A method for the preparation of a diglyceride

Abstract

The present application belongs to the field of light industry grease, and discloses a preparation method of diglyceride. The present application carries out pretreatment on the immobilized enzyme carrier, mixes free lipase and glycerol according to a certain proportion, adds the immobilized carrier into the mixed solution of the lipase and glycerol, and carries out immobilization through the adsorption method, so that the immobilized enzyme which has catalytic characteristics and can be used as a reaction substrate is prepared. The obtained immobilized enzyme can be used for the continuous production of diglyceride in a packed bed reactor. Compared with the traditional enzyme method for catalyzing glycerolysis reaction to prepare diglyceride, the immobilized enzyme obtained by the present application not only has catalytic effect, but also has enough glycerol adsorbed on the immobilized enzyme which can be used as a substrate to carry out glycerolysis reaction, so that the reaction efficiency is improved. Not only the problems of difficult recovery and poor reusability of free enzyme are solved, but also the problem of difficult continuous production due to the large viscosity of glycerol in production is solved.

Description

Technical Field

[0001] This invention belongs to the field of light industrial oils and fats, and specifically relates to a method for preparing diglycerides. Background Technology

[0002] Diacylglycerols are structural lipids in which one fatty acid is replaced by a hydroxyl group in a class of triglycerides. After ingestion, they are difficult to resynthesize into neutral fats in the human body and are ultimately released as CO2 and H2O, without causing fat accumulation, thus possessing high nutritional value. However, natural oils contain relatively little diglyceride. Current main methods for preparing diglycerides include chemical and enzymatic methods. Enzymatic catalysis, with its advantages of mild reaction conditions, high selectivity, environmental friendliness, and low energy consumption, has attracted increasing attention. However, in the process of lipase-catalyzed diglyceride production, the inability to reuse free enzymes leads to high costs, and water-soluble enzymes are difficult to separate from the reaction system, hindering control and continuous production. Therefore, immobilizing free lipases can improve enzyme stability and reusability.

[0003] Enzymatic catalytic glycerol hydrolysis is suitable for preparing diglycerides due to its low raw material cost, simple product processing, and minimal environmental pollution. Traditional enzymatic catalytic glycerol hydrolysis requires excess glycerol in the reaction system and has a high substrate viscosity, often requiring stirred reactors. This catalytic method causes significant mechanical damage to the enzyme, resulting in low enzyme reuse efficiency and difficulty in continuous production, leading to increased production costs. Therefore, this method has significant limitations in its application. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a method for preparing diglycerides. This invention pretreats an immobilized enzyme carrier, mixes free lipase with glycerol in a specific ratio, adds the immobilized carrier to the mixed solution of lipase and glycerol, and immobilizes the enzyme through adsorption. This prepares an immobilized enzyme that possesses both catalytic properties and can serve as a reaction substrate. The resulting immobilized enzyme can be used in a packed-bed reactor for continuous production of diglycerides. Compared to traditional enzymatic catalytic glycerol hydrolysis for diglyceride preparation, the immobilized enzyme obtained by this invention not only possesses catalytic activity but also adsorbs sufficient glycerol for use as a substrate in the glycerol hydrolysis reaction, thus improving reaction efficiency. This method not only solves the problems of difficulty in recovering and reusability of free enzymes but also addresses the issue of high glycerol viscosity during production, which hinders continuous production.

[0005] The objective of this invention is achieved through the following solution:

[0006] A method for preparing diglyceride includes the following steps:

[0007] (1) Mixing glycerol with free lipase: Mix lipase with glycerol to completely dissolve lipase in glycerol to obtain lipase-glycerol solution;

[0008] (2) Preparation of immobilized enzyme: Add resin carrier to lipase-glycerol solution, filter after adsorption, and then air dry at room temperature to obtain immobilized enzyme that has both catalytic activity and can be used as reaction substrate.

[0009] (3) The immobilized enzyme, which has both catalytic activity and can be used as a reaction substrate, is placed in the reaction column of a packed bed reactor. After heating the packed bed, reactants are added to the reaction column to initiate the enzyme-catalyzed reaction. The crude sample flowing out from the bottom of the reaction column is collected. Subsequently, free fatty acids and monoglycerides are removed by molecular distillation equipment to obtain oil with diglycerides as the main component.

[0010] The amount of lipase and glycerol used in step (1) is such that the volume fraction of lipase in the lipase-glycerol solution is 2.5-5%.

[0011] The enzyme in step (1) is a lipase that can catalyze glycerol hydrolysis, preferably Novozymes CALB enzyme.

[0012] To enhance the curing effect, the resin carrier mentioned in step (2) is preferably pretreated by the following steps before being added: Weigh the resin carrier, soak it in 95% ethanol for 24 hours, filter and wash until there is no alcohol odor, then soak it in 5% hydrochloric acid and 5% sodium hydroxide for 3 hours in sequence, wash it with water until neutral, and filter it dry for later use.

[0013] The resin carrier in step (2) can be at least one of macroporous resin, ion exchange resin, and epoxy resin, preferably at least one of LXTE-1000, LXTE-600, LXTE-603, LXTE-604, LX-1000EP and R103SX resin, and more preferably LXTE-1000 macroporous adsorption resin.

[0014] In step (2), the amounts of lipase-glycerol solution and resin carrier are such that the resin is always immersed in the lipase-glycerol solution; preferably, 10-20 mL of lipase-glycerol solution is added for every 1 g of resin.

[0015] The adsorption in step (2) is carried out on a constant temperature shaker at a temperature of 20-40°C, preferably 30°C, with a shaker speed of 100-300 r / min, preferably 150 r / min, and a reaction time of 0.5-5 h, preferably 2 h.

[0016] Step (3) specifically includes the following steps: a continuous packed bed reactor is used for the reaction. The immobilized enzyme, which has both catalytic activity and can be used as a reaction substrate, is placed in the reaction column of the packed bed reactor with a heat jacket. The reactants are placed in a substrate tank, and the substrate tank is connected to the reaction column by a constant flow pump. The reaction column is heated using the heat jacket. When the temperature reaches the reaction temperature, the constant flow pump connected to the reaction column is turned on, and the reaction flow rate is adjusted so that the reactants continuously enter the reaction column from the top of the reaction column to initiate the enzyme catalytic reaction. At the same time as the constant flow pump is turned on, the bottom of the reaction column is in an open state, and the outflow rate is consistent with the inflow rate of the reactants. After the reaction is completed, the constant flow pump is turned off, the heating is stopped, and all the crude samples flowing out from the bottom of the reaction column are collected. Subsequently, free fatty acids and monoglycerides are removed by molecular distillation equipment to obtain an oil with diglycerides as the main component.

[0017] The reactant in step (3) is triglyceride, preferably soybean oil or flaxseed oil.

[0018] The amounts of immobilized enzyme and reactant triglycerides used in step (3) satisfy the following: the mass of immobilized enzyme is 2%-6% of the mass of reactant triglycerides, preferably 4%.

[0019] The heating mentioned in step (3) refers to heating to 60-100℃, preferably 80℃;

[0020] The reaction flow rate described in step (3) is 1.0-3.0 mL / min.

[0021] The enzyme-catalyzed reaction time in step (3) is 10-30 min, preferably 20 min, starting from when the reactant triglyceride enters the top of the reaction column.

[0022] The resulting oil, which is mainly composed of diglycerides, contains more than 60% diglycerides.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] (1) In this invention, LXTE-1000 resin is used as the lipase immobilization carrier. Free lipase and glycerol are simultaneously immobilized on the carrier by adsorption, so that the immobilized enzyme prepared has both catalytic activity and can be used as a substrate for catalytic glycerol hydrolysis reaction, which increases the catalytic stability of the enzyme and can be recycled and reused many times, reducing production costs.

[0025] (2) The prepared immobilized enzyme can be used for continuous production of diglycerides in a packed bed reactor. This not only solves the problem of the difficulty in recycling free enzymes and increases the reusability of immobilized enzymes, but also solves the problem of the difficulty in continuous production of two-phase reactions. Attached Figure Description

[0026] Figure 1 The graph shows the enzyme activity and the content of diglycerides produced by different modified resin carriers.

[0027] Figure 2 This is the gas phase spectrum of glycerides from Example 1. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0029] Unless otherwise specified, all reagents used in the examples are commercially available.

[0030] The lipases used in the examples and comparative examples were purchased from Novozymes.

[0031] Enzyme activity assay:

[0032] Solution preparation: (1) Polyvinyl alcohol olive oil emulsion: Weigh 40 g of polyvinyl alcohol, add about 800 mL of distilled water, heat in a boiling water bath and stir constantly until the polyvinyl alcohol is completely dissolved. After cooling, make up to 1000 mL. Filter with clean double-layer gauze and keep the filtrate for later use. Take 150 mL of 4% polyvinyl alcohol solution, add 50 mL of olive oil, and treat with a high-speed homogenizer for 6 min (two treatments, 5 min apart, 3 min each time) to obtain a milky white polyvinyl alcohol olive oil emulsion. Prepare fresh before use.

[0033] (2) Phosphate buffer (100mM pH7.0): Prepare 1mol / L standard solutions of disodium hydrogen phosphate and sodium dihydrogen phosphate, mix them in a volume ratio of 57.7:43.3, and adjust the pH of the solution to 7.0.

[0034] (3) 0.05 mol / L sodium hydroxide standard solution: Weigh 2 g of sodium hydroxide, dissolve it in a small amount of water, and make up to 1 L.

[0035] (4) 1% phenolphthalein solution: Weigh 1g of phenolphthalein, dissolve it in 95% ethanol, and then make up to 100mL with 95% ethanol.

[0036] Determination of lipase activity by alkaline titration: Take two 50mL Erlenmeyer flasks with stoppers. Weigh 4mL of olive oil emulsion and 5mL of phosphate buffer into each flask (A) and (B), respectively. Add 15mL of 95% ethanol to flask A and preheat in a 40℃ water bath for 5min. Then add 1mL or 1g of diluted enzyme solution (100mM pH 7.0 phosphate buffer) to each flask (A and B). React at 200rpm for 15min. Immediately add 15mL of 95% ethanol to flask B to terminate the reaction. After the reaction, add 2 drops of 1% phenolphthalein solution and titrate with 0.05mol / L NaOH standard solution until the solution changes from colorless to pink and does not fade within 30s. Calculate the lipase activity units based on the volume of NaOH consumed. Repeat each experiment three times. Enzyme activity is defined as the amount of enzyme required per minute to catalyze the hydrolysis of a substrate into 1 μmol of fatty acid under specific reaction conditions. This unit of enzyme activity is expressed in U / mL (U / g). Enzyme activity is calculated using the following formula:

[0037]

[0038] Where: X is the specific enzyme activity, U / mg; V1: the volume of sodium hydroxide consumed by the experimental group (bottle B), mL; V0: the volume of sodium hydroxide consumed by the control group (bottle A), mL; c: sodium hydroxide concentration, mol / L; n: the dilution factor of the sample; 0.05: the conversion factor for the standard volume fraction of sodium hydroxide.

[0039] Immobilization efficiency (IE) determination: The immobilization efficiency is estimated by measuring the enzyme activity of the free enzyme in phosphate buffer solution before and after immobilization. The immobilization efficiency (IE) is calculated as follows:

[0040]

[0041] Where E0 is the initial lipase activity (U / ml), V0 is the initial volume of the enzyme solution (ml), and E f It is the lipase activity (U / ml) in the filtrate, V f This is the filter media volume (ml).

[0042] Gas chromatography determination of glycerol ester content: 50 mg of glycerol ester sample was dissolved completely in 5.0 mL of n-hexane. The solution was filtered through a filter membrane into a sample vial. After sample preparation, the sample was analyzed by gas chromatography (GC). GC conditions: DB-1ht capillary column (15 m × 0.25 mm × 0.1 μm), injection volume 1 μL, split ratio 40:1; column oven temperature 50 °C, flame ionization detector temperature 380 °C; carrier gas: N2, flow rate 4.17 mL / min. Staged temperature program detection: initial temperature 50 °C, held for 1 min, then increased at 50 °C / min to 100 °C, 80 °C / min to 220 °C, 30 °C / min to 290 °C, 50 °C / min to 330 °C, held for 2 min, and finally increased at 50 °C / min to 380 °C and held for 3 min. The area normalization method was used to quantitatively analyze free fatty acids, monoglycerides, diglycerides and triglycerides.

[0043] Example 1

[0044] (1) Weigh 2g of LXTE-1000 resin into a beaker, soak it in 95% ethanol for 24h, filter and wash until there is no alcohol smell, then soak it in 5% hydrochloric acid and 5% sodium hydroxide for 3h, wash it with deionized water until neutral, and filter it for later use.

[0045] (2) Add 1 mL of Novezyme CALB lipase solution to a 150 mL Erlenmeyer flask, then add 39 mL of glycerol and mix well to completely dissolve the lipase in the glycerol to obtain a lipase solution with a concentration of 2.5%.

[0046] (3) Take 40 mL of the lipase solution obtained in step (2) above into an Erlenmeyer flask, add 2 g of resin carrier, shake at 150 r / min on a constant temperature shaker at 30℃ for 2 h, then transfer it to a beaker, filter, air dry at room temperature, and determine its enzyme activity and immobilization rate.

[0047] (4) Place 85g of the immobilized enzyme prepared by the above method in a glass reaction column with a heat jacket to obtain an enzyme packed bed reactor. Connect the packed bed reaction column to the circulating water pipeline, turn on the constant temperature water bath, and set the temperature to 80℃. Take soybean oil triglycerides (purchased from Yihai Kerry Arawana Grain & Oil Food Co., Ltd.) and place them in the substrate tank. Connect the substrate tank, packed column, and product tank in sequence. After the heat jacket temperature reaches the set temperature, turn on the constant flow pump and set the flow rate to 1.0mL / min, so that the soybean oil triglycerides in the substrate tank continuously enter from the top of the reaction column, thereby initiating the glycerolysis reaction of triglycerides and glycerol under the action of enzyme catalysis. At the same time as the constant flow pump is turned on, the bottom of the reaction column is in the open state, and the outflow rate is the same as the flow rate of the reactants entering the reaction column. Start timing from the time the reactant triglycerides enter the top of the reaction column. After 20min of reaction, turn off the constant flow pump, stop the heating of the circulating water bath, collect the crude glycerolysis product in the product tank, and determine the mass fraction of glycerides by gas chromatography.

[0048] The immobilized enzyme had a specific activity of 283.33 U / g and an immobilization efficiency of 95.22%. Under these conditions, the packed-bed enzyme reactor catalyzed the glycerol hydrolysis of soybean oil, yielding 5.17% free fatty acids, 6.67% monoglycerides, 63.85% diglycerides, and 24.31% triglycerides, with a diglyceride to triglyceride ratio of 2.63. The free fatty acids and monoglycerides were subsequently removed by molecular distillation. The gas phase results of the glycerides after molecular distillation are shown below. Figure 2 As shown, after area normalization of each component, the mass fractions of free fatty acids (FFA) were 0.17%, monoglycerides (MAG) were 0.37%, diglycerides (DAG) were 69.58%, and triglycerides (TAG) were 29.88%. The diglyceride content can reach more than 60% of the total oil content, which can prepare diglycerides relatively efficiently.

[0049] Example 2

[0050] This embodiment is the same as that of Embodiment 1 except for the following technical features: In step (2), the amount of Novezyme CALB lipase added is 2 mL, the amount of glycerol added is 48 mL, and the mass fraction of lipase is 4%.

[0051] The immobilized enzyme had a specific activity of 263.72 U / g and an immobilization efficiency of 93.21%. Under these conditions, the packed-bed enzyme reactor catalyzed the glycerol hydrolysis of soybean oil, yielding 4.21% free fatty acids, 5.70% monoglycerides, 62.50% diglycerides, and 27.59% triglycerides. The mass ratio of diglycerides to triglycerides was 2.27. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.09% free fatty acids, 0.31% monoglycerides, 67.38% diglycerides, and 32.22% triglycerides. Although the diglyceride content was not as high as in Example 1, it still reached over 60% of the total oil content, demonstrating a relatively efficient method for preparing diglycerides.

[0052] Example 3

[0053] This embodiment is the same as that of Embodiment 1 except for the following technical features: In step (2), the amount of Novezyme CALB lipase added is 2 mL, the amount of glycerol added is 38 mL, and the mass fraction of lipase is 5%.

[0054] The immobilized enzyme had a specific activity of 239.74 U / g and an immobilization efficiency of 90.02%. Under these conditions, the glycerol hydrolysis of soybean oil catalyzed by the packed-bed enzyme reactor yielded 3.49% free fatty acids, 3.34% monoglycerides, 58.81% diglycerides, and 34.36% triglycerides. The mass ratio of diglycerides to triglycerides was 1.71. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.11% free fatty acids, 0.20% monoglycerides, 62.02% diglycerides, and 37.67% triglycerides. Although the diglyceride content was not as high as in Example 1, it still reached over 60% of the total oil content after fractional distillation, demonstrating a relatively efficient method for preparing diglycerides.

[0055] Example 4

[0056] This embodiment is the same as embodiment 1 except for the following technical features: in step (4), the constant flow pump flow rate is set to 2.0 mL / min.

[0057] The immobilized enzyme had a specific activity of 283.33 U / g and an immobilization efficiency of 95.22%. Under these conditions, the packed-bed enzyme reactor catalyzed the glycerol hydrolysis of soybean oil, yielding 3.85% free fatty acids, 2.50% monoglycerides, 58.91% diglycerides, and 34.74% triglycerides. The mass ratio of diglycerides to triglycerides was 1.70. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.02% free fatty acids, 0.04% monoglycerides, 61.90% diglycerides, and 38.04% triglycerides. The diglyceride content reached over 60% of the total oil content, demonstrating a relatively efficient method for diglyceride preparation.

[0058] Example 5

[0059] This embodiment is the same as embodiment 1 except for the following technical features: in step (4), the constant flow pump flow rate is set to 3.0 mL / min.

[0060] The immobilized enzyme had a specific activity of 283.33 U / g and an immobilization efficiency of 95.22%. Under these conditions, the packed-bed enzyme reactor catalyzed the glycerol hydrolysis of soybean oil, yielding 3.79% free fatty acids, 3.51% monoglycerides, 58.94% diglycerides, and 33.76% triglycerides. The mass ratio of diglycerides to triglycerides was 1.75. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.08% free fatty acids, 0.16% monoglycerides, 61.58% diglycerides, and 38.18% triglycerides. The diglyceride content reached over 60% of the total oil content, demonstrating a relatively efficient method for diglyceride preparation.

[0061] Example 6

[0062] This embodiment is the same as embodiment 1 except for the following technical features: the resin in step (1) was not pretreated.

[0063] The immobilized enzyme had a specific activity of 253.62 U / g and an immobilization efficiency of 92.23%. Under these conditions, the packed-bed enzyme reactor catalyzed the glycerol hydrolysis of soybean oil, yielding 5.21% free fatty acids, 5.89% monoglycerides, 60.50% diglycerides, and 28.40% triglycerides. The mass ratio of diglycerides to triglycerides was 2.13. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.14% free fatty acids, 0.27% monoglycerides, 66.05% diglycerides, and 33.54% triglycerides. Although the diglyceride content was not as high as in Example 1, it still reached over 60% of the total oil content, demonstrating a relatively efficient method for preparing diglycerides.

[0064] Comparative Example 1

[0065] This comparative example is the same as Example 1 except for the following technical features: in step (4), the constant flow pump flow rate is set to 4.0 mL / min.

[0066] The immobilized enzyme had a specific activity of 283.33 U / g and an immobilization efficiency of 95.22%. Under these conditions, the glycerol hydrolysis of soybean oil catalyzed in the packed-bed enzyme reactor yielded 5.85% free fatty acids, 7.50% monoglycerides, 31.91% diglycerides, and 54.74% triglycerides. The mass ratio of diglycerides to triglycerides was 0.58. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.20% free fatty acids, 0.41% monoglycerides, 35.83% diglycerides, and 63.56% triglycerides. The diglyceride content did not reach over 60% of the total oil content because the excessively high flow rate led to uneven contact between the soybean oil triglycerides and the immobilized enzyme, resulting in a lower yield.

[0067] Comparative Example 2

[0068] Except for the following technical features, this comparative example is the same as implementation 1: In step (1), 2g of each of the following carriers (Xi'an Lanxiao Technology New Material Co., Ltd.) LXTE-1000, LXTE-600, LXTE-603, LXTE-604, LX-1000EP (Yuanye Biotechnology Co., Ltd.), and R103SX (Guangzhou Tianrui New Material Technology Co., Ltd.) were selected, soaked in 95% ethanol, filtered and washed until no alcohol smell was found, and then soaked in 5% hydrochloric acid and 5% sodium hydroxide for 3 hours in sequence. They were then washed with deionized water until neutral and filtered dry for later use.

[0069] The results after immobilization of each resin are shown in the figure. Figure 1Of the six resins used for immobilization, LXTE-1000 showed the highest specific enzyme activity. Testing revealed that the immobilized lipase on LXTE-1000 resin catalyzed glycerol hydrolysis to yield 63.85% diglyceride by mass, while LXTE-600 resin yielded 24.32%, LXTE-603 resin 21.85%, LXTE-604 resin 29.82%, LX-1000EP resin 32.98%, and R103SX resin 21.57%.

[0070] Comparative Example 3

[0071] (1) Weigh 2g of LXTE-1000 (Xi'an Lanxiao Technology Co., Ltd.) resin, soak it in 95% ethanol, filter and wash until there is no alcohol smell, then soak it in 5% hydrochloric acid and 5% sodium hydroxide for 3 hours in sequence, wash it with deionized water until neutral, and filter it dry for later use.

[0072] (2) Add 2g of resin carrier, 39mL of 50mM, pH7.0 phosphate buffer solution and 1mL of lipase solution to a 150mL Erlenmeyer flask, mix well, seal the flask, and place it on a shaker at 30℃ and shake at 150r / min for 2h. After the adsorption is complete, take out the fixed enzyme, wash it 5 times with the same buffer solution, 30mL each time, and collect the carrier.

[0073] (3) Place 85g of the immobilized enzyme prepared by the above method in a glass reaction column with a heat jacket to obtain an enzyme packed bed reactor. Connect the packed bed reaction column to the circulating water pipeline, turn on the constant temperature water bath, and set the temperature to 80℃. Take soybean oil triglycerides and glycerol at a molar ratio of 1:10 and place them in the substrate tank. Mix them evenly by stirring. Connect the substrate tank, reaction column, and product tank in sequence. After the heat jacket temperature reaches the set temperature, turn on the constant flow pump and set the flow rate to 1.0mL / min so that soybean oil triglycerides and glycerol in the substrate tank continuously enter from the top of the reaction column, thereby initiating the glycerolysis reaction of triglycerides and glycerol under the action of enzyme catalysis. At the same time as the constant flow pump is turned on, the bottom of the reaction column is in the open state, and the outflow rate is consistent with the flow rate of the reactants entering the reaction column. Timing begins when the reactants triglycerides and glycerol enter the top of the reaction column. After 20 minutes of reaction, the constant flow pump is turned off, the circulating water bath heating is stopped, and the crude glycerol product in the product tank is collected. A small amount of the product is placed in a centrifuge tube and centrifuged at 10,000 r / min for 10 minutes. The upper oil layer is analyzed by gas chromatography to determine the mass fraction of glycerides.

[0074] The immobilized enzyme had a specific activity of 273.65 U / g and an immobilization efficiency of 94.32%. Under these conditions, the glycerol hydrolysis of soybean oil catalyzed in the packed-bed enzyme reactor yielded 4.85% free fatty acids, 9.50% monoglycerides, 21.31% diglycerides, and 64.34% triglycerides. The mass ratio of diglycerides to triglycerides was 0.33. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.12% free fatty acids, 0.39% monoglycerides, 23.88% diglycerides, and 75.61% triglycerides. The diglyceride content did not reach over 60% of the total oil content because glycerol and oil are immiscible and separate in the packed bed, leading to a reduced yield.

[0075] Comparative Example 4

[0076] 85g of Novezyme 435 immobilized enzyme was placed in a heat-shrinkable glass reaction column to obtain an enzyme-packed bed reactor. The packed bed reaction column was connected to a circulating water pipeline, and a constant temperature water bath was turned on and the temperature was set to 80℃. Soybean oil triglycerides and glycerol were placed in a substrate tank at a molar ratio of 1:10 and mixed evenly by stirring. The substrate tank, reaction column, and product tank were connected in sequence. After the heat-shrinkable tank reached the set temperature, a constant flow pump was turned on and the flow rate was set to 1.0mL / min, so that soybean oil triglycerides and glycerol in the substrate tank continuously entered from the top of the reaction column, thereby initiating the glycerolysis reaction of triglycerides and glycerol under enzyme catalysis. At the same time as the constant flow pump was turned on, the bottom of the reaction column was in the open state, and the outflow rate was the same as the flow rate of the reactants entering the reaction column. Timing begins when the reactants triglycerides and glycerol enter the top of the reaction column. After 20 minutes of reaction, the constant flow pump is turned off, the circulating water bath heating is stopped, and the crude glycerol product in the product tank is collected. A small amount of the product is placed in a centrifuge tube and centrifuged at 10,000 r / min for 10 minutes. The upper oil layer is analyzed by gas chromatography to determine the mass fraction of glycerides.

[0077] Testing revealed that under these conditions, the glycerol hydrolysis of soybean oil catalyzed in a packed-bed enzyme reactor yielded 5.82% free fatty acids, 9.96% monoglycerides, 25.77% diglycerides, and 58.45% triglycerides. The mass ratio of diglycerides to triglycerides was 0.44. Subsequent molecular distillation removed the free fatty acids and monoglycerides, resulting in 0.04% free fatty acids, 0.29% monoglycerides, 29.59% diglycerides, and 70.08% triglycerides. The diglyceride content did not reach over 60% of the total oil content because glycerol and oil are immiscible and separate in the packed bed, leading to a reduced yield.

[0078] This invention immobilizes glycerol and lipase by mixing and immobilizing the lipase, resulting in an immobilized lipase with enhanced stability and higher catalytic activity. This immobilized enzyme can be recovered, reused, and replenished with glycerol, solving the problems of difficult enzyme recovery and limited reuse in the glycerolysis reaction for diglyceride preparation. This significantly improves enzyme stability and reduces industrial production costs. Furthermore, the glycerol loading on the immobilized enzyme allows it to serve as a substrate for the glycerolysis reaction, enabling continuous, long-term diglyceride preparation in a packed-bed reactor. This eliminates the mechanical damage to the immobilized enzyme caused by stirring due to phase incompatibility in the glycerolysis reaction, reducing enzyme activity loss. It was also found that appropriate lipase concentration and packed-bed reaction time can regulate the enzyme's catalytic effect, control the progress of the glycerolysis reaction, and, under suitable conditions, bring the glycerolysis reaction to an equilibrium state, yielding the highest content of diglyceride.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing diglycerides, characterized in that... Includes the following steps: (1) Mixing glycerol with free lipase: Mix lipase with glycerol to completely dissolve lipase in glycerol to obtain lipase-glycerol solution; (2) Preparation of immobilized enzyme: Add resin carrier to lipase-glycerol solution, filter after adsorption, and then air dry at room temperature to obtain immobilized enzyme that has both catalytic activity and can be used as reaction substrate; (3) The immobilized enzyme, which has both catalytic activity and serves as a reaction substrate, is placed in the reaction column of a packed bed reactor. After heating the packed bed, reactants are added to the reaction column to initiate the enzyme-catalyzed reaction. The crude sample flowing out from the bottom of the reaction column is collected and then free fatty acids and monoglycerides are removed by molecular distillation equipment to obtain oil with diglycerides as the main component. The resin carrier in step (2) is LXTE-1000.

2. The method for preparing diglyceride according to claim 1, characterized in that: The amount of lipase and glycerol used in step (1) is such that the volume fraction of lipase in the lipase-glycerol solution is 2.5-5%; The lipase mentioned in step (1) is a lipase that catalyzes glycerol hydrolysis.

3. The method for preparing diglyceride according to claim 2, characterized in that: The lipase mentioned in step (1) is Novozymes CALB enzyme.

4. The method for preparing diglyceride according to claim 1, characterized in that: Before adding the resin carrier in step (2), it is pretreated by the following steps: weigh the resin carrier, soak it in 95% ethanol for 24 hours, filter and wash until there is no alcohol smell, then soak it in 5% hydrochloric acid and 5% sodium hydroxide for 3 hours in sequence, wash it with water until neutral, and filter it dry for later use.

5. The method for preparing diglyceride according to claim 1, characterized in that: In step (2), the amounts of lipase-glycerol solution and resin carrier are such that the resin is always immersed in the lipase-glycerol solution.

6. The method for preparing diglyceride according to claim 1, characterized in that: In step (2), the amounts of lipase-glycerol solution and resin carrier are such that 10-20 mL of lipase-glycerol solution is added for every 1 g of resin.

7. The method for preparing diglyceride according to claim 1, characterized in that: The adsorption in step (2) is carried out on a constant temperature shaker at a temperature of 20-40°C, a shaking speed of 100-300 r / min, and a reaction time of 0.5-5 h.

8. The method for preparing diglyceride according to claim 1, characterized in that: Step (3) specifically includes the following steps: a continuous packed bed reactor is used for the reaction. The immobilized enzyme, which has both catalytic activity and can be used as a reaction substrate, is placed in the reaction column of the packed bed reactor with a heat jacket. The reactants are placed in a substrate tank, and the substrate tank is connected to the reaction column by a constant flow pump. The reaction column is heated by the heat jacket. When the temperature reaches the reaction temperature, the constant flow pump connected to the reaction column is turned on, and the reaction flow rate is adjusted so that the reactants continuously enter the reaction column from the top of the reaction column to initiate the enzyme catalytic reaction. At the same time as the constant flow pump is turned on, the bottom of the reaction column is in the open state, and the outflow rate is consistent with the inflow rate of the reactants. After the reaction is completed, the constant flow pump is turned off, the heating is stopped, and all the crude samples flowing out from the bottom of the reaction column are collected. Subsequently, free fatty acids and monoglycerides are removed by molecular distillation equipment to obtain an oil with diglycerides as the main component.

9. The method for preparing diglyceride according to claim 8, characterized in that: The reactant mentioned in step (3) is triglyceride.

10. The method for preparing diglyceride according to claim 9, characterized in that: The reactants mentioned in step (3) are soybean oil or flaxseed oil.

11. The method for preparing diglyceride according to claim 8, characterized in that: The amounts of immobilized enzyme and reactant triglycerides used in step (3) are such that the mass of the immobilized enzyme is 2%-6% of the mass of the reactant triglycerides.

12. The method for preparing diglyceride according to claim 8, characterized in that: The heating mentioned in step (3) refers to heating to 60-100℃; The reaction flow rate described in step (3) is 1.0-3.0 mL / min; The enzyme-catalyzed reaction in step (3) takes 10-30 minutes, starting from when the triglyceride reactant enters the top of the reaction column.

13. The method for preparing diglyceride according to claim 1, characterized in that: The resulting oil, which is mainly composed of diglycerides, contains more than 60% diglycerides.

Images