A method for selectively producing 1, 3-diglyceride using immobilized enzyme

Abstract

The application discloses a method for selectively preparing 1,3-diglyceride by using immobilized enzyme, and belongs to the technical field of oil deep processing. The method comprises the following steps: taking oil, glycerol and a composite microsphere immobilized enzyme into a reactor, and obtaining a lipid mixture through reaction; and centrifuging the obtained lipid mixture to remove glycerol and the composite microsphere immobilized enzyme, and obtaining oil rich in diglyceride; wherein the composite microsphere immobilized enzyme is prepared by using shellac, silicon dioxide nanoparticles and lipase as raw materials. The method uses the composite microsphere immobilized enzyme as a catalyst and a reaction system emulsifier, fully mixes the oil and glycerol under the condition that no intense stirring and high-pressure homogenization treatment are needed, improves the content of diglyceride in the product, and the relative content of 1,3-DAG is higher than 40%.

Description

Technical Field

[0001] This invention belongs to the field of deep processing technology of oils and fats, specifically involving a method for preparing 1,3-glycerol diesters with high selectivity using immobilized enzymes. Background Technology

[0002] Diacylglycerol (DAG) is a diester product formed by the esterification of two hydroxyl groups of fatty acids and glycerol. It has various physiological functions, including regulating blood lipids, inhibiting visceral fat accumulation, and suppressing weight gain. Based on the different binding positions of the fatty acid and glycerol hydroxyl groups, diglycerides can be divided into two isomers: 1,3-DAG and 1,2-DAG. Studies have shown that 1,3-DAG, after being ingested by the human body, is hydrolyzed by digestive enzymes and then released as energy through β-oxidation. Unlike triglycerides and 1,2-DAG, it does not produce 2-monoglycerides in the body, which are then re-esterified to form triglycerides and accumulate there.

[0003] Diglyceride oil, as a novel food resource, must be prepared using lipase catalysis. Enzymatic preparation of diglycerides mainly includes hydrolysis, esterification, and glycerol hydrolysis. Esterification involves the direct esterification of free fatty acids with glycerol, a method that more readily produces 1,3-DAG. However, the high viscosity of glycerol and its poor miscibility with free fatty acids result in low catalytic efficiency.

[0004] To improve catalytic efficiency, enhanced emulsification is often employed to increase the reaction contact area. This mainly includes increasing stirring speed, raising reaction temperature, adding organic reagents, and glycerol pre-adsorption. However, excessively high stirring speed or reaction temperature can promote acyl transfer, reducing the proportion of 1,3-DAG in the product. Furthermore, solvent toxicity and operational safety limit its industrial application. For example, glycerol pre-adsorption involves mixing glycerol with adsorbents such as silica gel, diatomaceous earth, or cellulose before the enzymatic reaction, followed by subsequent ultrasonic or fixed-bed reaction column acceleration.

[0005] Therefore, it is still necessary to find a method that can increase the reaction contact area and further improve the catalytic efficiency of lipase and the content of 1,3-DAG in the product. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a method for selectively preparing 1,3-glycerol diesters using immobilized enzymes.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for selectively preparing 1,3-glycerol diesters using immobilized enzymes, comprising, Oil, glycerol, and immobilized enzyme in composite microspheres were placed in a reactor. After the reaction was completed, the glycerol and immobilized enzyme in composite microspheres were removed by centrifugation to obtain oil rich in diglycerides. Among them, the composite microsphere immobilized enzyme was prepared using shellac, silica nanoparticles and lipase as raw materials.

[0010] In a preferred embodiment of the method described in this invention, the molar ratio of the oil and glycerin is 10:1 to 1:20.

[0011] In a preferred embodiment of the method described in this invention, the relative content of 1,3-diglycerides in the oil rich in diglycerides is higher than 40%.

[0012] In a preferred embodiment of the method described in this invention, the amount of the immobilized enzyme in the composite microspheres is 3% to 11% of the total mass of the oil and glycerol, the reaction temperature is 25°C to 75°C, and the reaction time is 30 minutes to 48 hours.

[0013] As a preferred embodiment of the method described in this invention, the method for preparing the composite microsphere immobilized enzyme includes mixing and emulsifying an oil phase containing silica nanoparticles with an aqueous phase containing shellac and lipase, removing ethanol, and drying to obtain a composite microsphere immobilized enzyme loaded with lipase.

[0014] In a preferred embodiment of the method described in this invention, the method for preparing the aqueous phase containing shellac and lipase includes, Dissolve shellac in ethanol to obtain a shellac ethanol solution. The concentration of shellac in ethanol is 100-200 mg / mL. Lipase was then added to the shellac ethanol solution to obtain a shellac enzyme mixed solution, which yielded an aqueous phase.

[0015] In a preferred embodiment of the method described in this invention, the volume ratio of the oil phase to the water phase is 3 to 5:1.

[0016] In a preferred embodiment of the method described in this invention, the silica nanoparticles include hydrophilic silica nanoparticles and hydrophobic silica nanoparticles, and the concentration of the nanoparticles in the oil phase is 10-20 mg / mL.

[0017] In a preferred embodiment of the method described in this invention, the hydrophobic silica nanoparticles account for 100% to 20% of the total mass of nano-silica.

[0018] In a preferred embodiment of the method described in this invention, after centrifugation to remove glycerol and immobilized lipase, the method further includes washing to remove glycerol, and molecular distillation or extraction to remove free fatty acids and monoglycerides from the lipid mixture, resulting in a higher diglyceride content. Beneficial effects of this invention: (1) The present invention uses composite microsphere immobilized enzyme as catalyst and reaction system emulsifier to fully mix oil and glycerol without vigorous stirring and high pressure homogenization, thereby increasing the content of diglycerides in the product, and the diglycerides mainly exist as 1,3-DAG.

[0019] (2) This invention provides a method for selectively preparing 1,3-glycerol diester using immobilized enzymes. Using shellac, silica nanoparticles and lipase as raw materials, composite microsphere immobilized enzymes are prepared by emulsion template method. By changing the surface properties of the composite microsphere immobilized enzymes, it can act as both an emulsifier and a catalyst, thereby increasing the reaction contact area and improving the reaction efficiency. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 These are microstructure diagrams of emulsions formed under different conditions in Example 1 of the present invention; Figure 2 These are scanning electron microscope images of the enzyme immobilized in the composite microspheres formed in Example 1 of this invention; Figure 3 These are the water contact angle test results of the composite microspheres immobilized with enzymes prepared under different conditions in Example 1 of this invention; Figure 4 These are optical microscope images of the enzyme-stabilized water-in-oil Pickering emulsion immobilized on composite microspheres obtained in Example 1 of this invention. Figure 5 This is the high-performance liquid chromatogram of the present invention; Figure 6 This is a graph showing the changes in lipid composition of the product under different substrate molar ratios according to the present invention; Figure 7 This is a graph showing the change in the conversion rate of free fatty acids over time under different substrate molar ratios according to the present invention; Figure 8 This is a graph showing the changes in lipid composition of the product at different reaction temperatures according to the present invention; Figure 9This is a graph showing the change in the conversion rate of free fatty acids over time at different reaction temperatures according to the present invention. Detailed Implementation

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0022] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0023] Following the method of Wang et al. (Wang WF, Li T, Qin XL, et al. Production of lipase SMG1 and its application in synthesizing diacylglycerol [J]. Journal of Molecular Catalysis B: Enzymatic, 2012, 77(6): 87-91.), high-performance liquid chromatography with a differential refractive index detector (HPLC-RID) was used to quantitatively analyze the lipid components in the reaction system. The chromatographic conditions were as follows: Sepax HP-Silica column (4.6 mm × 250 mm × 5 μm), column temperature 30℃; sample concentration 10 mg / mL, injection volume 10 μL; mobile phase ratio of n-hexane:isopropanol:formic acid 15:1:0.003 (v / v / v), flow rate 1 mL / min. Each lipid component was qualitatively analyzed using standards. The sample concentration showed a linear relationship with the peak area, and the relative composition (acylglycerol composition) of each substance was expressed as a percentage by area normalization.

[0024] The formula for calculating fatty acid conversion (FA conversion) is shown below: The formula for calculating the selectivity of 1,3-DAG is shown below: Example 1 (1) Accurately weigh 0.6 g of nano silica (R974 (hydrophobic) and A200 (hydrophilic) silica nanoparticles were purchased from Evonik Industries AG, Germany) and mix them evenly with 40 mL of caprylic / capric glyceride as the oil phase. The proportions of hydrophobic silica nanoparticles were 100% (based on the total mass of silica, the sample number under this condition was M2), 99% (M5), 90% (M6), 80% (M7), 70% (M8), 60% (M9), 40% (M10) and 20% (M11).

[0025] (2) Accurately weigh 8.0g of shellac, add it to 40mL of anhydrous ethanol, and stir thoroughly until the shellac is completely dissolved to obtain a shellac ethanol solution.

[0026] (3) Prepare a 6% concentration of nano-iron oxide dispersion (nano-iron oxide dispersion (10-50 nm, 20% aqueous solution) purchased from Shanghai Aladdin Reagent Co., Ltd.), take 2 mL, add 1 g of free lipase and stir to dissolve (free lipase 5000 MM, sourced from Candida antarctica, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), mix evenly, add 8 mL of shellac ethanol solution, mix evenly to obtain shellac enzyme mixed solution, as the aqueous phase.

[0027] (4) Add the aqueous phase (shellacase mixture) from step (3) to the oil phase (mixture of nano silica and caprylic / capric triglyceride, oil-to-water volume ratio 4:1) from step (1), and emulsify for two minutes using a high-speed shear machine at 12000 rpm to obtain a water-in-oil emulsion. For details of the emulsion microstructure, see [link to relevant documentation]. Figure 1 ; (5) The emulsion was rotary evaporated at 45°C to remove ethanol, and a dispersion of nano-silica / shellac composite microspheres loaded with lipase was obtained. The dispersion was centrifuged and washed with n-hexane multiple times to remove the oil phase. The resulting solid product was then dried to obtain the composite microsphere immobilized enzyme.

[0028] Enzyme (M5) was immobilized on conductive adhesive by composite microspheres obtained with a hydrophobic silica nanoparticle ratio of 99%. The microspheres were then sputtered with gold under vacuum conditions, and the surface morphology of the enzyme-loaded microspheres was observed using a scanning electron microscope. The resulting microscopic images are shown below. Figure 2 As shown in the figure, the surface of the enzyme immobilized by the composite microspheres is covered with silica nanoparticles.

[0029] Hydrophilicity tests of enzymes immobilized on composite microspheres with different surface compositions, such as Figure 3 As shown, after contact angle measurement, the hydrophilicity of the enzyme immobilized in the composite microspheres increases as the proportion of hydrophobic silica nanoparticles decreases.

[0030] (6) Take the prepared immobilized lipase and disperse it evenly in toluene to a concentration of 10% (w / v). Add distilled water at an oil-to-water volume ratio of 2:1, and emulsify by vortexing or ultrasonication to obtain an emulsion. See the optical microscope image for details. Figure 4 .

[0031] The results showed that M2, M5, M6, M7 and M8 formed stable emulsions with smaller particle sizes, while M9 and M10 formed stable emulsions with larger particle sizes. M11 could not form a stable emulsion.

[0032] Example 2 Using caprylic acid and glycerol as raw materials, caprylic acid and glycerol were added to a reaction vessel at a molar ratio of 1:1. Then, 6 wt% (by weight of the caprylic acid and glycerol mixture) of a composite microsphere-immobilized enzyme (M5) was added to each. The mixture was vortexed and reacted at 45°C for 1 hour. The composition of the products was determined by high-performance liquid chromatography (HPLC). The obtained HPLC chromatograms are shown below. Figure 5 As shown in Table 1, the lipid composition is as follows.

[0033] Table 1

[0034] As shown in Table 1, under the conditions of using caprylic acid and glycerol as substrates, reacting at 45℃ for 1 h, and adding M5 at a total substrate mass of 6 wt%, the conversion rate of caprylic acid reached 74.52%, indicating that the composite microsphere immobilized enzyme has high esterification catalytic activity and can effectively promote the esterification reaction of fatty acids and glycerol in a short time.

[0035] In the product composition, the content of 1,3-diglyceride was 27.92%, which was significantly higher than that of 1,2-diglyceride (5.84%). The ratio of the two was approximately 4.78:1, indicating that the immobilized enzyme exhibited strong site selectivity during the catalytic process, which was more conducive to the formation of 1,3-diglyceride.

[0036] Meanwhile, the DAG selectivity was 40.14%, with a 1,3-DAG selectivity of 32.55%, further indicating that the obtained diglyceride product was mainly composed of 1,3-diglycerides, which is the main component of the target product. On the other hand, the triglyceride content in the product was only 0.81%, indicating that the degree of further esterification to generate highly esterified products in the system was low. The immobilized enzyme can inhibit the further conversion of diglycerides to triglycerides to a large extent, thus promoting the accumulation of diglycerides. In addition, the 1-monoglyceride content in the product was significantly higher than that of 2-monoglyceride, which also reflects the high reactivity preference of this immobilized enzyme for the primary hydroxyl site of the glycerol molecule.

[0037] In summary, the M5 composite microsphere immobilized enzyme not only has high catalytic efficiency, but also can increase the production ratio of 1,3-diglycerides and inhibit the formation of triglyceride byproducts, demonstrating good directional synthesis ability of 1,3-diglycerides.

[0038] Example 3 Using oleic acid and glycerol as raw materials, oleic acid and glycerol were added to a reaction vessel at molar ratios of 10:1, 2:1, 1:1, 1:4, 1:10, and 1:20, respectively. Then, 7% of the mass of the oleic acid and glycerol mixture of immobilized enzyme M5 on composite microspheres was added, and the mixture was vortexed. The reaction was carried out at a reaction temperature of 55℃ for 48 hours. Samples were taken at intervals, and the composition of the product was determined by high performance liquid chromatography. The conversion rate of fatty acids and the content of 1,3-DAG in the product were used as indicators to investigate the substrate molar ratio.

[0039] HPLC-RID analysis revealed the following changes in the relative composition of triglycerides (TAG), free fatty acids (FFA), 1,3-DAG, 1,2-DAG, 1-monoglyceride (1-MAG), and 2-monoglyceride (2-MAG) in the product: Figure 6 As shown, the change in oleic acid conversion rate is as follows: Figure 7 As shown.

[0040] from Figure 6 It was found that the substrate molar ratio significantly affected the product composition. With excess oleic acid, the system had a higher proportion of free fatty acids and a lower 1,3-DAG content. As the glycerol ratio increased, the free fatty acid content gradually decreased, while the 1,3-DAG content significantly increased. The highest 1,3-DAG content was observed under the 1:10 and 1:20 conditions, indicating that excess glycerol promotes 1,3-DAG formation and inhibits its further conversion to TAG. Simultaneously, the 1,3-DAG content was higher than 1,2-DAG under all conditions, indicating that the M5 composite microsphere immobilized enzyme exhibits a certain degree of 1,3-position selectivity.

[0041] from Figure 7 It can be seen that the fatty acid conversion rate increases rapidly in the initial stage of the reaction under different substrate molar ratios, and then tends to stabilize. With the increase of glycerol content, the overall fatty acid conversion rate increases, with the highest conversion rates at 1:10 and 1:20 conditions, indicating that excess glycerol can effectively promote the esterification reaction and improve the oleic acid conversion efficiency.

[0042] comprehensive Figure 6 and Figure 7 The results show that a higher glycerol ratio, especially 1:10 and 1:20, is more conducive to achieving high oleic acid conversion and high accumulation of 1,3-DAG.

[0043] Example 4 Oleic acid and glycerol were used as raw materials. Oleic acid and glycerol were added to the reaction vessel at a molar ratio of 1:8. Then, 7% of the mass of the oleic acid and glycerol mixture was added to the composite microsphere immobilized enzyme M5. The mixture was vortexed and reacted at 35℃, 45℃, 65℃ and 75℃ for 24 hours. Samples were taken at intervals, and the composition of the product was detected by high performance liquid chromatography. The fatty acid conversion rate and the content of 1,3-DAG in the product were used as indicators. The reaction temperature was investigated.

[0044] HPLC-RID analysis revealed the following changes in the relative composition of TAG, FFA, 1,3-DAG, 1,2-DAG, 1-MAG, and 2-MAG in the product: Figure 8 As shown, the change in oleic acid conversion rate is as follows: Figure 9 As shown.

[0045] Depend on Figure 8 It is evident that reaction temperature significantly affects product composition. At 35℃, 45℃, 65℃, and 75℃, the content of free fatty acids decreased rapidly with the reaction, while the contents of 1,3-DAG, 1,2-DAG, and monoglycerides gradually increased and stabilized in the later stages. Compared to 35℃, increasing the temperature significantly promoted the formation of 1,3-DAG, with faster formation and higher content at 45℃ and 65℃, indicating that moderate heating is beneficial for the accumulation of the target product. Although the reaction proceeded faster at 75℃, the distribution advantage of the target product did not improve significantly further. Under all temperature conditions, the content of 1,3-DAG was higher than that of 1,2-DAG, and the overall TAG content was lower, indicating that the immobilized enzyme M5 in the composite microspheres has a certain 1,3-position selectivity and can inhibit the further conversion of diglycerides to triglycerides.

[0046] Depend on Figure 9 It can be seen that under all temperature conditions, the oleic acid conversion rate increases rapidly in the initial stage of the reaction, then tends to stabilize, and the rate of increase in oleic acid conversion rate accelerates significantly with increasing temperature. The conversion rate increases more slowly at 35℃, while higher conversion levels can be reached in a shorter time at 45℃, 65℃, and 75℃, indicating that increasing the temperature is beneficial to improving the esterification reaction rate. Combined with... Figure 8 The results show that 45℃ and 65℃ not only have higher oleic acid conversion rates, but are also more conducive to the formation of 1,3-DAG, thus representing the optimal reaction temperature range.

[0047] Example 5 Oleic acid and glycerol were used as raw materials. Oleic acid and glycerol were added to a reaction vessel at a molar ratio of 1:8. Then, 7% (by mass) of the oleic acid and glycerol mixture of immobilized enzyme M5 on composite microspheres was added. The mixture was vortexed and reacted at 75℃ for 4.5 hours. The composition of the product was determined by high-performance liquid chromatography (HPLC). The relative compositions of TAG, FFA, 1,3-DAG, 1,2-DAG, 1-MAG, and 2-MAG in the product, as determined by HPLC-RID, are shown in Table 2.

[0048] After the reaction was completed, the oil phase was collected by centrifugation. 15 mL of 85% ethanol-water solution and 10 mL of n-hexane were added to 10 g of the oil phase, and then the mixture was transferred to a separatory funnel and shaken to separate.

[0049] The mixed solution separated into two layers: an upper hexane phase containing weakly polar DAG, and a lower 85% ethanol-water phase containing strongly polar MAG. The lower layer containing MAG was collected and washed twice with 10 mL of hexane. The solvent was removed by rotary evaporation at 45°C to obtain product 1. The composition of product 1 was determined by high-performance liquid chromatography (HPLC), and the results are shown in Table 2. The upper layer containing DAG was collected, and the solvent was removed by rotary evaporation at 45°C. The composition of product 2 was determined by HPLC, and the results are shown in Table 2.

[0050] Table 2

[0051] As shown in Table 2, the 1,3-DAG content in the esterification reaction product was 39.30%, which was higher than the 18.32% of 1,2-DAG, indicating that the immobilized enzyme M5 in the composite microspheres was conducive to the formation of 1,3-DAG. At the same time, the 1-monoglyceride content was 31.11%, indicating that the reaction product contained a large amount of monoglyceride components. The free fatty acid content was 3.70%, and the triglyceride content was 5.37%, indicating that the oleic acid in the raw material was fully converted, and the content of the by-product triglyceride was low.

[0052] After extraction with an 85% ethanol-water / n-hexane solvent system, the contents of 1,3-DAG and 1,2-DAG in product 2 were 49.33% and 22.62%, respectively, and the total diglyceride content was 71.95%, indicating that the extraction method can effectively enrich the diglyceride components, among which 1,3-DAG is still the main component.

[0053] The above results indicate that using the immobilized enzyme M5 on the composite microspheres to catalyze the esterification reaction of oleic acid and glycerol can yield esterified products rich in 1,3-DAG and monoglycerides. Further solvent extraction using an 85% ethanol-water / n-hexane system effectively separates the monoglyceride and diglyceride components, yielding product 1 rich in monoglycerides and product 2 rich in 1,3-DAG, respectively. This method not only demonstrates good directional synthesis of 1,3-DAG but also excellent product separation.

[0054] Comparative Example 1 Oleic acid and glycerol were used as raw materials. Oleic acid and glycerol were added to reaction vessels at molar ratios of 1:2, 1:4, and 1:8, respectively. Then, 7% (by mass) of free lipase (5000 MM, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) was added to the mixture. The mixture was vortexed and reacted at 35℃ for 24 hours. Samples were taken at intervals, and the composition of the product was determined by high-performance liquid chromatography (HPLC). The fatty acid conversion rate and the 1,3-DAG content of the product were used as indicators. The relative composition changes of TAG, FFA, 1,3-DAG, 1,2-DAG, 1-MAG, and 2-MAG in the product, as well as the oleic acid conversion rate, are shown in Table 3, as determined by HPLC-RID.

[0055] Table 3

[0056] A comparison of the results of Comparative Example 1 and Example 4 shows that, under similar reaction conditions, the composite microsphere immobilized enzyme M5 prepared by the method of this invention exhibits higher catalytic efficiency than the free lipase. Specifically, under different oleic acid to glycerol molar ratios, the overall oleic acid conversion rate is higher when using composite microsphere immobilized enzyme M5, and the 1,3-DAG content in the product is significantly increased. This indicates that preparing the lipase as a composite microsphere immobilized enzyme is beneficial to improving its catalytic activity and its ability to generate the target product 1,3-DAG.

[0057] Comparative Example 2 Using caprylic acid and glycerol as raw materials, caprylic acid and glycerol were added to a reaction vessel at a molar ratio of 1:1. Then, 6 wt% of the commercially immobilized lipase Novozym 435 (based on the mass of the caprylic acid and glycerol mixture) was added to each vessel. The mixture was vortexed and reacted at 45°C for 1 hour. The composition of the products was determined by high-performance liquid chromatography (HPLC). The obtained HPLC chromatograms are shown below. Figure 5 The lipid composition is shown in Table 4.

[0058] Table 4

[0059] A comparison of Comparative Example 2 and Example 2 shows that, under the same reaction conditions, the composite microsphere immobilized enzyme M5 prepared using the method of this invention exhibits better catalytic performance than the commercially available immobilized lipase Novozym 435. Specifically, when catalyzed by the composite microsphere immobilized enzyme M5, the fatty acid conversion rate was 74.52%, and the 1,3-DAG content in the product was 27.92%; while when catalyzed by Novozym 435, the fatty acid conversion rate was 78.81%, and the 1,3-DAG content in the product was 23.97%. These results indicate that although the fatty acid conversion rate is slightly higher under Novozym 435 catalysis, the composite microsphere immobilized enzyme M5 prepared using the method of this invention has a higher 1,3-DAG content in the product, and the 1,3-DAG selectivity is 32.55%, higher than the 25.89% of Novozym 435, indicating that the composite microsphere immobilized enzyme M5 is more conducive to the selective formation of 1,3-DAG. Therefore, compared with the commercially immobilized lipase Novozym 435, the composite microsphere immobilized enzyme prepared in this invention has better 1,3-DAG-directed synthesis ability when catalyzing esterification reactions to prepare diglycerides.

[0060] Comparative Example 3 Oleic acid and glycerol were used as raw materials. Oleic acid and glycerol were added to reaction vessels at molar ratios of 5:1, 2:1, 1:2, and 1:5, respectively. Then, 5% (by mass) of the oleic acid and glycerol mixture of immobilized microsphere enzyme M5 or commercially available immobilized lipase Novozym 435 (purchased from Novozymes (China) Biotechnology Co., Ltd.) was added. The mixture was vortexed and reacted for 4 hours. Samples were taken, and the composition of the product was determined by high-performance liquid chromatography (HPLC). The relative compositional changes of TAG, FFA, 1,3-DAG, 1,2-DAG, and MAG in the product, as determined by HPLC-RID, are shown in Tables 5 and 6.

[0061] The results show that, under the same conditions, the composite microsphere immobilized enzyme prepared in this patent increases the content of diglycerides in the product, and also increases the content of 1,3-diglycerides in the product diglycerides. As the substrate molar ratio changes from 5:1 to 1:5, the proportion of 1,3-diglycerides in the diglyceride product catalyzed by the composite microsphere immobilized enzyme gradually increases from 69.55% to 87.96%, gradually exceeding that of commercially immobilized lipases.

[0062] Table 5

[0063] Table 6

[0064] As shown in Tables 5 and 6, under the same reaction conditions, the diglyceride content in the product catalyzed by the composite microsphere immobilized enzyme M5 prepared in this patent is higher than that of the commercially immobilized lipase Novozym 435, and the proportion of 1,3-DAG in the obtained diglyceride is also higher. As the molar ratio of oleic acid to glycerol changes from 5:1 to 1:5, the proportion of 1,3-DAG in the diglyceride obtained by M5 catalysis increases from 69.55% to 87.96%, significantly higher than the 67.82% of Novozym 435. This indicates that the composite microsphere immobilized enzyme M5 is more conducive to increasing diglyceride yield and promoting the selective formation of 1,3-DAG, especially under conditions of excess glycerol.

[0065] This invention provides a method for the selective preparation of 1,3-DAG using immobilized enzymes. The method uses natural shellac, silica nanoparticles, and lipase as raw materials, and employs an emulsion template method to prepare composite microsphere immobilized enzymes. By regulating the surface properties of the composite microsphere immobilized enzymes, they possess both emulsifying and catalytic functions. They can act as emulsifiers to stabilize the reaction system and as catalysts to catalyze the substrate reaction, thereby increasing the interfacial contact area, improving reaction efficiency, and facilitating the selective formation of 1,3-DAG.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for selectively preparing 1,3-glycerol diesters using immobilized enzymes, characterized in that: include, Oil, glycerol, and immobilized enzyme in composite microspheres were placed in a reactor. After the reaction was completed, the glycerol and immobilized enzyme in composite microspheres were removed by centrifugation to obtain oil rich in diglycerides. Among them, the composite microsphere immobilized enzyme was prepared using shellac, silica nanoparticles and lipase as raw materials.

2. The method as described in claim 1, characterized in that: The molar ratio of the oil and glycerin is 10:1 to 1:

20.

3. The method as described in claim 2, characterized in that: The relative content of 1,3-diglycerides in the oil rich in diglycerides is higher than 40%.

4. The method as described in claims 1 to 3, characterized in that: The amount of enzyme added to the composite microsphere immobilized is 3% to 11% of the total mass of oil and glycerol, the reaction temperature is 25℃ to 75℃, and the reaction time is 30 minutes to 48 hours.

5. The method as described in claim 4, characterized in that: The preparation method of the composite microsphere immobilized enzyme includes mixing and emulsifying an oil phase containing silica nanoparticles with an aqueous phase containing shellac and lipase, removing ethanol, and drying to obtain a composite microsphere immobilized enzyme loaded with lipase.

6. The method as described in claim 5, characterized in that: The method for preparing the aqueous phase containing shellac and lipase includes, Dissolve shellac in ethanol to obtain a shellac ethanol solution. The concentration of shellac in ethanol is 100-200 mg / mL. Lipase was then added to the shellac ethanol solution to obtain a shellac enzyme mixed solution, which yielded an aqueous phase.

7. The method as described in claim 5 or 6, characterized in that: The volume ratio of the oil phase to the water phase is 3 to 5:

1.

8. The method as described in claim 7, characterized in that: The silica nanoparticles include hydrophilic silica nanoparticles and hydrophobic silica nanoparticles, and the concentration of the nanoparticles in the oil phase is 10-20 mg / mL.

9. The method as described in claim 8, characterized in that: The hydrophobic silica nanoparticles account for 100% to 20% of the total mass of the nano-silica.

10. The method as described in claim 1 or 9, characterized in that: After centrifugation to remove glycerol and immobilized lipase, the process further includes washing to remove glycerol, and molecular distillation or extraction to remove free fatty acids and monoglycerides from the lipid mixture, resulting in a higher diglyceride content.

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