5-10):1, place them in a rotary evaporation flask, dissolve at 60 °C, cool down to 45-55 °C, add immobilized lipase accounting for 5-15% of the total mass of the substrates, seal the rotary evaporation flask with plastic wrap, and react on a rotary evaporator at 80-150 r / min for 3-8 h;

[0035] (2) Immediately after the reaction, filter to separate the lipase and the reaction product. The whole process is completed under nitrogen protection and explosion-proof environment. The product needs to be stored under nitrogen and protected from light (adding 0.02% natural vitamin E antioxidant);

[0036] The sample was analyzed by high performance liquid chromatography - refractive index detector (HPLC - RID). It was dissolved in the solution with the mobile phase formula to prepare a 20 mg / ml solution, and passed through a 0.22 - um microporous membrane for testing. The chromatographic column was Thermo Hypersil GOLDAmino (4.6 mm×250 mm 5 um), the mobile phase was n - hexane; isopropanol; formic acid = 21:1:0.004, the column temperature was 30.0 °C, the flow rate was 0.8 ml / min, and the injection volume was 10 ul. The relative content of each component was analyzed by the area normalization method;

[0037] (3) The reaction product was added to a mixture of 85 - 88% ethanol aqueous solution and the sample volume ratio of (6 - 10):1, heated in a water bath at 60 °C until the upper and lower layer solutions were clear, allowed to stand for 1 h, the upper layer was separated by liquid separation, and the above operation was repeated more than once. The supernatant was de - solventized under reduced pressure;

[0038] The sample was dissolved in n - hexane to prepare a 5 mg / ml solution, passed through a 0.22 - um microporous membrane for testing, and analyzed by high performance liquid chromatography - evaporative light scattering detector (HPLC - ELSD). The chromatographic column was Thermo dionex C30 (4.6 mm×250 mm 5 um), the mobile phase A was acetonitrile, the mobile phase B was isopropanol, the column temperature was 30.0 °C, the flow rate was 1 ml / min, the injection volume was 10 ul, and the time program was: 0 - 10 min, the concentration of mobile phase B increased from 5% to 40% and was maintained for 50 min; 60 - 61 min, the concentration of mobile phase B increased from 40% to 75% and was maintained for 5 min. The drift tube temperature of the evaporative light scattering detector was 40 °C, the carrier gas pressure was 3.5 bar, and the photomultiplier gain was 6. The relative content of each component was analyzed by the area normalization method;

[0039] (4) The triglyceride product obtained by removing fatty acids, monoglycerides and diglycerides in the previous step was added with 6 - 15 times the volume of petroleum ether with a boiling range of 30 - 60 °C, placed at - 20 °C, and allowed to stand for 24 h to remove triglycerides with higher melting points such as PPP, OPP, and LPP generated during the reaction, further improving the purity of OPO, OPL, and LPL.

[0040] The content ratio of each component was determined by an evaporative light scattering detector, and the sample purity was detected by high performance liquid chromatography - evaporative light scattering detector (HPLC - ELSD).

[0041] Example 2

[0042] Enzymatic preparation of OPL (1 - oleoyl - 2 - palmitoyl - 3 - linoleoyl glycerol)

[0043] Oleic acid, linoleic acid and PPP were mixed in a molar ratio of (5 - 10):(5 - 10):1, and the reaction time was 2 - 6 h. The rest was the same as that of OPO.

[0044] Example 3

[0045] Enzymatic preparation of LPL (1,3-dilinoleoyl-2-palmitoyl glycerol triester)

[0046] Mix linoleic acid and PPP at a molar ratio of (5-10):1, with a reaction time of 2-6 h, and the rest is the same as OPO.

[0047] As Figure 1 and Figure 2 shown, by HPLC-RID analysis, triglycerides, diglycerides, monoglycerides and fatty acids have good resolution in liquid phase analysis. It can be used for the detection of the contents of fatty acids, monoglycerides, diglycerides and triglycerides. At the end of enzymatic synthesis, the triglyceride contents in the OPO, OPL and LPL synthesis products are 15.4%, 10.3% and 14.2% respectively. The experiment uses an ethanol concentration of 86%, a material-liquid ratio of 1:6, and 2 extraction times. The triglyceride concentration of 96.2% is obtained.

[0048] Further adopt the method of crystallization with petroleum ether solvent to reduce the triglycerides with higher triglyceride saturation. Because the melting points of triglycerides such as PPP, OPP, and LPP with higher saturation than OPO, OPL, and LPL are significantly higher than those of OPO, OPL, and LPL, they can be effectively removed by the solvent crystallization method. After 24 h of crystallization, a large amount of triglycerides with higher melting points crystallize and precipitate at the bottom. Immediately filter to obtain the supernatant. After determination by evaporative light scattering detector, it is found that the content of fats with higher melting points is significantly reduced. As Figure 4 shown, the content of specific structured lipids in triglycerides is further increased. Because the final step is to perform preparative liquid separation on specific structured lipids, it is hoped that the purity of each specific structured lipid can be increased as much as possible by the solvent method in the previous steps of the experiment. Because the quality of the mobile phase of preparative liquid is higher than that of the analytical pure reagent used in the previous solvent method, and its price is correspondingly much higher than that of analytical pure. Improving the purity of the sample as much as possible before using preparative liquid can not only save the preparative separation time, but also save the time of the mobile phase and the time of decompression rotary evaporation to remove the solvent after obtaining the sample in multiples. Figure 4 For the difference in the liquid phase diagrams before and after crystallization separation, it can be seen that the content of oils and fats with higher melting points is significantly reduced.

[0049] Figure 4 Among them: Figure a is the liquid phase diagram of OPO before and after separation, the upper part is before separation and the lower part is after separation; Figure b is the liquid phase diagram of OPL before and after separation; Figure c is the liquid phase diagram of LPL before and after separation.

[0050] Table 1 shows the content ratios of OPO, OPL, and LPL before and after solvent crystallization. After purification, OPO, OPL, and LPL were analyzed by high performance liquid chromatography. The liquid chromatography diagrams are shown in Figures 5 to 7 respectively, and the corresponding characterization substances are shown in Tables 2 to 4 respectively.

[0051] Table 1

[0052] OPO OPL LPL Purity before purification 54.7％ 35.4％ 70.6％ Purity after purification 74.3％ 47.5％ 89.8％

[0053] Table 2

[0054]

[0055]

[0056] Table 3

[0057] Peak # Retention time Area % Area Peak start Peak end 1 22.085 0.9592 355069 21.683 22.875 2 28.128 1.4402 533111 27.733 28.733 3 29.406 16.9491 6273817 28.800 32.717 4 33.394 2.4092 891778 32.850 34.317 5 35.057 47.5429 17598284 34.383 36.508 6 37.020 3.1956 1182857 36.508 37.508 7 40.245 0.5837 216058 39.767 40.833 8 42.509 23.0963 8549236 41.825 43.825 9 45.155 3.8238 1415391 44.550 45.950

[0058] Table 4

[0059] Peak # Retention time Area % Area Peak start Peak end 1 20.984 0.7381 149132 20.733 21.475 2 24.521 1.8432 372413 24.167 24.908 3 28.689 0.3532 71366 28.350 29.225 4 29.991 89.8474 18153333 29.358 35.225 5 35.895 3.8860 785157 35.292 36.775 6 37.872 3.3320 673228 37.317 38.667

[0060] As can be seen from Tables 1 to 4, the solvent crystallization method significantly improves the purity of specific structured lipids.

[0061] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. An enzymatic synthesis method of UPU type glyceride, characterized in that, It includes the following steps: S1. After dissolving the substrate in a rotary flask, cool it down to 45 - 55 °C, then add immobilized lipase accounting for 5 - 15% of the total mass of the substrate, seal the rotary evaporation flask with plastic wrap, and carry out a rotary reaction on a rotary evaporator; S2. Immediately after the reaction ends, filter to separate the lipase from the reaction product; S3. Use an ethanol / water system to carry out liquid - liquid extraction on the reaction product, and then reduce the pressure to remove the solvent from the supernatant after extraction; S4. Use the solvent crystallization method for purification to obtain high - purity UPU - type glycerides.

2. The enzymatic synthesis method of a UPU-type glyceride according to claim 1, characterized in that, The substrate described in step S1 is a mixture of fatty acids and PPP.

3. The enzymatic synthesis method of a UPU type glyceride according to claim 2, characterized in that, The fatty acid is one or more of oleic acid and linoleic acid.

4. The enzymatic synthesis method of a UPU-type glyceride according to claim 1, wherein The immobilized lipase described in step S1 is immobilized lipase ANL@MARE.

5. The enzymatic synthesis method of a UPU-type glyceride according to claim 1, characterized in that, During the rotary reaction in step S1, the rotation speed is 80 - 150 r / min, and the reaction time is 2 - 8 h.

6. The enzymatic synthesis method of a UPU-type glyceride according to claim 1, characterized in that, Both step S1 and step S2 are completed under nitrogen protection and explosion - proof environment. The reaction product needs to be stored under nitrogen and protected from light, and 0.02% natural vitamin E antioxidant is added during storage.

7. The enzymatic synthesis method of a UPU type glyceride according to claim 1, characterized in that, The specific operation of the liquid - liquid extraction described in step S3 is as follows: Add the reaction product to an ethanol aqueous solution with a concentration of 85 - 88%, mix the ethanol aqueous solution and the product in a volume ratio of (6 - 10):1, heat in a water bath at 60 °C to make the upper and lower layer solutions clear, let it stand for 1 h, separate and take the upper layer, repeat the above operation more than once, and then reduce the pressure to remove the solvent from the supernatant.

8. The enzymatic synthesis method of a UPU-type glyceride according to claim 1, characterized in that, The specific operation of the crystallization method described in step S4 is as follows: Add the triglyceride product after liquid - liquid extraction to petroleum ether with a boiling range of 30 - 60 °C in a volume of 6 - 15 times, place it at - 20 °C, and let it stand for 24 h to obtain high - purity UPU - type glycerides.

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