Method for preparing partial glyceride through glycerolysis reaction

文档序号：502671 发布日期：2021-05-28 浏览：24次中文

阅读说明：本技术 一种甘油解反应制备偏甘油酯的方法 (Method for preparing partial glyceride through glycerolysis reaction ) 是由王永华岳海鹏杨博王卫飞蓝东明罗日明于 2021-02-23 设计创作，主要内容包括：本发明公开了一种甘油解反应制备偏甘油酯的方法,包括以下步骤：(1)制备Lipase MAS1突变体的固定化脂肪酶；Lipase MAS1突变体为MAS1-H108A,其氨基酸序列如SEQ NO.2所示；(2)酶催化甘油解反应制备偏甘油酯,按质量份计,将1～10份植物油,1～4份的甘油,0～40份有机相试剂,与0.2～1.5份固定化脂肪酶混合,在40～55℃的温度下反应1～6小时,得到产物混合物；(3)产物混合物经分离得到偏甘油酯。本发明工艺简单,反应速率快,生成物含量高,不存在副反应。(The invention discloses a method for preparing partial glyceride through glycerolysis reaction, which comprises the following steps: (1) preparing immobilized Lipase of Lipase MAS1 mutant; the Lipase MAS1 mutant is MAS1-H108A, and the amino acid sequence is shown in SEQ NO. 2; (2) preparing partial glyceride through enzymatic glycerolysis reaction, mixing 1-10 parts by mass of vegetable oil, 1-4 parts by mass of glycerol, 0-40 parts by mass of an organic phase reagent and 0.2-1.5 parts by mass of immobilized lipase, and reacting at the temperature of 40-55 ℃ for 1-6 hours to obtain a product mixture; (3) separating the product mixture to obtain partial glyceride. The invention has simple process, high reaction rate, high product content and no side reaction.)

1. A method for preparing partial glyceride through glycerolysis reaction is characterized by comprising the following steps:

(1) preparing immobilized Lipase of a Lipase MAS1 mutant, wherein the Lipase MAS1 mutant is MAS1-H108A, and the amino acid sequence of the immobilized Lipase is shown in SEQ NO. 2;

(2) preparing partial glyceride through an enzymatic glycerolysis reaction, wherein the partial glyceride comprises the following components in parts by mass: mixing 1-10 parts of vegetable oil, 1-4 parts of glycerol, 0-40 parts of an organic phase reagent and 0.2-1.5 parts of immobilized Lipase of Lipase MAS1 mutant, and reacting at the temperature of 40-55 ℃ for 1-6 hours to obtain a product mixture;

(3) separating the product mixture to obtain partial glyceride.

2. The method according to claim 1, wherein the immobilization carrier is ECR8285, ECR8806, XAD1180N or ECR 1030.

3. The method according to claim 2, wherein the preparation of the immobilized lipase in step (1) comprises the following steps in parts by mass: taking 3-10 parts of immobilized carrier, 6-20 parts of Tris-HCL buffer solution by MASs and 0.3-1.5 parts of Lipase MAS1 mutant, mixing, stirring and adsorbing for 3-5 hours at 25-35 ℃, then filtering and drying to obtain immobilized Lipase of Lipase MAS1 mutant.

4. The method according to claim 3, wherein the immobilization carrier is pretreated by: firstly, soaking for 24 hours in 95 percent ethanol, and washing to be neutral by using distilled water; soaking the mixture in 5% HCl for 24 hr, and washing with distilled water to neutrality; soaking in 3% NaOH solution for 24 hr, and washing with distilled water to neutral.

5. The method of claim 3, wherein the Tris-HCl buffer has a concentration of 0.03 to 0.05mol/L and a pH of 7.3 to 7.7.

6. The method according to claim 1 or 2 or 3 or 4 or 5, wherein the moisture content in the reaction system of the step (2) is not higher than 0.05%; the addition of the immobilized lipase is 2 to 15 percent of the total mass of the substrate; the molar ratio of the vegetable oil to the glycerol is 1: (1-5); the adding amount of the organic phase reagent is 1.5-4 times of the total mass of the substrate.

7. The method according to claim 5, wherein the organic phase reagent in the step (2) is tert-butyl alcohol, and the organic phase reagent is 15-30 parts.

8. The method of claim 7, wherein the reaction of step (2) is carried out at a gas bath shaker rotation speed of 150 ± 50 rpm/min.

9. The method according to claim 8, wherein the separation in the step (3) is that the mixture is subjected to high-speed centrifugal separation to obtain upper-layer grease, and then the upper-layer grease is subjected to multi-stage molecular distillation to obtain a high-purity partial glyceride product. The rotation speed of the centrifugal separation is 8000-12000 r/min, the duration is 3-5 min, the molecular distillation evaporation temperature is 140 +/-20 ℃, and the vacuum pressure is 10 +/-5 Pa.

10. The method according to claim 9, wherein the vegetable oil is one or more of soybean oil, sesame oil, palm oil, peanut oil, corn oil, sunflower oil, coconut oil, olive oil, linseed oil, camellia oil, palm kernel oil, cottonseed oil, safflower oil, and rice oil.

Technical Field

The invention relates to a method for preparing partial glyceride through glycerolysis reaction.

Background

Partial glyceride is a general name of diglyceride and monoglyceride, and both diglyceride and monoglyceride can be used as additives for food. Monoglyceride can be used as an emulsifier in the fields of foods, pharmaceuticals and cosmetics, and diglyceride is a novel healthy oil which is certified as safe, free from toxic and side effects, and effective in preventing fat accumulation and regulating body fat. Generally, the production of partial glyceride can be divided into chemical method and biological enzyme method, and the chemical method has the disadvantages of more by-products, large pollution and high reaction energy consumption, although the chemical method has low cost and is easy to carry out large-scale production. The problems are well solved by the biological enzyme method, the partial glyceride produced by the enzyme method has high reaction efficiency, no side reaction, energy conservation and environmental protection, and the processed oil product with excellent color, sense and taste can be obtained.

The method for producing the grease mainly comprises a chemical method and a biological enzyme method at present, because the chemical method has high energy consumption, partial pollution to the environment, potential safety hazards and other problems, the biological enzyme method for preparing the grease becomes a research hotspot of people in recent years, the biological enzyme method for preparing the grease mainly comprises an esterification method, a hydrolysis method and a glycerolysis method, although the esterification method and the hydrolysis method can produce a mixture of diglyceride and monoglyceride, the hydrolysis method can produce a large amount of fatty acid and glycerol byproducts, the content of the diglyceride and the monoglyceride in a system is lower, the production cost of the esterification method is higher, the generation rate of the product is lower, and the production requirement of a factory is not met, so the glycerolysis method with high raw material utilization rate, simple and convenient operation and high product generation rate is selected for production.

The system for preparing the grease by the biological enzyme method generally comprises a solvent-free system, an organic solvent system and supercritical CO₂Systems, etc., but because supercritical CO is used₂The systems are expensive and are therefore generally not produced by this process. In a solvent-free system, the viscosity of glycerol and the property of insolubility of glycerol with grease influence the reaction, and in an organic solvent system, the dissolution of glycerol and vegetable grease can be improved, and the mass transfer effect between substances can be improved. Therefore, the development of a new glycerolysis process to improve the production efficiency and generate high content of partial glyceride has important significance for factory production.

Disclosure of Invention

The invention aims to overcome the defects of the existing partial glyceride production process, and develops a method for preparing partial glyceride by immobilized lipase, which improves the yield of partial glyceride in a solvent system and does not generate byproducts.

The purpose of the invention is realized by the following technical scheme:

a method for preparing partial glyceride by glycerolysis reaction comprises the following steps:

(1) preparing immobilized Lipase of a Lipase MAS1 mutant, wherein the Lipase MAS1 mutant is MAS1-H108A, and the amino acid sequence of the immobilized Lipase is shown in SEQ NO. 2;

(2) preparing partial glyceride by an enzymatic glycerolysis reaction in parts by mass; mixing 1-10 parts of vegetable oil, 1-4 parts of glycerol, 0-40 parts of an organic phase reagent and 0.2-1.5 parts of immobilized Lipase of Lipase MAS1 mutant, and reacting at the temperature of 40-55 ℃ for 1-6 hours to obtain a product mixture;

(3) separating the product mixture to obtain partial glyceride.

Preferably, the immobilization carrier is ECR8285, ECR8806, XAD1180N, XAD1180 or ECR 1030.

Preferably, the preparation of the immobilized lipase in the step (1) comprises the following steps of: taking 3-10 parts of immobilized carrier, 6-20 parts of Tris-HCL buffer solution by MASs and 0.3-1.5 parts of Lipase MAS1 mutant, mixing, stirring and adsorbing for 3-5 hours at 25-35 ℃, then filtering and drying to obtain immobilized Lipase of Lipase MAS1 mutant.

Preferably, the immobilization support is pretreated as follows: firstly, soaking for 24 hours in 95 percent ethanol, and washing to be neutral by using distilled water; soaking the mixture in 5% HCl for 24 hr, and washing with distilled water to neutrality; soaking in 3% NaOH solution for 24 hr, and washing with distilled water to neutral.

Preferably, the concentration of the Tris-HCL buffer solution is 0.03-0.05 mol/L, and the pH value is 7.3-7.7.

Preferably, the moisture content in the reaction system of the step (2) is not higher than 0.05%; the addition of the immobilized lipase is 2 to 15 percent of the total mass of the substrate; the molar ratio of the vegetable oil to the glycerol is 1: (1-5); the adding amount of the organic phase reagent is 1.5-4 times of the total mass of the substrate.

Preferably, the organic phase reagent of step (2) is tert-butanol; 15-30 parts of an organic phase reagent.

Preferably, the reaction in step (2) is carried out at a gas bath shaker rotation speed of 150. + -. 50 rpm/min.

Preferably, the separation in the step (3) means that the mixture is subjected to high-speed centrifugal separation to obtain upper-layer grease, and then the high-purity partial glyceride product can be obtained through multi-stage molecular distillation.

Preferably, the rotation speed of the centrifugal separation is 8000-12000 r/min, the duration is 3-5 min, the molecular distillation evaporation temperature is 140 +/-20 ℃, and the vacuum pressure is 10 +/-5 Pa.

Preferably, the vegetable oil is one or a mixture of several of soybean oil, sesame oil, palm oil, peanut oil, corn oil, sunflower oil, coconut oil, olive oil, linseed oil, camellia oil, palm kernel oil, cottonseed oil, safflower oil and rice oil.

The amino acid sequence of the free lipase MAS1 is shown in SEQ NO. 1. Each mutant sequence is obtained by single-site mutation on the basis, and the amino acid sequence of MAS1-H108A is shown as SEQ NO. 2.

Compared with the prior art, the invention has the beneficial effects that:

(1) the immobilized Lipase of the Lipase MAS1 mutant has strong biocatalytic activity and high glycerol tolerance, can be subjected to glycerolysis reaction in an organic solvent system at 40-55 ℃, does not produce by-products such as fatty acid and the like, simplifies the procedures related to separation and purification in the later production stage, and is convenient for production operation.

(2) The method carries out the glycerolysis reaction in a system with a solvent, has high reaction degree, high reaction rate, high content of the generated partial glyceride, no side reaction, reusability of the immobilized enzyme and low cost consumption, and has good industrial prospect.

Detailed Description

The present invention will be described in further detail with reference to specific examples. In the following, several examples of the invention are provided to further illustrate the invention, in which all percentages are by mass, without limiting the scope of the invention thereto. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Firstly, soaking the immobilized carrier in 95% ethanol for 24 hours, and washing the immobilized carrier to be neutral by using distilled water; soaking the mixture in 5% HCl for 24 hr, and washing with distilled water to neutrality; soaking in 3% NaOH solution for 24 hr, washing with distilled water to neutral, and storing in refrigerator at 4 deg.C.

In some embodiments, the preparation of the immobilized Lipase of the Lipase MAS1 mutant in the step (1) is controlled by selecting any one of the immobilized vectors XAD1180, XAD1180N, DA-201, ECR1030, ECR8285 and ECR8806 and any one of the Lipase MAS1 mutants MAS1-C260S, MAS1-C261S, MAS1-H108W, MAS1-V202F, MAS1-V233F and MAS1-H108A to be combined. The immobilized lipase MAS1-H108A can prepare partial glyceride with the highest content after the lipase MAS1-H108A is combined with an immobilized carrier ECR 8285.

The source of the various enzymes used in the examples below or literature prepared by reference are as follows:

triglyceride lipase MAS1-H108A was prepared with reference to the following references: the study on the preparation of the plum line immobilized lipase MAS1-H108A and the application of the catalytic synthesis of triglyceride rich in alpha-linolenic acid [ D ]. university of southern China, 2019.

Triglyceride lipase MAS1-C260S, MAS1-C261S, MAS1-H108W, MAS1-V202F and MAS1-V233F were prepared by referring to Zhaozhenxin, study of the relationship between the structure of lipase MAS1 and heat resistance and substrate selectivity [ D ]. university of south China, 2017.

Example 1 (solvent free System)

3g of ECR1030 resin, 6g of Tris-HCL buffer solution and 0.3g of free lipase MAS1-V202F are mixed and stirred for 3 hours at 25 ℃, then the mixture is filtered and dried to obtain immobilized lipase MAS1-V202F, and the immobilized lipase is put into a refrigerator at 4 ℃ for standby.

8.28g of soybean oil, 1.72g of glycerol and 1g of the immobilized lipase MAS1-V202F were put in a flask, and the flask was placed in a gas bath shaker at a temperature of 40 ℃ and a rotation speed of 150rpm/min for 5 hours, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was tested to determine a triglyceride content of 27.36%, a diglyceride content of 28.92%, and a monoglyceride content of 42.06%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 140 ℃ and the pressure of 10Pa to obtain a light phase component with the monoglyceride content of 88.3% and the diglyceride content of 10.7%; the content of triglyceride in the heavy phase component was 56.68%, the content of diglyceride was 43.32%, and the conversion of triglyceride was 71.46%.

Example 2 (solvent free System)

5g of XAD1180 resin, 10g of Tris-HCL buffer solution and 1g of free lipase MAS1-H108W are mixed and stirred for 3 hours at 30 ℃, then the mixture is filtered and dried to obtain immobilized lipase MAS1-H108W, and the immobilized lipase MAS1-H108W is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.38g of glycerol and 1g of the immobilized lipase MAS1-H108W were put in a flask, and the flask was placed in a gas bath shaker at a temperature of 50 ℃ and a rotation speed of 150rpm/min for 4 hours, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglycerides 17.9%, the content of diglycerides 28.2%, and the content of monoglycerides 53.9%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 98.5% and the diglyceride content of 1.5%; the content of triglyceride in the heavy phase component was 44.47%, the content of diglyceride was 55.53%, and the conversion of triglyceride was 81.33%.

Example 3 (solvent free System)

10g of ECR8806 resin, 20g of Tris-HCl buffer solution and 1.5g of free lipase MAS1-V233F are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized lipase MAS1-V233F, and the immobilized lipase is put into a refrigerator at 4 ℃ for standby.

6.58g of soybean oil, 3.42g of glycerol and 1g of the immobilized lipase MAS1-V233F were put in a flask, and the flask was placed in a gas bath shaker at a temperature of 60 ℃ and a rotation speed of 150rpm/min for 6 hours, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglycerides 20.4%, diglycerides 30.8%, and monoglycerides 48.8%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 97.7 percent and the diglyceride content of 2.3 percent; the content of triglyceride in the heavy phase component was 42.73%, the content of diglyceride was 57.27%, and the conversion of triglyceride was 78.82%.

Example 4 (solvent free System)

10g of ECR8285 resin, 20g of Tris-HCL buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized lipase MAS1-H108A, and the mixture is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.38g of glycerol and 1g of the immobilized lipase MAS1-H108A were put in a flask, and the flask was placed in a gas bath shaker at a temperature of 60 ℃ and a rotation speed of 150rpm/min for 3 hours, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglycerides 11.4%, diglycerides 38.8%, and monoglycerides 49.8%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.4% and the diglyceride content of 0.6%; the content of triglyceride in the heavy phase component was 22.73%, the content of diglyceride was 77.27%, and the conversion of triglyceride was 88.11%.

Example 5 (with solvent System)

7.62g of soybean oil, 2.37g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 2 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was tested to determine the content of triglycerides 4.38%, the content of diglycerides 38.92%, and the content of monoglycerides 54.03%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.6% and the diglyceride content of 0.4%; the content of triglyceride in the heavy phase component was 10.35%, the content of diglyceride was 89.65%, and the conversion of triglyceride was 95.43%.

Example 6 (with solvent System)

Mixing 3g of XAD1180N resin, 6g of Tris-HCL buffer solution and 0.3g of free lipase MAS1-H108W, mixing and stirring at 30 ℃ for 5 hours, filtering and drying to obtain immobilized lipase MAS1-H108W, and placing in a refrigerator at 4 ℃ for later use.

8.28g of soybean oil, 1.72g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108W were placed in a flask, and placed in a gas bath shaker to react for 3 hours at a temperature of 40 ℃ and a rotation speed of 150rpm/min, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglyceride 10.76%, the content of diglyceride 34.33%, and the content of monoglyceride 52.69%. Performing molecular distillation on the upper layer grease after centrifugation at 160 ℃ and 15Pa to obtain a light phase component with the monoglyceride content of 99.4% and the diglyceride content of 0.6%; the content of triglyceride in the heavy phase component was 25.12%, the content of diglyceride was 74.88%, and the conversion of triglyceride was 88.78%.

Example 7 (with solvent System)

6g of DA-201 resin, 12g of Tris-HCl buffer solution and 0.6g of free lipase MAS1-C260S are mixed and stirred for 6 hours at the temperature of 30 ℃, then the mixture is filtered and dried to obtain immobilized lipase MAS1-C260S, and the immobilized lipase MAS1-C260S is put into a refrigerator at the temperature of 4 ℃ for standby.

8.28g of soybean oil, 1.72g of glycerol, 20ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-C260S were placed in a flask, and placed in a gas bath shaker to react for 4 hours at a temperature of 60 ℃ and a rotation speed of 150rpm/min, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglycerides of 17.61%, the content of diglycerides of 29.29%, and the content of monoglycerides of 54.48%. Performing molecular distillation on the upper layer grease after centrifugation at 160 ℃ and 15Pa to obtain a light phase component with the monoglyceride content of 99.1% and the diglyceride content of 0.9%; the content of triglyceride in the heavy phase component was 42.64%, the content of diglyceride was 57.36%, and the conversion of triglyceride was 81.63%.

Example 8 (with solvent System)

6g of DA-201 resin, 12g of Tris-HCl buffer solution and 0.6g of free lipase MAS1-C261S are mixed and stirred for 6 hours at the temperature of 30 ℃, then the mixture is filtered and dried to obtain immobilized lipase MAS1-C261S, and the immobilized lipase MAS1-C261S is put into a refrigerator at the temperature of 4 ℃ for standby.

8.28g of soybean oil, 1.72g of glycerol, 20ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-C261S were placed in a flask, and placed in a gas bath shaker to react for 4 hours at a temperature of 60 ℃ and a rotation speed of 150rpm/min, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglyceride 18.16%, the content of diglyceride 27.30%, and the content of monoglyceride 54.54%. Performing molecular distillation on the upper layer grease after centrifugation at 160 ℃ and 15Pa to obtain a light phase component with the monoglyceride content of 99.4% and the diglyceride content of 0.6%; the content of triglyceride in the heavy phase component was 44.45%, the content of diglyceride was 55.55%, and the conversion of triglyceride was 81.06%.

Example 9 (with solvent System)

10g of XAD1180N resin, 20g of Tris-HCL buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred at 35 ℃ for 4 hours, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.37g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 2 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was tested to determine the content of triglyceride 10.38%, the content of diglyceride 34.90%, and the content of monoglyceride 52.05%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.6% and the diglyceride content of 0.4%; the content of triglyceride in the heavy phase component was 20.35%, the content of diglyceride was 79.65%, and the conversion of triglyceride was 89.17%.

Example 10 (with solvent System)

10g of XAD1180 resin, 20g of Tris-HCL buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.37g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 2 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglyceride 12.76%, the content of diglyceride 29.72%, and the content of monoglyceride 57.52%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.7% and the diglyceride content of 0.3%; the content of triglyceride in the heavy phase component was 42.96%, the content of diglyceride was 57.04%, and the conversion of triglyceride was 86.69%.

Example 11 (with solvent System)

10g of DA-201 resin, 20g of Tris-HCl buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.37g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 2 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglycerides 15.76%, diglycerides 27.24%, and monoglycerides 57.00%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.6% and the diglyceride content of 0.4%; the content of triglyceride in the heavy phase component was 45.72%, the content of diglyceride was 54.28%, and the conversion of triglyceride was 83.56%.

Example 12 (with solvent System)

10g of ECR1030 resin, 20g of Tris-HCL buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.37g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 2 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was tested to determine a triglyceride content of 8.76%, a diglyceride content of 32.20%, and a monoglyceride content of 59.04%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.7% and the diglyceride content of 0.3%; the content of triglyceride in the heavy phase component was 19.12%, the content of diglyceride was 80.88%, and the conversion of triglyceride was 90.86%.

Example 13 (with solvent System)

10g of ECR8806 resin, 20g of Tris-HCL buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

7.62g of soybean oil, 2.37g of glycerol, 25ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 2 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was tested to determine the content of triglyceride was 9.41%, the content of diglyceride was 31.55%, and the content of monoglyceride was 59.04%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 150 ℃ and the pressure of 13Pa to obtain a light phase component with the monoglyceride content of 99.7% and the diglyceride content of 0.3%; the content of triglyceride in the heavy phase component was 24.33%, the content of diglyceride was 75.67%, and the conversion of triglyceride was 90.18%.

Example 14 (with solvent System)

10g of ECR8285 resin, 20g of Tris-HCL buffer solution and 1.5g of free lipase MAS1-H108A are mixed and stirred for 4 hours at 35 ℃, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

8.28g of soybean oil, 1.72g of glycerol, 25ml of tert-butanol and 1.5g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at 40 ℃ for 3 hours at a rotation speed of 150rpm/min of the gas bath shaker, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was examined to determine the content of triglycerides 5.76%, the content of diglycerides 38.33%, and the content of monoglycerides 53.69%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 160 ℃ and the pressure of 15Pa to obtain a light phase component with the monoglyceride content of 98.4% and the diglyceride content of 1.6%; the content of triglyceride in the heavy phase component was 13.12%, the content of diglyceride was 86.88%, and the conversion rate of triglyceride was 94.64%.

Example 15 (with solvent System)

6g of ECR1030 resin, 12g of Tris-HCL buffer solution and 0.6g of free lipase MAS1-H108A are mixed and stirred at 30 ℃ for 6 hours, then the mixture is filtered and dried to obtain immobilized enzyme MAS1-H108A, and the immobilized enzyme MAS1-H108A is put into a refrigerator at 4 ℃ for standby.

8.28g of soybean oil, 1.72g of glycerol, 20ml of tert-butyl alcohol and 1g of the immobilized lipase MAS1-H108A were placed in a flask, placed in a gas bath shaker, and reacted at a temperature of 60 ℃ and a rotation speed of 150rpm/min for 4 hours, and the reaction was stopped. And pouring the reactant into a centrifugal tube, and carrying out centrifugal separation for 3min at 10000rpm/min to obtain an upper oil phase which is the mixture rich in the partial glyceride. The upper oil phase was tested to determine the triglyceride content of 7.61%, the diglyceride content of 39.29%, and the monoglyceride content of 54.48%. Performing molecular distillation on the upper layer grease after centrifugation at the temperature of 160 ℃ and the pressure of 15Pa to obtain a light phase component with the monoglyceride content of 97.9 percent and the diglyceride content of 2.1 percent; the content of triglyceride in the heavy phase component was 16.64%, the content of diglyceride was 83.36%, and the conversion rate of triglyceride was 92.91%.

Sequence listing

<110> university of southern China's science

Guangzhou Yonghuate medical Nutrition technology Co Ltd

<120> method for preparing partial glyceride by glycerolysis reaction

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 265

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Thr Ala Thr Ala Ala Thr Pro Ala Ala Glu Ala Thr Ser Arg Gly

1 5 10 15

Trp Asn Asp Tyr Ser Cys Lys Pro Ser Ala Ala His Pro Arg Pro Val

20 25 30

Val Leu Val His Gly Thr Phe Gly Asn Ser Ile Asp Asn Trp Leu Val

35 40 45

Leu Ala Pro Tyr Leu Val Asn Arg Gly Tyr Cys Val Phe Ser Leu Asp

50 55 60

Tyr Gly Gln Leu Pro Gly Val Pro Phe Phe His Gly Leu Gly Pro Ile

65 70 75 80

Asp Lys Ser Ala Glu Gln Leu Asp Val Phe Val Asp Lys Val Leu Asp

85 90 95

Ala Thr Gly Ala Pro Lys Ala Asp Leu Val Gly His Ser Gln Gly Gly

100 105 110

Met Met Pro Asn Tyr Tyr Leu Lys Phe Leu Gly Gly Ala Asp Lys Val

115 120 125

Asn Ala Leu Val Gly Ile Ala Pro Asp Asn His Gly Thr Thr Leu Leu

130 135 140

Gly Leu Thr Lys Leu Leu Pro Phe Phe Pro Gly Val Glu Lys Phe Ile

145 150 155 160

Ser Asp Asn Thr Pro Gly Leu Ala Asp Gln Val Ala Gly Ser Pro Phe

165 170 175

Ile Thr Lys Leu Thr Ala Gly Gly Asp Thr Val Pro Gly Val Arg Tyr

180 185 190

Thr Val Ile Ala Thr Lys Tyr Asp Gln Val Val Thr Pro Tyr Arg Thr

195 200 205

Gln Tyr Leu Asp Gly Pro Asn Val Arg Asn Val Leu Leu Gln Asp Leu

210 215 220

Cys Pro Val Asp Leu Ser Glu His Val Ala Ile Gly Thr Ile Asp Arg

225 230 235 240

Ile Ala Phe His Glu Val Ala Asn Ala Leu Asp Pro Ala Arg Ala Thr

245 250 255

Pro Thr Thr Cys Ala Ser Val Ile Gly

260 265

<210> 2