A preparation method and application of diglyceride

Abstract

The present invention relates to the field of food technology, and in particular to a method for preparing diglyceride and its application. The method comprises the following steps: firstly hydrolyzing the oil and fat under the action of a first lipase, adding a solubilizing agent, and obtaining a hydrolyzate; then mixing the hydrolyzate with glycerol, and performing an esterification reaction with a second lipase; wherein the first lipase is prepared by compounding lipase Lipozyme RM and Antarctic Candida lipase B. The present invention can specifically synthesize 1,3-diglyceride that is beneficial to the cardiovascular system of the body, and has a high yield and good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation technology, and in particular to a preparation method and application of diglyceride. Background Art

[0002] Diacylcerol is a fat molecule with only two fatty acid chains. Diacylcerol can be used as an emulsifier, a fat plasticity improver or as a matrix for food, medicine, cosmetics, etc.

[0003] Recent studies have found that diglycerides and triglycerides have different absorption and metabolism patterns. Diglycerides are more easily converted into energy and utilized in the body, while triglycerides are more likely to accumulate in the body. Eating fats and oils containing diglycerides has the effect of inhibiting weight gain. Therefore, diglyceride fats and oils can be eaten as a healthy fat.

[0004] Diacylcerol can be produced by a variety of processes. Many patents in the relevant field have disclosed corresponding production methods, such as Chinese invention patent applications CN112513235A, CN116042736A, CN105400837A, etc., wherein CN105400837A discloses an enzyme-catalyzed preparation method of diacylglycerol, comprising the following steps: (1) partial hydrolysis of oil: partially hydrolyzing edible oil by enzyme-catalyzed hydrolysis, and obtaining a hydrolyzate after dehydration, so that the content of diacylglycerol in the hydrolyzate is between 30.0wt% and 35.0wt%, and the content of free fatty acids is between 26.0wt% and 30.0wt%; (2) re-esterification: adding glycerol in an amount of 2.5 to 4 times the mass of the hydrolyzate obtained in step (1), synthesizing diacylglycerol by esterification reaction by enzyme-catalyzed esterification, and removing excess glycerol to obtain the edible diacylglycerol.

[0005] Chinese invention patent application CN116042736A discloses an enzymatic production process for diglycerides, which adopts enzymatic glycerolysis to prepare diglyceride oil, that is, under the catalysis of lipase, oil and glycerol are reacted to prepare diglycerides; before enzymatic glycerolysis, the reaction substrate is homogenized to improve the catalytic efficiency of lipase in the reaction system, thereby achieving the purpose of reducing reaction time, reducing the amount of enzyme added, and increasing the diglyceride content in the product, and also achieving the purpose of reducing production costs.

[0006] The existing technology has conducted certain explorations and studies on the preparation of diglycerides and achieved certain results, but many processes are difficult to be truly applied on a large scale, and the product yield needs to be further improved. In addition, studies have shown that 1,3-DG is the one that truly has a good regulatory effect on the body, such as lowering blood lipids and losing weight. However, due to the lack of specificity of the reaction, most of the resulting products are mixtures of 1,2- and 1,3-DG, and the ratio is usually 7:3-6:4. Therefore, studying a preparation method with a higher 1,3-DG content is an urgent problem to be solved in this field. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a method for preparing diglyceride to solve the problems of low 1,3-DG content and low yield.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] A method for preparing diglyceride comprises the following steps:

[0010] (1) partially hydrolyzing the oil and fat by adding a solvent under the action of a first lipase to obtain a hydrolyzate;

[0011] (2) mixing the hydrolyzate with glycerol and subjecting it to an esterification reaction using a second lipase;

[0012] The first lipase is prepared by compounding lipase Lipozyme RM and Antarctic Candida lipase B in a mass ratio of 1:1-3.

[0013] Preferably, the solvent is a mixed solution of lecithin, ethanol and water in a mass ratio of 0.01-0.05:0.1-0.2:1.

[0014] Preferably, the hydrolysis temperature is 40-65°C and the hydrolysis time is 3-8h.

[0015] Preferably, the first lipase is used in an amount of 1-10% of the mass of the oil.

[0016] Preferably, the amount of the solvent used is 10-50% of the mass of the oil.

[0017] Preferably, the second lipase is lipase Lipozyme435.

[0018] Preferably, the mass ratio of hydrolyzate to glycerol is 1:0.5-2.

[0019] Preferably, the esterification reaction temperature is 40-60° C., and the reaction time is 5-10 h.

[0020] Preferably, the oil is selected from vegetable oil;

[0021] Preferably, the vegetable oil is selected from one or more of linseed oil, soybean oil, rapeseed oil, peanut oil, corn oil, sunflower seed oil, camellia oil, coconut oil, palm oil, palm kernel oil, olive oil, olive pomace oil, walnut oil, rice bran oil, rice oil, cottonseed oil, perilla seed oil, safflower seed oil, grape seed oil, tea seed oil, peony seed oil, sesame oil, wheat germ oil, Acer truncatum seed oil, Xanthoceras sorbifolia oil, sea buckthorn seed oil, DHA algae oil, medium-chain triglycerides, medium- and long-chain fatty acid edible oil, hemp oil, prickly ash seed oil, prickly ash oil, pumpkin seed oil, chili oil, almond oil, and sacha inchi oil;

[0022] Or, the fat is selected from animal oil.

[0023] Preferably, the vegetable oil is selected from corn oil, soybean oil, palm oil, peanut oil, olive oil or avocado.

[0024] The present invention also provides application of the preparation method in preparing 1,3 diglyceride.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] After repeated research, the present invention screened out the best preparation process, which uses a composite lipase to perform a hydrolysis reaction in a specific biphasic solvent, then adds glycerol, performs an esterification reaction under the action of a second lipase, and finally separates the oil phase by high-speed centrifugation. This method has a high yield for preparing diglycerides, and more importantly, can increase the proportion of 1,3-diglycerides that are beneficial to the body's metabolism. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific implementation modes.

[0028] The following raw materials are all conventional raw materials in the art, among which the supplier of Candida antarctica lipase B is Hangzhou Chuangke Biotechnology Co., Ltd., and the model is Lipozyme CALB. The supplier of lipase Lipozyme RM is Novozymes.

[0029] Example 1

[0030] The preparation method of diglyceride in this embodiment comprises the following steps:

[0031] (1) 5% of the first lipase (compounded by lipase Lipozyme RM and Candida antarctica lipase B in a mass ratio of 1:2) was added to the soybean oil, and then a solvent (a mixed solution of lecithin, ethanol and water in a mass ratio of 0.02:0.1:1) with a mass ratio of 25% of the soybean oil was added, and hydrolyzed at 55°C for 5 hours. The oil phase was taken as the hydrolyzate after standing and stratification;

[0032] (2) Add 1 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 and react at 50℃ for 8h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC conditions are as follows: chromatographic column: Waters C18 column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0033] Example 2

[0034] (1) Add 2% of the first lipase (prepared by Lipozyme RM and Candida antarctica lipase B in a mass ratio of 1:2) to corn oil, then add 15% of a solvent (a mixed solution of lecithin, ethanol and water in a mass ratio of 0.05:0.2:1), partially hydrolyze at 65°C for 4 hours, let stand and separate the layers, and take the oil phase as the hydrolyzate;

[0035] (2) Add 0.5 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 and perform esterification reaction at 40℃ for 5h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The high performance liquid chromatography conditions are as follows: chromatographic column: Waters C18 chromatographic column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0036] Example 3

[0037] (1) Add 7% of the first lipase (compounded by lipase Lipozyme RM and Candida antarctica lipase B in a mass ratio of 1:3) to olive oil, then add 45% of a solvent (a mixed solution of lecithin, ethanol and water in a mass ratio of 0.01:0.2:1), partially hydrolyze at 40°C for 8 hours, let stand and separate the layers, and take the oil phase as the hydrolyzate;

[0038] (2) Add 2 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 and react at 60℃ for 10h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC conditions are as follows: chromatographic column: Waters C18 column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0039] Example 1-1

[0040] The details are as follows:

[0041] (1) Add 5% of the first lipase Lipozyme RM to the soybean oil and mix, then add a solvent (a mixed solution of lecithin, ethanol and water with a mass ratio of 0.02:0.1:1) with a mass ratio of 25% of the soybean oil, hydrolyze at 55°C for 5 hours, let stand and separate the layers, and take the oil phase as the hydrolyzate;

[0042] (2) Add 1 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 to react at 50℃ for 8h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC conditions are as follows: chromatographic column: Waters C18 chromatographic column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0043] Example 1-2

[0044] The details are as follows:

[0045] (1) 5% of the first lipase Antarctic Candida lipase B was added to soybean oil, and then a solvent (a mixed solution of lecithin, ethanol and water in a mass ratio of 0.02:0.1:1) was added to the mixture, and the mixture was hydrolyzed at 55°C for 5 hours. The mixture was allowed to stand for stratification and the oil phase was taken as the hydrolyzate;

[0046] (2) Add 1 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 to react at 50℃ for 8h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC conditions are as follows: chromatographic column: Waters C18 chromatographic column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0047] Examples 1-3

[0048] The solvent is different from that in Example 1, as follows:

[0049] (1) 5% of the first lipase (compounded by lipase Lipozyme RM and Candida antarctica lipase B in a mass ratio of 1:2) was added to the soybean oil, and then a solvent (a mixed solution of ethanol and water in a mass ratio of 0.1:1) with a mass ratio of 25% of the soybean oil was added, and the mixture was hydrolyzed at 55°C for 5 hours. The mixture was allowed to stand for stratification and the oil phase was taken as the hydrolyzate;

[0050] (2) Add 1 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 to react at 50℃ for 8h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC conditions are as follows: chromatographic column: Waters C18 chromatographic column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0051] Examples 1-4

[0052] The solvent is water. The details are as follows:

[0053] (1) 5% of the first lipase (compounded by lipase Lipozyme RM and Candida antarctica lipase B in a mass ratio of 1:2) was added to the soybean oil and mixed, and then 25% of water by mass of the soybean oil was added, and hydrolyzed at 55°C for 5 h. The oil phase was taken as the hydrolyzate after standing for stratification;

[0054] (2) Add 1 times the amount of glycerol to the hydrolyzate and mix, add the second lipase Lipozyme435 to react at 50℃ for 8h. After the reaction, centrifuge at 5500rad for 30min to separate the oil and water, take the oil phase, and determine the content by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC conditions are as follows: chromatographic column: Waters C18 column (250mm×4.6 mm, 5μm); mobile phase is acetonitrile-isopropanol with a volume ratio of 95:5, isocratic elution; flow rate 1.0 mL / min; column temperature 35℃. Evaporative light scattering detector conditions: ELSD drift tube temperature 90℃, N2 flow rate 2.2 L / min.

[0055] Results: The contents of diglycerides prepared by different processes are shown in Table 1.

[0056] Table 1

[0057]

[0058] Result analysis:

[0059] Embodiments 1, 2, and 3 of the present invention adopt the preferred preparation process, and the content of diglyceride finally obtained is between 85-90%, and the content of 1,3-diacerol can reach 59-65%. It has a higher yield. Embodiments 1-1 and 1-2 use a single lipase Lipozyme RM or Antarctic Candida lipase B to carry out a two-phase solvent glycerol hydrolysis reaction. The content of diglyceride prepared is lower than that of the specific composite enzyme. The two enzymes have a more obvious promoting effect on the specific generation of diglyceride and 1,3-diacerol in this system. Embodiments 1-3 and 1-4 use different solvent systems. Embodiment 1-3 does not add lecithin auxiliary agent, and Embodiment 1-4 only uses a single-phase water solvent system, and the yield of diglyceride is not ideal.

[0060] Conclusion: The preparation process of the present invention can prepare diglyceride products with better quality.

[0061] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride, characterized in that: The steps include: (1) Adding a solvent to the oil under the action of a first lipase to react and obtain a hydrolyzate; (2) mixing the hydrolyzate with glycerol and subjecting it to an esterification reaction using a second lipase; The first lipase is prepared by compounding lipase Lipozyme RM and Candida antarctica lipase B in a mass ratio of 1:1-3; The solvent is a mixed solution of lecithin, ethanol and water in a mass ratio of 0.01-0.05:0.1-0.2:1; The reaction temperature in step (1) is 40-65°C and the reaction time is 3-8h; The dosage of the first lipase is 1-10% of the mass of the oil; The amount of the solvent used is 10-50% of the mass of the oil; The second lipase is lipase Lipozyme435.

2. The preparation method according to claim 1, characterized in that: The mass ratio of hydrolyzate to glycerol is 1:0.5-2.

3. The preparation method according to claim 1, characterized in that: The temperature of the esterification reaction is 40-60°C, and the reaction time is 5-10h.

4. The preparation method according to claim 1, characterized in that: The oil is selected from vegetable oil; The vegetable oil is selected from one or more of linseed oil, soybean oil, rapeseed oil, peanut oil, corn oil, sunflower seed oil, camellia oil, coconut oil, palm oil, palm kernel oil, olive oil, olive pomace oil, walnut oil, rice bran oil, rice oil, cottonseed oil, perilla seed oil, safflower seed oil, grape seed oil, tea seed oil, peony seed oil, sesame oil, wheat germ oil, maple seed oil, Xanthoceras sorbifolia oil, sea buckthorn seed oil, DHA algae oil, medium-chain triglycerides, medium- and long-chain fatty acid edible oil, hemp oil, prickly ash seed oil, prickly ash oil, pumpkin seed oil, chili oil, almond oil, and sacha inchi oil; Or, the fat is selected from animal oil.

5. Use of the preparation method according to any one of claims 1 to 4 in the preparation of 1,3-diaceryl.

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