Method for preparing diglyceride by enzyme method

Abstract

The invention provides a method for preparing diglyceride by an enzyme method. The method comprises the following steps: (1) mixing substrate oil and glycerol to obtain a reaction mixture; (2) carrying out low-temperature homogenization treatment on the reaction mixture at a low temperature of-5-10 DEG C, and controlling the temperature of the treated sample at 10-25 DEG C; (3) allowing the reaction mixture to stand, and (4) allowing the reaction mixture in the step (3) to enter a filled enzyme reaction column for reaction. The method provided by the invention solves the problem of homogeneity of raw materials for enzymatic glycerolysis packed bed reaction, and improves the diglyceride index in the product, so that the diglyceride index in the product can meet the DAG content limit requirement (40%) in new resource food diglyceride in China.

Description

Technical Field

[0001] The present application relates to the field of oils and fats, and specifically to a method for preparing diglyceride. Background Art

[0002] Diglyceride (DAG) is a structural lipid in which one fatty acid in triglyceride (TAG) is replaced by a hydroxyl group. DAG is a natural component of oils and fats and an intermediate product of fat metabolism in the body. It is a generally recognized as safe (GRAS) food ingredient. Since the metabolic pathways of DAG and TAG in the body are different, the beneficial functions of DAG have been of concern since the 1980s. A large number of animal experiments and clinical trials have proved that dietary DAG has the effects of reducing visceral fat, inhibiting weight gain, and lowering blood lipids.

[0003] There are many kinds of DAG synthesis processes, which can be carried out by chemical methods or enzymatic methods. Compared with chemical methods, enzymatic methods have mild reaction conditions and are the mainstream processes for DAG synthesis in recent years. The main paths for synthesizing DAG by lipase catalysis are as follows: (1) Esterification of fatty acids and glycerol; (2) Enzymatic glycerolysis of TAG and glycerol; (3) Partial hydrolysis of TAG; (4) Transesterification of TAG and MAG. Esterification and glycerolysis are two commonly used means in the industry. The esterification method has a long path and high cost and is commonly used as the synthesis path for high-content DAG; enzymatic glycerolysis is currently the most commonly used method for synthesizing low-content DAG in the industry. However, since one of the substrates is glycerol, which has strong polarity, poor miscibility with oil, and is in a non-uniform phase, the degree of homogeneity of the raw materials not only affects the reaction efficiency, but also excessive glycerol will have a great impact on the catalytic activity of the enzyme. To reduce the impact of glycerol on enzyme activity and reaction efficiency in enzymatic glycerolysis reactions, reduce enzyme activity loss, and improve the repeated service life of the enzyme, various improvement measures have been proposed by predecessors. Since this highly viscous and immiscible substrate is generally not recommended for reaction in a packed bed, the improvement is mainly aimed at batch reactions. For example, (1) Upgrading the immobilization carrier of the enzyme can solve the problem of a large amount of glycerol adsorbing the enzyme into clusters, but the homogeneity of the raw materials is still challenged; (2) Adding organic solvents or ionic liquids and other means to create a co-solvent environment for the substrates, but the use and recovery of solvents in the actual industrialization process have very high requirements for factory design and construction; (3) Adsorbing glycerol onto the carrier silica gel in advance before the reaction and then adding it to the reaction system, and glycerol is gradually released, but how to separate it in industrialization remains to be discussed; (4) Adding emulsifiers to the raw material system, but the use of additives in the product is strictly restricted by regulations, and the presence of emulsifiers may affect the clarity and transparency of the product; (5) Designing a bubbling reactor to make the raw materials and the enzyme fully contact. The packed bed reactor is the most commonly preferred for enzyme reaction industrialization, with high efficiency, easy operation, and simple structure. However, different from batch reactions, in batch reactions, the raw materials and the enzyme can be fully contacted by stirring and shaking, while in the packed bed reaction, the mixing degree of glycerol and oil in the raw materials is poor and non-uniform. Even with stirring or homogenization, glycerol is extremely easy to stratify and deposit at the bottom of the raw materials. After transportation, the stratification phenomenon of glycerol and oil is more serious, resulting in the inability of oil, glycerol, and the enzyme to fully contact and react when passing through the column bed. The unreacted glycerol is gradually adsorbed by the enzyme, which affects the enzyme activity and the DAG yield is low, affecting the industrialization efficiency. Summary of the Invention

[0004] The inventors of the present invention have found through a large number of experimental studies that by treating the raw material substrates, the raw material substrates are reacted in a packed bed reactor to prepare diglyceride. This method effectively solves the problem of the homogeneity of the raw materials in the packed bed reaction of enzymatic glycerolysis, improves the diglyceride index in the product, and enables the diglyceride index in the product to meet the DAG content limit requirement (40%) of new resource food diglyceride in China.

[0005] In the first aspect of the present invention, a method for enzymatically preparing a diglyceride composition is provided, and the method includes the following steps:

[0006] (1) Mixing a substrate oil and glycerol to obtain a reaction mixture;

[0007] (2) Performing cryogenic homogenization treatment on the reaction mixture, where the low temperature is -5 to 10 °C, and controlling the temperature of the treated sample at 10 - 25 °C;

[0008] (3) Allowing the reaction mixture to stand;

[0009] (4) Feeding the reaction mixture in step (3) into a packed enzyme reaction column for reaction to obtain a reaction product.

[0011] The substrate oil is an oil that is liquid at room temperature.

[0012] In one or more specific embodiments of the present invention, the substrate oil is selected from one or more of soybean oil, corn oil, rice bran oil, sunflower oil, rapeseed oil, peanut oil, wheat germ oil, walnut oil, olive oil, linseed oil, sesame oil, tea oil, safflower oil, tiger nut oil, palm olein, medium and long chain fatty acid esters, rice bran oil, palm kernel olein, cottonseed oil, perilla oil, castor oil, coconut oil, olive oil, Chinese tallow tree seed oil, almond oil, apricot kernel oil, evening primrose oil, hazelnut oil, pumpkin seed oil, walnut oil, grape seed oil, borage oil, seabuckthorn oil, tomato seed oil, pumpkin seed oil, macadamia nut oil.

[0013] In one or more specific embodiments of the present invention, in the medium and long chain fatty acid esters, the medium chain fatty acid is a straight-chain saturated fatty acid with 6 to 12 carbon atoms.

[0014] In one or more specific embodiments of the present invention, in the medium and long chain fatty acid esters, the long chain fatty acid is a monounsaturated long chain fatty acid or a polyunsaturated long chain fatty acid, preferably a monounsaturated long chain fatty acid.

[0015] In one or more specific embodiments of the present invention, the long chain fatty acid is a straight-chain saturated or unsaturated fatty acid with 14 to 24 carbon atoms.

[0016] In one or more specific embodiments of the present invention, the molar ratio of the substrate oil to glycerol is 2:1 - 1:1.

[0017] In one or more specific embodiments of the present invention, the cryogenic homogenization treatment is carried out under an ice-water bath, a salt bath, dry ice, or liquid nitrogen for homogenization treatment.

[0018] In one or more specific embodiments of the present invention, the homogenization speed of the homogenization treatment is 10000 - 20000 rpm.

[0019] In one or more specific embodiments of the present invention, the homogenization time of the homogenization treatment is 5 - 15 min.

[0020] In one or more specific embodiments of the present invention, based on the total mass of the reaction mixture, the reaction mixture in step (1) contains 0.25 - 0.75% of water.

[0021] In one or more specific embodiments of the present invention, the reaction in step (4) is a glycerolysis reaction.

[0022] In one or more specific embodiments of the present invention, the temperature of the glycerolysis reaction is 50 - 70 °C.

[0023] In one or more specific embodiments of the present invention, the feeding rate of the reaction mixture in the glycerolysis reaction is 0.5 - 1 times the number of milliliters per hour of the mass of the enzyme in grams.

[0024] In one or more specific embodiments of the present invention, the enzyme filled in the packed enzyme reaction column is lipase.

[0025] In one or more specific embodiments of the present invention, the lipase is selected from one or more mixtures of lipases from porcine pancreatic lipase, Thermomyces lanuginosus, Mucor miehei, Pseudomonas fluorescens, Aspergillus niger, Rhizomucor miehei, Candida lipolytica, Rhizopus sp., Candida antarctica, and their genetically modified strains; preferably, the lipase is one or more of the lipases from Thermomyces lanuginosus, Rhizomucor miehei, Rhizopus oryzae, and Candida antarctica.

[0026] In one or more specific embodiments of the present invention, the injection temperature of the reaction mixture in step (4) is above room temperature, preferably 20 - 70 °C.

[0027] In one or more specific embodiments of the present invention, the DAG index of the diglyceride composition is 40 - 50%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

[0028] In one or more specific embodiments of the present invention, the DAG index of the diglyceride composition is 40-49%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

[0029] In a second aspect of the present invention, there is provided a diglyceride composition prepared by the method described in the first aspect of the present invention.

[0030] In one or more specific embodiments of the present invention, the DAG index of the diglyceride composition is 40-50%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

[0031] In one or more specific embodiments of the present invention, the DAG index of the diglyceride composition is 40-49%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

[0032] In a third aspect of the present invention, there is provided an oil and fat composition comprising the diglyceride composition described in the present invention.

[0033] In a fourth aspect of the present invention, there is provided a method for improving the stability of a raw material substrate, the method comprising the following steps:

[0034] (1) Mixing a substrate oil and glycerol to obtain a reaction mixture;

[0035] (2) Performing low-temperature homogenization treatment on the reaction mixture, the low temperature being -5 to 10°C, and controlling the temperature of the treated sample at 10-25°C;

[0036] (3) Allowing the reaction mixture to stand.

[0037] Technical effects

[0038] Compared with the prior art, the raw materials treated by the method of the present invention do not separate layers for a long time, glycerol does not precipitate, and after entering the column, the reduction in enzyme activity caused by the encapsulation of the enzyme due to excessive glycerol is reduced, and vegetable oil, glycerol and the enzyme can uniformly contact and react, improving the DAG yield, thereby solving the problem of the homogeneity of the raw materials in the enzymatic glycerolysis packed bed reaction, increasing the diglyceride index in the product, and enabling the diglyceride index in the product to meet the DAG content limit requirement (40%) for new resource foods of diglyceride in China. Specific embodiments

[0039] In the present invention, unless otherwise specified, percentages (%) or parts refer to weight percentages or weight parts relative to the composition.

[0040] In the present invention, unless otherwise specified, the components involved or their preferred components can be combined with each other to form new technical solutions.

[0041] In the present invention, unless otherwise specified, all the embodiments and preferred embodiments mentioned herein can be combined with each other to form new technical solutions.

[0042] In the present invention, unless otherwise specified, all the technical features and preferred features mentioned herein can be combined with each other to form new technical solutions.

[0043] In the present invention, unless otherwise stated, the sum of the contents of the components in the composition is 100%.

[0044] In the present invention, unless otherwise stated, the sum of the parts of the components in the composition can be 100 parts by weight.

[0045] In the present invention, unless otherwise specified, the numerical range "a - b" represents an abbreviated representation of any real number combination between a and b, where both a and b are real numbers.

[0046] In the present invention, unless otherwise specified, the integer numerical range "a - b" represents an abbreviated representation of any integer combination between a and b, where both a and b are integers. For example, the integer numerical range "1 - N" represents 1, 2... N, where N is an integer.

[0047] In the present invention, unless otherwise specified, "their combination" refers to a multi-component mixture of the said respective elements, such as a two-component, three-component, four-component, and up to the maximum possible multi-component mixture.

[0048] If not specifically indicated, the term "a" used in this specification means "at least one".

[0049] If not specifically indicated, the basis of the percentages (including weight percentages) described in the present invention is the total weight of the said composition.

[0050] The "ranges" disclosed herein are in the form of a lower limit and an upper limit. There can be one or more lower limits, and one or more upper limits respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower limit and upper limit define the boundary of a particular range. All ranges that can be defined in this way are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range.

[0051] In this article, unless otherwise specified, each reaction step can be carried out sequentially or non-sequentially. For example, other steps can be included between each reaction step, and the reaction steps can also be reversed in order. Preferably, the reaction methods herein are carried out sequentially.

[0052] The technical solution of the present invention will be further specifically described as follows:

[0053] <First aspect>

[0054] A method for preparing a diglyceride composition by an enzymatic method, the method comprising the following steps:

[0055] (1) Mixing a substrate oil and glycerol to obtain a reaction mixture;

[0056] (2) Performing low-temperature homogenization treatment on the reaction mixture, the low temperature being -5 to 10 °C, and controlling the temperature of the treated sample at 10 - 25 °C;

[0057] (3) Allowing the reaction mixture to stand,

[0058] (4) The reaction mixture in step (3) enters a packed enzyme reaction column for reaction.

[0059] Preferably, the substrate oil is an oil that is liquid at room temperature.

[0060] In the present invention, the preparation of the diglyceride is obtained by a glycerolysis reaction of triglyceride in the oil with glycerol, so oils containing triglyceride can all be used as the substrate of the present invention. In one or more specific embodiments of the present invention, the substrate oil is selected from one or more of soybean oil, corn oil, rice bran oil, sunflower oil, rapeseed oil, peanut oil, wheat germ oil, walnut oil, olive oil, linseed oil, sesame oil, tea oil, safflower oil, tiger nut oil, palm olein, medium and long-chain fatty acid esters, rice bran oil, palm kernel olein, cottonseed oil, perilla oil, castor oil, coconut oil, olive oil, Chinese tallow tree seed oil, almond oil, apricot kernel oil, evening primrose oil, hazelnut oil, pumpkin seed oil, walnut oil, grape seed oil, borage oil, seabuckthorn oil, tomato seed oil, pumpkin seed oil, macadamia nut oil.

[0061] In one or more specific embodiments of the present invention, the medium-chain fatty acid in the medium and long-chain fatty acid esters is a straight-chain saturated fatty acid having 6 to 11 carbon atoms.

[0062] In one or more specific embodiments of the present invention, the long-chain fatty acid in the medium and long-chain fatty acid esters is a monounsaturated long-chain fatty acid or a polyunsaturated long-chain fatty acid, preferably the long-chain fatty acid is a monounsaturated long-chain fatty acid.

[0063] In one or more specific embodiments of the present invention, the long-chain fatty acid is a straight-chain saturated or unsaturated fatty acid having 14 to 24 carbon atoms.

[0064] In one or more specific embodiments of the present invention, the molar ratio of the substrate oil to glycerol is 2:1 - 1:1.

[0065] In one or more specific embodiments of the present invention, homogenization treatment is adopted to make glycerol and oil mix evenly; and it is stably maintained for a relatively long time, such as 1 - 10 h, or 1 - 7 h, or 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h.

[0066] In one or more specific embodiments of the present invention, the low - temperature homogenization treatment is carried out under an ice - water bath, a brine bath, dry ice, or liquid nitrogen.

[0067] In one or more specific embodiments of the present invention, the purpose of the homogenization treatment is to make the substrate glycerol and oil mix evenly and maintain stability for more than 1 h. The time and speed of homogenization can be adjusted according to the amount of materials, etc. For example, the homogenization speed is 10000 - 20000 rpm, and the homogenization time is 5 - 15 min.

[0068] In one or more specific embodiments of the present invention, based on the total mass of the reaction mixture, the reaction mixture in step (1) contains 0.25 - 0.75% water, or 0.25 - 0.5% water, or 0.5 - 0.75% water. If the amount of water is too high, on the one hand, it will reduce the system stability; on the other hand, it will exacerbate the degree of hydrolysis, resulting in an increase in the formation of free fatty acids.

[0069] In one or more specific embodiments of the present invention, the material after homogenization treatment is stored in a raw material storage tank and left standing without stirring. If the material is stirred, its stability will deteriorate.

[0070] In one or more specific embodiments of the present invention, the temperature in the raw material storage tank is room temperature - 70 °C, or 20 - 70 °C. In one or more specific embodiments of the present invention, the reaction in step (4) is a glycerolysis reaction.

[0071] In one or more specific embodiments of the present invention, the temperature of the glycerolysis reaction is 50 - 70 °C.

[0072] In one or more specific embodiments of the present invention, the feeding rate of the reaction mixture in the glycerolysis reaction is 0.5 - 1 times the number of grams of the enzyme mass per hour in milliliters.

[0073] In one or more specific embodiments of the present invention, the enzyme filled in the packed enzyme reaction column is lipase. In the present invention, the function of the enzyme filled in the enzyme reaction column is to accelerate the glycerolysis reaction, so common lipases in the art can achieve the effects of the present invention.

[0074] In one or more specific embodiments of the present invention, the enzyme filled in the packed enzyme reaction column can be immobilized lipase.

[0075] In one or more specific embodiments of the present invention, the lipase is selected from one or more mixtures of lipases from porcine pancreatic lipase, Thermomyces lanuginosus, Mucor miehei, Pseudomonas fluorescens, Aspergillus niger, Rhizomucor miehei, Candida lipolytica, Rhizopus sp., Candida antarctica, and their genetically modified strains; preferably, the lipase is one or more of the lipases from Thermomyces lanuginosus, Rhizomucor miehei, Rhizopus oryzae, and Candida antarctica.

[0076] In one or more specific embodiments of the present invention, the injection temperature of the reaction mixture in step (4) is above room temperature, preferably 20 - 70 °C.

[0077] In one or more specific embodiments of the present invention, the injection temperature of the reaction mixture in step (4) is 20 - 60 °C, or 60 - 70 °C.

[0078] In one or more specific embodiments of the present invention, after the reaction in step (4), there is also a step of separation and purification.

[0079] In one or more specific embodiments of the present invention, the purpose of the separation and purification is to further purify the purity of diglyceride in the product. Examples of the steps include one or more of molecular distillation, short-path distillation, evaporation concentration, and stripping.

[0080] <Second aspect>

[0081] A diglyceride composition, which is prepared by the method described in the first aspect of the present invention.

[0082] In one or more specific embodiments of the present invention, the DAG index of the diglyceride composition is 40 - 50%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

[0083] In one or more specific embodiments of the present invention, the DAG index of the diglyceride composition is 40-49%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride, or the DAG index is 40-48.5%.

[0084] <The third aspect>

[0085] An oil and fat composition, the composition comprising the diglyceride composition of the present invention.

[0086] <The fourth aspect>

[0087] A method for improving the stability of a raw material substrate, the method comprising the following steps:

[0088] (1) Mixing the substrate oil and glycerol to obtain a reaction mixture;

[0089] (2) Performing low-temperature homogenization treatment on the reaction mixture, the low temperature being -5 to 10°C, and controlling the temperature of the treated sample at 10-25°C;

[0090] (3) Allowing the reaction mixture to stand.

[0091] In one or more specific embodiments of the present invention, the substrate oil is selected from one or more of soybean oil, corn oil, rice oil, sunflower oil, rapeseed oil, peanut oil, wheat germ oil, walnut oil, olive oil, linseed oil, sesame oil, tea oil, safflower oil, tiger nut oil, palm olein, medium and long chain fatty acid esters, rice oil, palm kernel olein, cottonseed oil, perilla oil, castor oil, coconut oil, olive oil, Chinese tallow tree seed oil, almond oil, apricot kernel oil, evening primrose oil, hazelnut oil, pumpkin seed oil, walnut oil, grape seed oil, borage oil, seabuckthorn oil, tomato seed oil, pumpkin seed oil, macadamia nut oil.

[0092] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods, reagents and materials used in the examples,

[0093] Unless otherwise specified, they are conventional methods, reagents and materials in the art. The raw material compounds in the examples can all be obtained through commercial channels.

[0094] Lipase: RM IM (self-made)

[0095] Preparation process:

[0096] Rhizomucor miehei lipase solution (RML), produced by the production strain BC4 (deposit number CGMCC No. 18825), is prepared by Aspergillus oryzae fermentation. The fermentation method refers to Patent CN201410822387 (a method for fermenting lipase). The obtained fermentation broth is pretreated by adding 2 times of pure water and 5% diatomaceous earth RS300, and then prepared into a liquid enzyme through plate and frame filtration, vertical plate and frame secondary filtration, ultrafiltration concentration, and sterile filtration for subsequent enzyme immobilization. The solvent of the lipase solution is 10-

[0097] 100 mM phosphate buffer at pH 5.5 - 6.5, without preservatives and protectants.

[0098] Set the cooling water temperature < 15°C, and pass cooling water into the jacket of an 800 L adsorption tank. Add 400 kg of RML lipase solution (10 mg / g, 60000 U / g) into the 800 L adsorption tank, then add 60 kg of Tween 80, and stir for 60 min at a stirring motor frequency of 20 Hz through a ribbon agitator. After adding 200 kg of ion exchange resin LX1000HAA, continue stirring for 22 h, and separate the solid-liquid mixture using a bag filter centrifuge. The solid material is added to a 420 L fluidized bed dryer in batches. Use a Sartorius MA150 moisture analyzer to measure the moisture content of the immobilized enzyme irregularly. When the moisture content drops to about 12%, it is bagged and stored, and weighed. The transesterification activity of the self-made immobilized enzyme is about 450 IUN / g.

[0099] Sunflower oil: Purchased from the refined first-grade sunflower oil of the Jinlongyu brand

[0100] Packed bed reactor: Julabo Technology (Beijing) Co., Ltd.

[0101] Stable time:

[0102] Take a total of 10 ml of samples from the upper, middle, and lower parts of the container every 1 h and place them in a 15 ml centrifuge tube to stand still. Observe the proportion of glycerol after the final stratification of the sample. The percentage of the proportion of glycerol in the sample to the proportion of glycerol in the original sample is the glycerol retention ratio. When the glycerol retention ratio in the raw material system ≥ 80% is regarded as stable. When it is observed that the raw material system is unstable, the stable time of the sample is considered to be the current time minus 1; for example, when it is observed that it is unstable at the 2nd h, the stable time is recorded as 1 h.

[0103] Calculation of DAG index:

[0104] Referring to CN201510167583.1, define glycerol diester / (glycerol diester + triglyceride) as the glycerol diester index. This index is the ratio of the amount of glycerol diester in the reaction product to the total amount of glycerol diester and triglyceride in the reaction product. The amounts of glycerol diester and triglyceride are tested by gas chromatography.

[0105] The operation procedures of the examples and comparative examples are as follows:

[0106] Weigh the raw materials (reaction substrates, and the oil is sunflower oil) in a certain proportion (as shown in Table 1), homogenize (15,000 rpm, 10 min) or not, and place them in the raw material storage tank, controlling the temperature and stirring speed of the raw material storage tank; load 150 g of lipase (RM enzyme) into the enzyme reaction column in a dry method, and heat the enzyme reaction column to a reaction temperature of 60 °C; the raw materials can be pumped into the enzyme reaction column for reaction by controlling different flow rates with a peristaltic pump, and the reaction products enter the collection bottle. After the reaction reaches stability in 4 h, analyze the DAG content every 2 h, and take the average value of the stable time period as the DAG content under this condition. The process parameters in each example and comparative example are shown in Table 1.

[0107] Among them, the judgment basis for the uniform stability of the raw materials is that the glycerol retention ratio in the sample is above 80%. The glycerol retention ratio is calculated by taking 10 ml of the upper, middle, and lower layer mixed samples in the raw material bottle at different times, and calculating the percentage of the glycerol contained in the 10 ml sample in the theoretical glycerol in the raw material after the sample stands still and the glycerol completely precipitates. The stable time of the raw materials and the DAG index in the products obtained by treating each example and comparative example are shown in Table 2.

[0108] Table 1 Process parameters of examples and comparative examples

[0109]

[0110]

[0111] Table 2 Stable time of raw materials and DAG index of products in each example and comparative example

[0112]

[0113] The experimental results show that if the substrates are not homogenized at low temperature as in Comparative Examples 3 and 6, the substrate raw materials of oil and glycerol are unstable and cannot pass through the lipase column; if the substrates are stirred without standing after homogenization as in Comparative Examples 4 and 5, the substrate raw materials of oil and glycerol are also unstable and cannot pass through the lipase column; if the substrates are not homogenized as in Comparative Examples 7 and 8, the substrates will immediately stratify. If the molar ratio of oil to glycerol in the substrate is too low or too high, or the injection flow rate is too high, the diglyceride index in the finally obtained composition is lower than that in the examples.

[0114] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing diglyceride by enzymatic method, the method comprising the following steps: (1) Mixing the substrate oil and glycerol to obtain a reaction mixture; (2) Performing low-temperature homogenization treatment on the reaction mixture, the low temperature being -5 to 10 °C, and controlling the temperature of the treated sample at 10 - 25 °C; (3) Allowing the reaction mixture to stand; (4) Feeding the reaction mixture in step (3) into a packed enzyme reaction column for reaction.

2. The method according to claim 1, characterized in that The method satisfies one or more of the following conditions: (a) The substrate oil is an oil that is liquid at room temperature; and / or (b) The substrate oil is selected from one or more of soybean oil, corn oil, rice oil, sunflower oil, rapeseed oil, peanut oil, wheat germ oil, walnut oil, olive oil, linseed oil, sesame oil, tea oil, safflower oil, cyperus esculentus oil, palm olein, medium and long chain fatty acid esters, rice oil, palm kernel olein, cottonseed oil, perilla oil, castor oil, coconut oil, olive oil, Chinese tallow tree seed oil, almond oil, apricot kernel oil, evening primrose oil, hazelnut oil, pumpkin seed oil, walnut oil, grape seed oil, borage oil, seabuckthorn oil, tomato seed oil, pumpkin seed oil, macadamia nut oil; and / or (c) In the medium and long chain fatty acid esters, the medium chain fatty acid is a straight-chain saturated fatty acid having 6 to 12 carbon atoms; and / or (d) In the medium and long chain fatty acid esters, the long chain fatty acid is a monounsaturated long chain fatty acid or a polyunsaturated long chain fatty acid, preferably a monounsaturated long chain fatty acid; and / or (e) The long chain fatty acid is a straight-chain saturated or unsaturated fatty acid having 14 to 24 carbon atoms.

3. The method according to claim 1, wherein The molar ratio of the substrate oil to glycerol is 2:1 - 1:

1.

4. The method according to claim 1, characterized in that, The method satisfies one or more of the following conditions: (f) The low-temperature homogenization treatment is carried out in an ice-water bath, a salt bath, dry ice, or liquid nitrogen; and / or (g) The homogenization speed of the homogenization treatment is 10000 - 20000 rpm; and / or (h) The homogenization time of the homogenization treatment is 5 - 15 min; and / or (i) Based on the total mass of the reaction mixture, the reaction mixture in step (1) contains 0.25 - 0.75% of water; and / or (j) The reaction in step (4) is a glycerolysis reaction; preferably, the temperature of the glycerolysis reaction is 50 - 70 °C; and / or (k) In the glycerolysis reaction, the feeding speed of the reaction mixture is 0.5 - 1 times the number of grams of enzyme mass per hour in milliliters; and / or (l) The enzyme filled in the filling enzyme reaction column is lipase. Preferably, the lipase is selected from lipases of porcine pancreatic lipase, Thermomyces lanuginosus, Mucor miehei, Pseudomonas fluorescens, Aspergillus niger, Rhizomucor miehei, Candida lipolytica, Rhizopus sp., Candida antarctica and their genetically modified strains; preferably, the lipase is one or more of lipases from Thermomyces lanuginosus, Rhizomucor miehei, Rhizopus oryzae, Candida antarctica; and / or (m) The injection temperature of the reaction mixture in step (4) is above room temperature, preferably 20-70 °C.

5. The method according to claim 1, wherein The DAG index of the diglyceride composition is 40-50%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

6. The method according to claim 1, characterized in that, The DAG index of the diglyceride composition is 40-49%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

7. A diglyceride composition, characterized in that The DAG index of the diglyceride composition is 40-50%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride. The composition is prepared by the method according to any one of claims 1-6.

8. The composition according to claim 7, wherein The DAG index of the diglyceride composition is 40-49%, and the DAG index is the ratio of the weight of diglyceride to the total weight of diglyceride and triglyceride.

9. An oil and fat composition, characterized in that, The composition comprises the diglyceride composition according to claim 7 or 8; or comprises the composition prepared by the method according to any one of claims 1-6.

10. A method for improving the stability of a raw material substrate, the method comprising the following steps: (1) Mixing a substrate oil and glycerol to obtain a reaction mixture; (2) Performing low-temperature homogenization treatment on the reaction mixture, the low temperature being -5 to 10 °C, and controlling the temperature of the treated sample at 10-25 °C; (3) Allowing the reaction mixture to stand.

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