A method for enzymatic production of diglyceride vegetable oil

Abstract

The present application relates to the technical field of enzyme synthesis of target compounds, and particularly relates to a method for preparing diglyceride vegetable oil by enzyme method, which comprises the following steps: mixing vegetable oil and glycerol according to a mass ratio of 3:1, then adding 5%-10% of a composite enzyme resin to the mixed liquid in terms of the mass of the vegetable oil, oscillating and treating at 40-60 DEG C for 8-16h, and then performing solid-liquid separation to obtain the diglyceride vegetable oil; wherein the composite enzyme resin takes macroporous resin AB-8 as a carrier, the carrier pores are fixed with partial glyceride lipase, the carrier exterior is coated with a gel layer, and the gel layer is dispersed with Candida antarctica lipase A. The method for preparing the diglyceride vegetable oil by enzyme method provided by the present application shortens the original method for obtaining the diglyceride vegetable oil crude product in steps to one step, belongs to a one-pot operation, and the diglyceride content of the diglyceride vegetable oil produced finally is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzymatic synthesis of target compounds, and in particular to a method for preparing diglyceride vegetable oil by enzymatic method. BACKGROUND

[0002] Diglyceride is a product obtained by replacing the glycerol skeleton with two fatty acids after glycerol esterification, has multiple physiological functions such as reducing blood lipids, reducing visceral fat, improving metabolism, promoting digestion, enhancing immunity, etc., and helps to control weight and prevent obesity-related diseases. In addition, diglyceride can also be used as a food additive, a pharmaceutical raw material and a chemical raw material, etc., and has a wide application prospect in the fields of food, medicine and chemical industry.

[0003] Enzymatic method is a commonly used method for preparing diglyceride at present, has advantages such as mild reaction conditions, high product quality, small environmental pollution, few by-products, simple product post-treatment and being applicable to preparation of structural esters; meanwhile, enzymatic hydrolysis also has disadvantages such as difficulty in controlling reaction and many by-products. In recent years, researchers have found that the use of different lipases for preparing diglyceride can improve the yield of diglyceride to a certain extent. For example, CN 118652945 A provides a process for producing diglyceride by complex enzymatic method, which comprises the following steps:

[0004] (1) performing first enzymatic hydrolysis on plant oil, water, Candida lipase, Mucor lipase and glycerol to obtain product 1;

[0005] (2) performing second enzymatic hydrolysis on product 1, phospholipase A1 and partial glyceride lipase, filtering and centrifuging, collecting the upper light phase to prepare crude diglyceride;

[0006] (3) purifying the crude diglyceride by molecular distillation to obtain the product.

[0007] The method performs two enzymatic hydrolysis by using specific enzymatic hydrolysis sequence and enzyme types, obtains diglyceride with high purity, and further purifies the diglyceride by molecular distillation, but also has problems such as difficulty in separation, instability of enzyme activity and need for transferring the feed liquid. SUMMARY

[0008] In view of the technical problems of difficulty in recovering enzymes for producing triglyceride using non-immobilized enzymes, and involving feed liquid transfer and complicated steps for producing triglyceride using complex enzymes in steps, the present application provides a method for preparing diglyceride vegetable oil by enzymatic method.

[0009] The technical scheme of the present application is as follows:

[0010] A method for preparing diglyceride vegetable oil by enzymatic method, comprising the following steps:

[0011] The plant oil and glycerol are mixed according to a mass ratio of 3:1, 5%-10% of the composite enzyme resin is added to the mixed liquid in terms of the mass of the plant oil, and the mixed liquid is treated by oscillation at 40-60 DEG C for 8-16h; after the treatment, solid-liquid separation is performed to obtain a diglyceride plant oil;

[0012] The composite enzyme resin takes macroporous resin AB-8 as a carrier, the carrier pores are fixed with partial glyceride lipase, the carrier is coated with a gel layer, and the gel layer is dispersed with Candida antarctica lipase A.

[0013] Further, the plant oil is corn oil.

[0014] Further, the oscillation speed of the mixed liquid mixed with the composite enzyme resin is 100-300 rpm.

[0015] Further, the diglyceride content in the diglyceride plant oil is greater than or equal to 55%.

[0016] Further, the composite enzyme resin is prepared by the following method:

[0017] (1) 5g of macroporous resin AB-8 is added to 50mL of a diluent of partial glyceride lipase, and adsorption is performed by oscillation at room temperature; solid-liquid separation is performed to obtain a carrier with the pores fixed with partial glyceride lipase, and the carrier is stored in cold storage;

[0018] (2) 1g of Candida antarctica lipase A is added to 50mL of water, and after stirring, 1g of kaolin is added; after further stirring, 4g of polyvinyl alcohol and 2g of sodium alginate are added to obtain a gel;

[0019] (3) the carrier after the solid-liquid separation in step (1) is added to the gel prepared in step (2), and after stirring and mixing, the mixture is dried at 40 DEG C.

[0020] Further, before the adsorption of the macroporous resin AB-8 in step (1), the macroporous resin AB-8 is first soaked in PBS buffer for 6h.

[0021] Further, the diluent of the partial glyceride lipase in step (1) is prepared by adding 2g of partial glyceride lipase to 50mL of PBS buffer.

[0022] The present application has the following beneficial effects:

[0023] The application provides a method for preparing diglyceride vegetable oil by an enzymatic method, and the method is a one-pot operation by designing a special composite enzyme resin, and the method is shortened to one step from the original method for obtaining a diglyceride vegetable oil crude product in steps. First, the raw material is subjected to a first enzymatic reaction with Candida antarctica lipase A distributed in a gel layer on the surface of a macroporous resin, and then subjected to a second enzymatic reaction with a partial glyceride lipase adsorbed in pores of the resin. The smooth progress of the two enzymatic reactions is closely related to the selection of the lipases and the mass transfer capacity of the gel layer. The two enzymes used in the application can maintain good activity in the resin or the gel layer, and experimental results prove that the content of diglyceride in the diglyceride vegetable oil is obviously improved through the cooperation of the two enzymes; if the mass transfer capacity is too fast, the effect of the first enzymatic reaction cannot be guaranteed, and if the mass transfer capacity is too slow, the cycle of the whole production process is prolonged. The application selects a suitable gel layer modifier (kaolin) to obtain a suitable gel layer mass transfer resistance, so that the content of diglyceride in the diglyceride vegetable oil product obtained in the final production is high. DETAILED DESCRIPTION

[0024] In order for those skilled in the art to better understand the technical solutions in the application, the technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the application.

[0025] The macroporous resin AB-8 used in the specific embodiment of the application is purchased from Langfang Miaoyang Chemical Co., Ltd., the particle size range is 60-16 mesh, the specific surface area is greater than or equal to 480 m 2 / g, and the water content is 65%-75%; the partial glyceride lipase is purchased from Sigma-Aldrich Company, the number is SAE0065, the enzyme activity is greater than or equal to 100 LCLU / g; and the Candida antarctica lipase A is purchased from Sigma-Aldrich Company, the number is 62287, and the specific activity is greater than or equal to 2 U / mg.

[0026] Embodiment 1

[0027] S1: Preparation of the composite enzyme resin

[0028] (1) The macroporous resin AB-8 is soaked in a PBS buffer for 6 h, then 5 g of the macroporous resin AB-8 is added into 50 mL of a PBS diluent with a partial glyceride lipase concentration of 1 g / 100 mL, the mixture is subjected to adsorption oscillation at room temperature for 3 h, solid-liquid separation is performed, and the carrier with the partial glyceride lipase fixed in the pores is obtained and stored in a refrigerator;

[0029] (2) 1 g Candida antarctica lipase A was added to 50 mL water, 1 g kaolin was added after stirring, 4 g polyvinyl alcohol and 2 g sodium alginate were added after continuous stirring to obtain a gel;

[0030] (3) The carrier after solid-liquid separation in step (1) was added to the gel prepared in step (2), and after stirring and mixing, it was dried at 40°C to obtain a composite enzyme resin.

[0031] S2: Enzymatic preparation of diglyceride vegetable oil

[0032] After mixing corn oil and glycerol at a mass ratio of 3:1, 5% of the composite enzyme resin prepared in the above step S1 was added to the mixed liquid, and the mixture was treated by oscillation at 40°C for 8 h, the oscillation speed was 100 rpm, after the end of the treatment, the mixture was subjected to solid-liquid separation, and a diglyceride vegetable oil was obtained. The diglyceride content of the sample was 58.8%.

[0033] Example 2

[0034] S1: Preparation of composite enzyme resin

[0035] (1) Macroporous resin AB-8 was soaked in PBS buffer for 6 h, then 5 g of macroporous resin AB-8 was added to 50 mL of PBS diluent with a concentration of 1 g / 100 mL of partial glyceride lipase, and the mixture was oscillated at room temperature for 3 h to adsorb the partial glyceride lipase. After solid-liquid separation, a carrier with partial glyceride lipase fixed in the pores was obtained and stored in a refrigerator;

[0036] (2) 1 g Candida antarctica lipase A was added to 50 mL water, 1 g kaolin was added after stirring, 4 g polyvinyl alcohol and 2 g sodium alginate were added after continuous stirring to obtain a gel;

[0037] (3) The carrier after solid-liquid separation in step (1) was added to the gel prepared in step (2), and after stirring and mixing, it was dried at 40°C to obtain a composite enzyme resin.

[0038] S2: Enzymatic preparation of diglyceride vegetable oil

[0039] After mixing corn oil and glycerol at a mass ratio of 3:1, 8% of the composite enzyme resin prepared in the above step S1 was added to the mixed liquid, and the mixture was treated by oscillation at 55°C for 12 h, the oscillation speed was 200 rpm, after the end of the treatment, the mixture was subjected to solid-liquid separation, and a diglyceride vegetable oil was obtained. The diglyceride content of the sample was 59.4%.

[0040] Example 3

[0041] S1: Preparation of composite enzyme resin

[0042] (1) soak macroporous resin AB-8 in PBS buffer for 6 hours, then add 5 g macroporous resin AB-8 into 50 mL PBS diluent with a concentration of 1 g / 100 mL of partial glyceride lipase, adsorb for 3 hours at room temperature, separate solid and liquid, and obtain a carrier with partial glyceride lipase fixed in the pores, which is stored in cold storage;

[0043] (2) add 1 g Candida antarctica lipase A into 50 mL water, stir, then add 1 g kaolin, continue to stir, and then add 4 g polyvinyl alcohol and 2 g sodium alginate to obtain a gel;

[0044] (3) add the carrier after solid-liquid separation in step (1) into the gel prepared in step (2), stir and mix, and then dry at 40°C to obtain a composite enzyme resin.

[0045] S2: Enzymatic preparation of diglyceride vegetable oil

[0046] Mix corn oil and glycerol according to a mass ratio of 3:1, then add 10% of the composite enzyme resin prepared in step S1 above into the mixed solution, and treat at 60°C for 16 hours at a stirring speed of 300 rpm, then separate solid and liquid to obtain diglyceride vegetable oil, and measure the diglyceride content of the sample to be 57.6%.

[0047] Comparative Example 1

[0048] S1: Preparation of composite enzyme resin

[0049] (1) soak macroporous resin AB-8 in PBS buffer for 6 hours, then add 5 g macroporous resin AB-8 into 50 mL PBS diluent with a concentration of 1 g / 100 mL of partial glyceride lipase, adsorb for 3 hours at room temperature, separate solid and liquid, and obtain a carrier with partial glyceride lipase fixed in the pores, which is stored in cold storage;

[0050] (2) add 1 g Candida antarctica lipase A into 50 mL water, stir, then add 4 g polyvinyl alcohol and 2 g sodium alginate to obtain a gel;

[0051] (3) add the carrier after solid-liquid separation in step (1) into the gel prepared in step (2), stir and mix, and then dry at 40°C.

[0052] S2: Enzymatic preparation of diglyceride vegetable oil

[0053] Mix corn oil and glycerol according to a mass ratio of 3:1, then add 5% of the composite enzyme resin prepared in step S1 above into the mixed solution, and treat at 40°C for 8 hours at a stirring speed of 100 rpm, then separate solid and liquid to obtain diglyceride vegetable oil, and measure the diglyceride content of the sample to be 50.2%.

[0054] Comparing Example 1 and Comparative Example 1, it can be found that the mass transfer capacity of the gel layer without kaolin is too strong, the effect of the first enzymatic hydrolysis process in the gel layer is not ideal, and the content of diglycerides in the diglyceride vegetable oil is affected.

[0055] Comparative Example 2

[0056] S1: Preparation of composite enzyme resin

[0057] (1) Soak macroporous resin AB-8 in PBS buffer for 6 hours, then add 5g macroporous resin AB-8 to 50mL of PBS diluted with 1g / 100mL of metaglycerol lipase, shake at room temperature for 3 hours to adsorb, and then separate solid and liquid to obtain a carrier with metaglycerol lipase immobilized in the pores. Store in cold for later use.

[0058] (2) Add 1g of Candida antarctica lipase A to 50mL of water, stir, add 3g of kaolin, continue stirring, add 4g of polyvinyl alcohol and 2g of sodium alginate to obtain a gel;

[0059] (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.

[0060] S2: Enzymatic preparation of diglyceride vegetable oil

[0061] After mixing corn oil and glycerol at a mass ratio of 3:1, 5% of the composite enzyme resin prepared in step S1 above was added to the mixture based on the mass of vegetable oil. The mixture was shaken at 40°C for 8 hours at a shaking speed of 100 rpm. After the mixture was shaken, solid-liquid separation was performed to obtain diglyceride vegetable oil. The diglyceride content was measured to be 51.5% after sampling.

[0062] Comparing Example 1 and Comparative Example 2, it can be found that when too much kaolin is added, the mass transfer capacity of the gel layer is significantly reduced, which hinders the secondary enzymatic hydrolysis of the raw materials entering the pores of the macroporous resin. The utilization rate of glycerol lipase is low, which affects the content of diglycerides in the diglyceride vegetable oil.

[0063] Comparative Example 3

[0064] S1: Preparation of composite enzyme resin

[0065] (1) Soak macroporous resin AB-8 in PBS buffer for 6 hours. Then add 5g macroporous resin AB-8 to 50mL of enzyme PBS dilution solution. The enzyme PBS dilution solution contains 1g / 100mL of metaglycerol lipase and 1g / 100mL of Candida antarcticis lipase A. Shake at room temperature for 3 hours to adsorb. Separate solid and liquid to obtain a carrier with metaglycerol lipase and Candida antarcticis lipase immobilized in the pores. Store in cold for later use.

[0066] S2: Enzymatic preparation of diglyceride vegetable oil

[0067] After mixing corn oil and glycerol at a mass ratio of 3:1, 5% of the composite enzyme resin prepared in step S1 above was added to the mixture based on the mass of vegetable oil. The mixture was shaken at 40°C for 8 hours at a shaking speed of 100 rpm. After the mixture was shaken, solid-liquid separation was performed to obtain diglyceride vegetable oil. The diglyceride content was measured to be 48.8% after sampling.

[0068] Removing the outer gel layer of the composite enzyme resin and simultaneously adsorbing Candida antarcticis lipase inside the resin pores will affect the enzymatic hydrolysis sequence of the raw materials. Comparing the diglyceride content of Example 1 and Comparative Example 3, it can be seen that the specific enzymatic hydrolysis sequence of the two lipases of the present invention is beneficial to increasing the diglyceride content in vegetable oil.

[0069] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for preparing diglyceride vegetable oil by enzymatic process, characterized in that, Includes the following steps: After mixing vegetable oil and glycerin at a mass ratio of 3:1, 5% to 10% of compound enzyme resin was added to the mixture based on the mass of vegetable oil. The mixture was shaken at 40 to 60°C for 8 to 16 hours. After the mixture was shaken, solid-liquid separation was performed to obtain diglyceride vegetable oil. Among them, the composite enzyme resin uses macroporous resin AB-8 as a carrier, with glycerol lipase immobilized in the pores of the carrier, and a gel layer coated on the outside of the carrier, with Candida antarcticis lipase A dispersed in the gel layer. The composite enzyme resin was prepared using the following method: (1) Add 5g of macroporous resin AB-8 to 50mL of dilution of metaglycerol lipase, shake at room temperature to adsorb, and separate solid and liquid to obtain a carrier with metaglycerol lipase immobilized in the pores. Store in cold for later use. (2) Add 1g of Candida antarctica lipase A to 50mL of water, stir, add 1g of kaolin, continue stirring, add 4g of polyvinyl alcohol and 2g 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.

2. The method as described in claim 1, characterized in that, The vegetable oil is corn oil.

3. The method as described in claim 1, characterized in that, The oscillation speed for mixing the mixture with the composite enzyme resin is 100~300 rpm.

4. The method as described in claim 1, characterized in that, Diglycerides: Vegetable oils contain 55% or more diglycerides.

5. The method as described in claim 1, characterized in that, Step (1) Before the macroporous resin AB-8 is adsorbed, it is first soaked in PBS buffer for 6 hours.

6. The method as described in claim 1, characterized in that, The dilution of metaglycerol lipase in step (1) was prepared by adding 2g of metaglycerol lipase to 50mL of PBS buffer.

7. The method as described in claim 1, characterized in that, Includes the following steps: (1) Soak macroporous resin AB-8 in PBS buffer for 6 hours, then add 5g macroporous resin AB-8 to 50mL of PBS diluted with 1g / 100mL of metaglycerol lipase, shake at room temperature for 3 hours to adsorb, and then separate solid and liquid to obtain a carrier with metaglycerol lipase immobilized in the pores. Store in cold for later use. (2) Add 1g of Candida antarctica lipase A to 50mL of water, stir, add 1g of kaolin, continue stirring, add 4g of polyvinyl alcohol and 2g 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 composite enzyme resin; (4) After mixing corn oil and glycerol in a mass ratio of 3:1, add 8% compound enzyme resin to the mixture based on the mass of vegetable oil. Shake the mixture at 55°C for 12 hours at a shaking speed of 200 rpm. After the mixture is finished, separate the solid and liquid to obtain diglyceride vegetable oil.