Method for preparing diglyceride vegetable oil by enzyme method

Abstract

The invention relates to the technical field of enzymatic synthesis of a target compound, in particular to a method for preparing diglyceride vegetable oil by an enzymatic method, which comprises the following steps: mixing vegetable oil and glycerol according to a mass ratio of 3: 1, adding 5-10% of compound enzyme resin into the mixed material liquid according to the mass of the vegetable oil, oscillating at 40-60 DEG C for 8-16 hours, and performing solid-liquid separation after the oscillation treatment is finished, thereby obtaining the diglyceride vegetable oil. The diglyceride vegetable oil is obtained; wherein the compound enzyme resin takes macroporous resin AB-8 as a carrier, partial glyceride lipase is fixed in pores of the carrier, the carrier is coated with a gel layer, and candida antarctica lipase A is dispersed in the gel layer. According to the method for preparing the diglyceride vegetable oil through the enzyme method, the original method for obtaining the diglyceride vegetable oil crude product step by step is shortened to one step, the method belongs to one-pot operation, and the diglyceride content of the finally produced diglyceride vegetable oil is high.

Description

Technical Field

[0001] The present invention relates to the technical field of enzymatic synthesis of target compounds, and particularly to a method for preparing diglyceride vegetable oil by enzymatic method. Background Art

[0002] Diglyceride is a product obtained by esterifying glycerol with two fatty acids instead of the glycerol backbone, and has various physiological functions such as reducing blood lipid, reducing visceral fat, improving metabolism, promoting digestion, and enhancing immunity, which helps to control body weight and prevent obesity-related diseases. In addition, diglyceride can also be used as a food additive, pharmaceutical raw material, chemical raw material, etc., and has broad application prospects in the fields of food, medicine, and chemical industry.

[0003] The enzymatic method is a commonly used method for preparing diglyceride at present, which has the advantages of mild reaction conditions, high product quality, small environmental pollution, few by-products, simple product post-treatment, and can be used for preparing structured esters; at the same time, the enzymatic method also has deficiencies such as difficult reaction control and many by-products. In recent years, researchers have found that using different lipases in combination to prepare diglyceride can improve the yield of diglyceride to a certain extent. For example, CN 118652945 A provides a process for producing diglyceride by a combined enzymatic method, which includes the following steps: (1) Using vegetable oil, water, Candida lipase, Mucor lipase and glycerol as raw materials for the first enzymatic hydrolysis to obtain product 1; (2) Then using product 1, phospholipase A1 and monoglyceride lipase as raw materials for the second enzymatic hydrolysis, filtering and centrifuging, and collecting the upper light phase to prepare crude diglyceride; (3) Purifying the crude diglyceride by molecular distillation to obtain the product.

[0004] This method obtains diglyceride with a relatively high purity through two enzymatic hydrolyses using a specific enzymatic hydrolysis sequence and types of enzymes, and further purifies the diglyceride by molecular distillation, but there are also problems such as difficult separation, unstable enzyme activity, and need for material liquid transfer. Summary of the Invention

[0005] Aiming at the technical problems of difficult enzyme recovery in the production of triglyceride using non-immobilized enzymes, and the step of using combined enzymes to produce triglyceride step by step involves material liquid transfer, the steps are relatively cumbersome and waste human energy, the present invention provides a method for preparing diglyceride vegetable oil by enzymatic method.

[0006] The technical solution of the present invention is as follows: A method for preparing diglyceride vegetable oil by enzymatic method, which includes the following steps: After mixing vegetable oil and glycerol in a mass ratio of 3:1, 5% - 10% of composite enzyme resin is added to the mixed material liquid based on the mass of the vegetable oil, and the mixture is oscillated at 40 - 60 °C for 8 - 16 h. After that, solid-liquid separation is carried out to obtain diacylglycerol vegetable oil; Among them, the composite enzyme resin uses macroporous resin AB-8 as the carrier, partial glyceride lipase is fixed in the pores of the carrier, and the outside of the carrier is coated with a gel layer, and Candida antarctica lipase A is dispersed in the gel layer.

[0007] Furthermore, the vegetable oil is corn oil.

[0008] Furthermore, the oscillation speed of the mixed material liquid and the composite enzyme resin is 100 - 300 rpm.

[0009] Furthermore, the content of diacylglycerol in the diacylglycerol vegetable oil is greater than or equal to 55%.

[0010] Furthermore, the composite enzyme resin is prepared by the following method: (1) Add 5 g of macroporous resin AB-8 to 50 mL of the dilution solution of partial glyceride lipase, oscillate and adsorb at room temperature, and carry out solid-liquid separation to obtain a carrier with partial glyceride lipase fixed in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir, then add 1 g of kaolin, continue to stir, and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and dry at 40 °C.

[0011] Furthermore, before the macroporous resin AB-8 in step (1) is adsorbed, it is first soaked in PBS buffer solution for 6 h.

[0012] Furthermore, the dilution solution of partial glyceride lipase in step (1) is prepared by adding 2 g of partial glyceride lipase to 50 mL of PBS buffer solution.

[0013] The beneficial effects of the present invention are as follows: The method for preparing diglyceride vegetable oil by enzymatic method provided by the present invention shortens the original method of obtaining crude diglyceride vegetable oil in steps to one step by designing a composite enzyme resin with a special structure, belonging to a one-pot operation. First, the raw material undergoes a primary enzymatic hydrolysis reaction with Candida antarctica lipase A distributed in the gel layer on the surface of the macroporous resin, and then undergoes a secondary enzymatic hydrolysis reaction with partial glyceride lipase adsorbed in the pores of the resin. The smooth progress of the two enzymatic hydrolysis reactions is closely related to the selection of lipase and the mass transfer ability of the gel layer. The two enzymes used in the present invention can maintain good activity in the resin or gel layer, and the experimental results prove that through the cooperation of the two enzymes, the content of diglyceride in the diglyceride vegetable oil is significantly increased; if the mass transfer ability is too fast, the effect of the primary enzymatic hydrolysis cannot be guaranteed, and if the mass transfer ability is too slow, the cycle of the entire production process will be prolonged. The present invention selects a suitable gel layer modifier (kaolin) to obtain an appropriate gel layer mass transfer resistance, ensuring a high diglyceride content in the finally produced diglyceride vegetable oil product. Detailed implementation manners

[0014] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0015] The macroporous resin AB-8 used in the specific implementation manner of the present invention is purchased from Langfang Miaoyang Chemical Co., Ltd., with a particle size range of 60~16 mesh, a specific surface area ≥ 480m 2 / g, and a water content of 65%~75%; the partial glyceride lipase is purchased from Sigma-Aldrich, with the number SAE0065 and an enzyme activity ≥ 100 LCLU / g; the Candida antarctica lipase A is purchased from Sigma-Aldrich, with the number 62287 and a specific activity ≥ 2 U / mg.

[0016] Example 1 S1: Preparation of composite enzyme resin (1) Soak the macroporous resin AB-8 with PBS buffer solution for 6 h, then add 5 g of macroporous resin AB-8 to 50 mL of PBS dilution solution with a partial glyceride lipase concentration of 1 g / 100 mL, shake and adsorb at room temperature for 3 h, separate the solid and liquid to obtain a carrier with partial glyceride lipase fixed in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir, add 1 g of kaolin, continue to stir, and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and then dry at 40 °C to obtain the composite enzyme resin.

[0017] S2: Enzymatic preparation of diglyceride vegetable oil Mix corn oil and glycerol in a mass ratio of 3:1. Based on the mass of the vegetable oil, add 5% of the composite enzyme resin prepared in the above step S1 to the mixed material liquid, oscillate and process at 40 °C for 8 h, with an oscillation speed of 100 rpm. After completion, perform solid-liquid separation to obtain diglyceride vegetable oil. After sampling, the diglyceride content is measured to be 58.8%.

[0018] Example 2 S1: Preparation of composite enzyme resin (1) Soak the macroporous resin AB-8 with PBS buffer for 6 h. Then add 5 g of macroporous resin AB-8 to 50 mL of PBS dilution solution with a partial glyceride lipase concentration of 1 g / 100 mL, oscillate and adsorb at room temperature for 3 h, perform solid-liquid separation to obtain a carrier with partial glyceride lipase immobilized in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir, then add 1 g of kaolin, continue to stir, and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and then dry at 40 °C to obtain the composite enzyme resin.

[0019] S2: Enzymatic preparation of diglyceride vegetable oil Mix corn oil and glycerol in a mass ratio of 3:1. Based on the mass of the vegetable oil, add 8% of the composite enzyme resin prepared in the above step S1 to the mixed material liquid, oscillate and process at 55 °C for 12 h, with an oscillation speed of 200 rpm. After completion, perform solid-liquid separation to obtain diglyceride vegetable oil. After sampling, the diglyceride content is measured to be 59.4%.

[0020] Example 3 S1: Preparation of composite enzyme resin (1) Soak the macroporous resin AB-8 with PBS buffer for 6 h. Then add 5 g of macroporous resin AB-8 to 50 mL of PBS dilution solution with a partial glyceride lipase concentration of 1 g / 100 mL, oscillate and adsorb at room temperature for 3 h, perform solid-liquid separation to obtain a carrier with partial glyceride lipase immobilized in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir, then add 1 g of kaolin, continue to stir, and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and then dry at 40 °C to obtain the composite enzyme resin.

[0021] S2: Preparation of diglyceride vegetable oil by enzymatic method Mix corn oil and glycerol in a mass ratio of 3:1. Based on the mass of the vegetable oil, add 10% of the composite enzyme resin prepared in the above step S1 to the mixed material liquid, oscillate and treat at 60 °C for 16 h, with an oscillation speed of 300 rpm. After completion, perform solid-liquid separation to obtain diglyceride vegetable oil. After sampling, the diglyceride content is measured to be 57.6%.

[0022] Comparative Example 1 S1: Preparation of composite enzyme resin (1) Immerse the macroporous resin AB-8 in PBS buffer solution for 6 h. Then add 5 g of macroporous resin AB-8 to 50 mL of PBS dilution solution with a concentration of 1 g / 100 mL of partial glyceride lipase, and oscillate and adsorb at room temperature for 3 h. Perform solid-liquid separation to obtain a carrier with partial glyceride lipase fixed in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir, and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and then dry at 40 °C.

[0023] S2: Preparation of diglyceride vegetable oil by enzymatic method Mix corn oil and glycerol in a mass ratio of 3:1. Based on the mass of the vegetable oil, add 5% of the composite enzyme resin prepared in the above step S, oscillate and treat at 40 °C for 8 h, with an oscillation speed of 100 rpm. After completion, perform solid-liquid separation to obtain diglyceride vegetable oil. After sampling, the diglyceride content is measured to be 50.2%.

[0024] By comparing Example 1 and Comparative Example 1, it can be found that the mass transfer capacity of the gel layer without adding kaolin is too strong, and the effect of the primary enzymatic hydrolysis process in the gel layer is not ideal, which affects the content of diglyceride in the diglyceride vegetable oil.

[0025] Comparative Example 2 S1: Preparation of composite enzyme resin (1) Immerse the macroporous resin AB-8 in PBS buffer solution for 6 h. Then add 5 g of macroporous resin AB-8 to 50 mL of PBS dilution solution with a concentration of 1 g / 100 mL of partial glyceride lipase, and oscillate and adsorb at room temperature for 3 h. Perform solid-liquid separation to obtain a carrier with partial glyceride lipase fixed in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water. After stirring, add 3 g of kaolin. After continuous stirring, add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel. (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2). After stirring and mixing, dry at 40 °C.

[0026] S2: Enzymatic preparation of diglyceride vegetable oil Mix corn oil and glycerol according to a mass ratio of 3:1. Based on the mass of the vegetable oil, add 5% of the composite enzyme resin prepared in the above step S1 to the mixed material liquid. Oscillate and process at 40 °C for 8 h, and the oscillation speed is 100 rpm. After completion, perform solid-liquid separation to obtain diglyceride vegetable oil. After sampling, the diglyceride content is measured to be 51.5%.

[0027] By comparing Example 1 and Comparative Example 2, it can be found that when too much kaolin is added, the mass transfer ability of the gel layer is significantly reduced, which hinders the entry of raw materials into the pores of the macroporous resin for secondary enzymatic hydrolysis. The utilization rate of partial glyceride lipase is low, affecting the diglyceride content in the diglyceride vegetable oil.

[0028] Comparative Example 3 S1: Preparation of composite enzyme resin (1) Immerse the macroporous resin AB-8 in PBS buffer for 6 h. Then add 5 g of macroporous resin AB-8 to 50 mL of the PBS dilution of the enzyme. The PBS dilution of the enzyme contains 1 g / 100 mL of partial glyceride lipase and 1 g / 100 mL of Candida antarctica lipase A. Oscillate and adsorb at room temperature for 3 h, perform solid-liquid separation to obtain a carrier with partial glyceride lipase and Candida antarctica lipase immobilized in the pores, and store it refrigerated for later use.

[0029] S2: Enzymatic preparation of diglyceride vegetable oil Mix corn oil and glycerol according to a mass ratio of 3:1. Based on the mass of the vegetable oil, add 5% of the composite enzyme resin prepared in the above step S1 to the mixed material liquid. Oscillate and process at 40 °C for 8 h, and the oscillation speed is 100 rpm. After completion, perform solid-liquid separation to obtain diglyceride vegetable oil. After sampling, the diglyceride content is measured to be 48.8%.

[0030] Removing the gel layer outside the composite enzyme resin and simultaneously adsorbing Candida antarctica lipase inside the resin pores will affect the enzymatic hydrolysis order of the raw materials. By comparing the diglyceride contents of Example 1 and Comparative Example 3, it can be seen that the specific enzymatic hydrolysis order of the two lipases in the present invention is beneficial to increasing the diglyceride content in the diglyceride vegetable oil.

[0031] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, and all should be covered within the protection scope of the present invention.

Claims

1. A method for preparing diacylglycerol vegetable oil by enzymatic method, characterized in that, It includes the following steps: After mixing vegetable oil and glycerol according to a mass ratio of 3:1, based on the mass of the vegetable oil, 5% - 10% of composite enzyme resin is added to the mixed material liquid, and it is oscillated at 40 - 60 °C for 8 - 16 h. After that, solid-liquid separation is carried out to obtain diacylglycerol vegetable oil; Among them, the composite enzyme resin uses macroporous resin AB-8 as the carrier, partial glyceride lipase is fixed in the pores of the carrier, and the outside of the carrier is coated with a gel layer, and Candida antarctica lipase A is dispersed in the gel layer.

2. The method according to claim 1, wherein The vegetable oil is corn oil.

3. The method according to claim 1, wherein The oscillation speed of the mixed material liquid and the composite enzyme resin during mixing is 100 - 300 rpm.

4. The method according to claim 1, wherein The content of diacylglycerol in the diacylglycerol vegetable oil is greater than or equal to 55%.

5. The method according to claim 1, characterized in that, The composite enzyme resin is prepared by the following method: (1) Add 5 g of macroporous resin AB-8 to 50 mL of the dilution solution of partial glyceride lipase, oscillate and adsorb at room temperature, and carry out solid-liquid separation to obtain a carrier with partial glyceride lipase fixed in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir and then add 1 g of kaolin, continue to stir and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and dry at 40 °C.

6. The method according to claim 5, wherein Before the macroporous resin AB-8 in step (1) is adsorbed, it is first soaked in PBS buffer solution for 6 h.

7. The method according to claim 5, wherein The dilution solution of partial glyceride lipase in step (1) is prepared by adding 2 g of partial glyceride lipase to 50 mL of PBS buffer solution.

8. The method according to claim 1, wherein It includes the following steps: (1) Soak macroporous resin AB-8 in PBS buffer solution for 6 h, then add 5 g of macroporous resin AB-8 to 50 mL of PBS dilution solution with a partial glyceride lipase concentration of 1 g / 100 mL, oscillate and adsorb at room temperature for 3 h, carry out solid-liquid separation to obtain a carrier with partial glyceride lipase fixed in the pores, and store it refrigerated for later use; (2) Add 1 g of Candida antarctica lipase A to 50 mL of water, stir and then add 1 g of kaolin, continue to stir and then add 4 g of polyvinyl alcohol and 2 g of sodium alginate to obtain a gel; (3) Add the carrier after solid-liquid separation in step (1) to the gel prepared in step (2), stir and mix, and dry at 40 °C to obtain the composite enzyme resin; (4) After mixing corn oil and glycerol according to a mass ratio of 3:1, based on the mass of the vegetable oil, 8% of the composite enzyme resin is added to the mixed material liquid, oscillate at 55 °C for 12 h, the oscillation speed is 200 rpm, and after completion, solid-liquid separation is carried out to obtain diacylglycerol vegetable oil.