A preparation method of high-purity diglyceride oil and its product

Abstract

The invention discloses a preparation method of high-purity diglyceride oil and a product thereof. The method comprises the following steps: using lipase to catalyze a glycerol hydrolysis reaction between glycerol and edible oil to obtain a glycerol hydrolysis product. During the glycerol hydrolysis reaction, in order to improve the solubility of glycerol and oil, glycerol is added in stages, and the solubility of glycerol and oil is coordinated with the solubilization effect of monoglyceride and the mixing effect of high-pressure homogenization, so as to improve the solubility of glycerol and oil and improve the reaction efficiency. The activity of partial glyceride lipase Lipase G50 is improved by coordinated pH value adjustment and non-ionic surface imprinting. The imprinted Lipase G50 is used as a catalyst and free fatty acids are used as acyl donors to catalyze the esterification reaction between monoglyceride and free fatty acids in the glycerol hydrolysis product under a certain vacuum degree. The free fatty acids are removed by molecular distillation, so as to finally obtain a high-purity diglyceride product.

Description

Technical Field

[0001] The invention belongs to the technical field of oils and fats, and specifically relates to a preparation method of high-purity diglyceride oil and a product thereof. Background Art

[0002] Diacylcerol (DAG) is a type of structural lipid in which one fatty acid in triglyceride is replaced by a hydroxyl group. DAG is a trace component of natural plant oils and fats and an endogenous intermediate product of fat metabolism in the body. It is a generally recognized as safe (GRAS) food ingredient. In recent years, it has been found that dietary DAG has the effects of reducing visceral fat, inhibiting weight gain, and lowering blood lipids, and has therefore received widespread attention. DAG has many advantages such as safety, nutrition, good processing suitability, and high human compatibility. It is a multifunctional additive that has been widely used in food, medicine, chemical (cosmetics) and other industries.

[0003] The main methods for synthesizing diacylglycerol are chemical method and enzyme catalysis method. The chemical method mainly uses an alkaline catalyst to react glycerol with oil for glycerol hydrolysis. The advantage of the chemical method is that the reaction route is simple, the time is short, the cost is relatively low, and it is easy to scale up, but the disadvantages are also very obvious. There are strict requirements for temperature, air pressure and other conditions during the reaction process, and side reactions are prone to occur; the chemical catalysts used may not only pollute the environment, but also easily cause consumers to worry about food safety; in addition, the target product obtained by the chemical method has poor flavor and color. Compared with the chemical method, the bioenzymatic preparation has better results in terms of yield, purity, reaction time and safety. However, further research and improvement are needed to apply the enzymatic method to the industrial production of DAG. The most important element in the enzyme-catalyzed synthesis of DAG is lipase. Lipase is mainly derived from fat-containing animals and plants, eukaryotic organisms such as fungi, and prokaryotes such as bacteria. It is a type of enzyme with multiple catalytic abilities. It can catalyze the hydrolysis, alcoholysis, esterification, transesterification, and reverse synthesis of triglycerides under certain conditions. It is characterized by its specificity, high efficiency, and greenness. However, in actual reaction systems, due to differences from biological systems, lipases often face the characteristics of low activity and reduced specificity.

[0004] At present, the methods for enzymatic synthesis of DAG mainly include glycerol hydrolysis, esterification and hydrolysis, as well as a combination of various methods. Enzymatic glycerol hydrolysis is widely used due to its high product yield and convenient separation. However, there are still some disadvantages in the enzymatic glycerol hydrolysis: (1) the poor compatibility between glycerol and oil leads to reduced reaction efficiency; (2) since the enzymatic glycerol hydrolysis reaction is a reversible reaction, in addition to diglycerol, a large amount of monoglycerol is produced in the reaction system. The traditional method uses molecular distillation to remove monoglycerol, which produces a considerable amount of glycidyl esters and chloropropanol esters, thus causing safety problems; (3) the catalytic activity of the enzyme is low. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing high-purity diglyceride oil.

[0008] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing high-purity diglyceride oil, comprising:

[0009] Using lipase to catalyze the glycerol hydrolysis reaction between glycerol and edible oil to obtain a glycerol hydrolysis product, wherein during the glycerol hydrolysis reaction, glycerol is added in stages;

[0010] Using imprinted Lipase G50 as a catalyst and free fatty acids as acyl donors, the monoglyceride in the glycerol hydrolysis product was catalyzed to undergo esterification with free fatty acids under vacuum, and the free fatty acids were removed by molecular distillation to finally obtain a high-purity diglyceride product.

[0011] As a preferred embodiment of the preparation method of the present invention, the conditions of the glycerol hydrolysis reaction are as follows: the reaction temperature is 50-70°C, the molar ratio of glycerol to edible oil is 1.5-74:1, and the amount of lipase added is 10-20wt% based on the weight of the edible oil.

[0012] As a preferred embodiment of the preparation method of the present invention, the stepwise addition of glycerol comprises:

[0013] In the first stage, 10-30% glycerol is added, homogenized at 20-40 MPa for 3-10 min, lipase is added, the stirring speed is 500-800 rpm, the reaction is carried out for 0.5-1.5 h, and the lipase is filtered out;

[0014] In the second stage, 30-50% glycerol is added, homogenized at 20-40 MPa for 3-10 min, lipase is added again to react for 0.5-1.5 h, the stirring speed is 500-800 rpm, and the lipase is filtered out;

[0015] In the third stage, 20-60% glycerol is added, homogenized at 20-40 MPa for 3-10 minutes, and lipase is added for reaction for 6-9 hours with a stirring rate of 600 rpm.

[0016] As a preferred embodiment of the preparation method of the present invention, the glycerol is food grade glycerol, the lipase is commercial lipase, including but not limited to Novozym 435, Lipozyme RM IM and NS 40086, and the edible oils include but not limited to sunflower oil, soybean oil and linseed oil.

[0017] As a preferred embodiment of the preparation method of the present invention, wherein: the preparation method of obtaining the glycerol hydrolysis product further comprises:

[0018] After the glycerol decomposition reaction, the temperature is heated to 85-95° C. and stirred slowly at a rotation speed of 10-30 rpm for 0.5-1.5 h to separate the glycerol and glyceride. The glyceride is the glycerol decomposition product.

[0019] As a preferred embodiment of the preparation method of the present invention, the preparation method of the imprinted Lipase G50 comprises:

[0020] Prepare a buffer solution with a pH value of 5 to 6, add 15 to 30% of Lipase G50 by mass, stir at 25° C. for 30 to 45 minutes, filter and vacuum dry to remove moisture, and obtain pH imprinted Lipase G50;

[0021] A nonionic surfactant is added to isopropanol at a concentration of 20 to 100 mg / mL, 15 to 30% of pH-imprinted Lipase G50 is added, the mixture is stirred at 25°C for 30 to 45 minutes, freeze-dried for 12 to 36 hours, excess imprinted template is eluted with a non-polar solvent, and the solvent is removed by vacuum drying to obtain pH and surfactant dual-imprinted Lipase G50.

[0022] As a preferred embodiment of the preparation method of the present invention, the non-ionic surfactant includes Tween 80, Tween 60 and Tween 40, and the non-polar solvent includes n-hexane and octane.

[0023] As a preferred embodiment of the preparation method of the present invention, the esterification reaction comprises:

[0024] The molar ratio of free fatty acids to glycerol backbone of glyceride is 2-4:1, the stirring speed is 600-800 rpm, the temperature is 30-50° C., the amount of imprinted lipase Lipase G50 added is 4-8 wt %, the vacuum degree is 10-30 mbar, and the reaction time is 6-10 h;

[0025] The free fatty acids are fatty acids derived from edible oils and fats, including but not limited to sunflower oil, soybean oil and linseed oil.

[0026] As a preferred embodiment of the preparation method of the present invention, the conditions for removing fatty acids by molecular distillation are: distillation temperature is 150-170°C, pressure is 2-5Pa, and condenser temperature is 25-35°C.

[0027] Another object of the present invention is to overcome the deficiencies in the prior art and provide a high-purity diglyceride oil product prepared by a method for preparing high-purity diglyceride oil, wherein the diglyceride content of the product is greater than 90%, the glycidyl ester content is less than 2 mg / Kg, and the chloropropanol ester content is less than 0.5 mg / Kg.

[0028] Beneficial effects of the present invention:

[0029] (1) In the lipase-catalyzed glycerol hydrolysis reaction, the present invention improves the solubility of glycerol and edible oils and fats and improves the reaction efficiency by adding glycerol in stages, coordinating the volume expansion of monoglycerides and high-pressure homogenization mixing; the further beneficial effect of adding glycerol in stages is that since monoglycerides and diglycerides are continuously generated during the reaction process, the amount of glycerol added can be precisely and quantitatively controlled to enable glycerol and oils to be better mixed;

[0030] (2) The present invention is based on the memory characteristics of biomacromolecules such as enzymes, that is, the characteristics of the enzyme in the aqueous solution will be maintained after the water is removed by a certain method. Therefore, by adjusting the optimal reaction pH and combining the effect of non-ionic surfactants to double-imprint partial glyceride lipase Lipase G50, its activity and stability are greatly improved; by utilizing the substrate selectivity of Lipase G50, by using Lipase G50 to catalyze the esterification reaction between monoglyceride in the glycerol hydrolysis product and free fatty acids, the monoglyceride in the reaction product is converted into diglyceride, thereby increasing the yield of diglyceride and the purity of diglyceride in the product. At the same time, only fatty acids need to be removed in the later molecular distillation stage, so that milder removal conditions can be used. Therefore, compared with the traditional process, the harmful substances in the final product, including glycidyl esters and propylene glycol esters, are also greatly reduced. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0034] Determination of Lipase G50 esterification activity: Glycerol and oleic acid were added to a batch reactor at a molar ratio of 1:1, and 4% Lipase G50 was added. The reaction was carried out at a temperature of 30°C and a stirring rate of 600 rpm for 1 hour. The initial activity of the enzyme was evaluated based on the esterification rate (%) of fatty acids in the system after 1 hour of reaction.

[0035] Example 1

[0036] Add sunflower oil into a batch reactor, fill it with nitrogen for protection, raise the temperature to 60°C, add 1.5% monoglyceride by mass, and stir to fully dissolve the monoglyceride in the sunflower oil;

[0037] Glycerol was added in three stages according to a molar ratio of glycerol to sunflower oil of 4:1. In the first stage, 10% glycerol was added, homogenized at 20 MPa for 10 minutes to fully mix the glycerol and sunflower oil, and lipase Novozym 435 was added, the amount of lipase added was 20wt% of the weight of the sunflower oil, and stirring was started at a speed of 600 rpm. The reaction was carried out for 1 hour and the lipase was filtered out. In the second stage, 30% glycerol was added, homogenized at 20 MPa for 10 minutes, the lipase was put in again for reaction for 1 hour, the stirring speed was 600 rpm, and the lipase was filtered out. In the third stage, 60% glycerol was added, homogenized at 20 MPa for 10 minutes, the lipase was put in for reaction for 6 hours, and the stirring speed was 600 rpm.

[0038] After the reaction, the system temperature was heated to 95°C and slowly stirred at a speed of 20 rpm for 0.5 h to accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, obtain glycerol hydrolysis product, and recover glycerol as raw material for new glycerol hydrolysis reaction.

[0039] Example 2

[0040] Add soybean oil into a batch reactor, fill it with nitrogen for protection, raise the temperature to 70°C, add 1% monoglyceride by mass, and stir to fully dissolve the monoglyceride in sunflower oil;

[0041] Glycerol was added in three stages according to a molar ratio of glycerol to soybean oil of 2:1. In the first stage, 20% glycerol was added and homogenized at 30 MPa for 6 minutes to fully mix the glycerol and soybean oil. Lipozyme RM IM was added in an amount of 15wt% of the soybean oil. Stirring was started at a speed of 800 rpm. The reaction was carried out for 0.5 h and the lipase was filtered out. In the second stage, 40% glycerol was added and homogenized at 30 MPa for 6 minutes. The lipase was put in again for reaction for 0.5 h at a stirring speed of 800 rpm and the lipase was filtered out. In the third stage, 40% glycerol was added and homogenized at 30 MPa for 6 minutes. The lipase was put in for reaction for 9 h at a stirring speed of 800 rpm.

[0042] After the reaction, the system temperature was heated to 90°C and slowly stirred at a speed of 30 rpm for 1 h to accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, obtain glycerol hydrolysis product, and recover glycerol as raw material for new glycerol hydrolysis reaction.

[0043] Example 3

[0044] Add linseed oil into a batch reactor, fill it with nitrogen for protection, raise the temperature to 50°C, add 0.5% monoglyceride by mass, and stir to fully dissolve the monoglyceride in the sunflower oil;

[0045] Glycerol was added in three stages according to a molar ratio of glycerol to linseed oil of 1.5:1. In the first stage, 30% glycerol was added and homogenized at 40 MPa for 3 minutes to fully mix the glycerol and linseed oil. Lipase NS 40086 was added in an amount of 10 wt% of the linseed oil, and stirring was started at a speed of 500 rpm. The reaction lasted for 1.5 hours and the lipase was filtered out. In the second stage, 50% glycerol was added and homogenized at 40 MPa for 3 minutes. The lipase was put in again to react for 1.5 hours at a stirring speed of 500 rpm and the lipase was filtered out. In the third stage, 20% glycerol was added and homogenized at 40 MPa for 3 minutes. The lipase was put in to react for 8 hours at a stirring speed of 500 rpm.

[0046] After the reaction, the system temperature was heated to 85°C and slowly stirred at a speed of 10 rpm for 1.5 h to accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, obtain glycerol hydrolysis product, and recover glycerol as raw material for new glycerol hydrolysis reaction.

[0047] Table 1. Glyceride composition of the glycerol hydrolysis products in Examples 1 to 3

[0048] Example 1 Example 2 Example 3 Monoglyceride (%) 71.5 56.7 51.2 Diacylceride (%) 25.5 36.7 39.9 Triglyceride (%) 3.0 6.6 8.9

[0049] Example 4

[0050] Biomacromolecule enzymes are soft in aqueous solution, rigid in non-aqueous solution, and have memory properties in their structure.

[0051] The optimal reaction pH value of the enzyme is adjusted by the aqueous solution state, and the lid of the active center of the lipase is opened by a surfactant, and the moisture is removed by freeze-drying to fix its conformation, which will help it maintain its active conformation and improve its catalytic efficiency.

[0052] Partial glyceride lipase Lipase G50 has substrate specificity and is only active on monoglycerides and diglycerides, but does not react to triglycerides. Using partial glyceride lipase Lipase G50 to catalyze the esterification reaction between monoglyceride and fatty acids in the glyceride product to generate diglyceride under a certain vacuum degree will greatly increase the yield of diglyceride in the reaction and improve the purity of diglyceride in the product. Partial glyceride lipase Lipase G50 has an optimal reaction pH value, at which the lipase has maximum reaction activity.

[0053] Therefore, by screening the optimal pH value of Lipase G50 and removing water by freeze-vacuum drying, Lipase G50 can maintain the optimal reaction conformation.

[0054] Therefore, buffer solutions with pH values ​​of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8 were prepared, Lipase G50 was added thereto, mixed thoroughly to achieve ion exchange equilibrium, and then the solution was freeze-dried in vacuum for 2 h to remove moisture, and its esterification activity was analyzed.

[0055] Table 2. Esterification activity of Lipase G50 at different pH values

[0056]

[0057]

[0058] It can be seen from the above table that among the Lipase G50 with fixed conformation after pH treatment, the esterification activity of Lipase G50 with a pH value between 4.5-6.5 is higher than that of the untreated one, and the esterification activity of Lipase G50 treated with a pH value of 5-6 is significantly higher than that of other pH values.

[0059] Example 5

[0060] A buffer solution with a pH value of 6 was prepared, and 30% of Lipase G50 by mass was added, and the mixture was stirred at 25°C for 30 minutes. After filtering, the mixture was dried in a vacuum to remove moisture, and pH-imprinted Lipase G50 was obtained. The non-ionic surfactant Tween 80 was dissolved in acetone at a concentration of 100 mg / mL, and 30% of pH-imprinted Lipase G50 was added. The mixture was stirred at 25°C for 30 minutes, filtered, and freeze-dried for 36 hours to obtain pH- and surfactant-double-imprinted lipase Lipase G50. The non-polar solvent octane was used to elute the excess imprinting template on the surface of the lipase, and the n-hexane was removed by vacuum drying to obtain the imprinted lipase.

[0061] Example 6

[0062] A buffer solution with a pH value of 5.5 was prepared, and 20% of Lipase G50 by mass was added, and the mixture was stirred at 25°C for 40 minutes. The mixture was filtered and then dried in a vacuum to remove moisture, thereby obtaining pH-imprinted Lipase G50. A nonionic surfactant Tween 60 was dissolved in acetone at a concentration of 60 mg / mL, and 20% of pH-imprinted Lipase G50 was added. The mixture was stirred at 25°C for 40 minutes, filtered, and freeze-dried for 24 hours to obtain pH- and surfactant-double-imprinted lipase Lipase G50. The non-polar solvent octane was used to elute the excess imprinting template on the surface of the lipase, and the octane was removed by vacuum drying to obtain the imprinted lipase.

[0063] Example 7

[0064] A buffer solution with a pH value of 5 was prepared, and 15% of Lipase G50 by mass was added. The mixture was stirred at 25°C for 45 minutes, filtered, and vacuum dried to remove moisture to obtain pH-imprinted Lipase G50. The nonionic surfactant Tween 40 was dissolved in acetone at a concentration of 20 mg / mL, 15% of pH-imprinted Lipase G50 was added, the mixture was stirred at 25°C for 45 minutes, filtered, and freeze-dried for 12 hours to obtain pH- and surfactant-double-imprinted lipase Lipase G50. The non-polar solvent octane was used to elute the excess imprinting template on the surface of the lipase, and the n-hexane was removed by vacuum drying to obtain the imprinted lipase.

[0065] Table 3. Esterification activity of imprinted lipase Lipase G50 in Examples 5-7

[0066]

[0067]

[0068] It can be seen from the above table that the esterification activity of lipase after double imprinting with pH value and non-polar surfactant is greatly improved.

[0069] Example 8

[0070] The glyceride product in Example 1 was added to a batch reactor, and free sunflower oil fatty acids were added based on the amount of the glyceride glycerol backbone, the molar ratio of free fatty acids to the glyceride glycerol backbone was 4:1, and stirring was started at a speed of 600 rpm. The temperature was raised to 50°C, and the double imprinted lipase Lipase G50 in Example 5 was added in an amount of 8wt% of the substrate, and vacuum was started at a vacuum degree of 10 mbar. The reaction was carried out for 6 hours to obtain a glyceride product, in which the monoglyceride content of the product was 0.7%, the diglyceride content was 95.9%, the triglyceride content was 3.4%, and the diglyceride yield was 95.9%.

[0071] The fatty acids are removed by molecular distillation, and the conditions for the removal of fatty acids by molecular distillation are: distillation temperature is 160°C, pressure is 3Pa, and condenser temperature is 25°C; the content of diglyceride in the obtained product is 96.3%, the content of glycidyl ester is 1.39mg / Kg, and the content of chloropropanol ester is 0.33mg / Kg.

[0072] Example 9

[0073] The glyceride product in Example 2 was added to a batch reactor, and free soybean oil fatty acids were added based on the amount of the glyceride glycerol backbone, with a molar ratio of free fatty acids to the glyceride glycerol backbone of 3:1, and stirring was started at a speed of 800 rpm. The temperature was raised to 40°C, and the double imprinted lipase Lipase G50 in Example 6 was added in an amount of 6wt% of the substrate, and vacuum was started at a vacuum degree of 20 mbar. The reaction was carried out for 8 hours to obtain a glyceride product, in which the monoglyceride content of the product was 0.5%, the diglyceride content was 92.6%, the triglyceride content was 6.9%, and the diglyceride yield was 92.6%.

[0074] The fatty acids are removed by molecular distillation, and the conditions for removing fatty acids by molecular distillation are: distillation temperature is 150°C, pressure is 2Pa, and condenser temperature is 30°C; the content of diglyceride in the obtained product is 92.8%, the content of glycidyl ester is 1.16mg / Kg, and the content of chloropropanol ester is 0.21mg / Kg.

[0075] Example 10

[0076] The glyceride product in Example 3 was added to a batch reactor, and free linseed oil fatty acids were added based on the amount of the glyceride glycerol backbone, with a molar ratio of free fatty acids to the glyceride glycerol backbone of 2:1, and stirring was started at a speed of 700 rpm. The temperature was raised to 30° C., and the double imprinted lipase Lipase G50 in Example 7 was added in an amount of 4 wt % of the substrate, and vacuum was started at a vacuum degree of 30 mbar. The reaction was carried out for 10 hours to obtain a glyceride product, in which the monoglyceride content of the product was 0.6%, the diglyceride content was 90.5%, the triglyceride content was 9.1%, and the diglyceride yield was 90.5%.

[0077] The fatty acids are removed by molecular distillation, and the conditions for the removal of fatty acids by molecular distillation are: distillation temperature is 170°C, pressure is 5Pa, and condenser temperature is 35°C; the content of diglyceride in the obtained product is 90.7%, the content of glycidyl ester is 1.71mg / Kg, and the content of chloropropanol ester is 0.44mg / Kg.

[0078] Comparative Example 1

[0079] The reaction conditions of Example 2 were adopted in the glycerol decomposition reaction stage, glycerol was not added in stages, it was not homogenized and monoglyceride was not added, and other conditions were consistent with Example 2.

[0080] Soybean oil was added to a batch reactor, nitrogen was filled for protection, the temperature was raised to 70°C, glycerol was added according to a molar ratio of glycerol to soybean oil of 2:1, lipase was added, the amount of lipase added was 15wt% of the weight of soybean oil, and stirring was started, the stirring speed was 800rpm, and the reaction time was 10h. After the reaction was completed, the system temperature was heated to 90°C, slowly stirred, the speed was 30rpm, and the stirring time was 1h, the stratification of glycerol and glyceride was accelerated, glycerol was separated from glyceride, and glycerol was recovered as a raw material for new glycerol decomposition reaction. The composition of the obtained glyceride is shown in Table 4.

[0081] Comparative Example 2

[0082] The reaction conditions of Example 2 were adopted in the glycerol decomposition reaction stage, glycerol was not added in stages, and it was not homogenized. Other conditions were consistent with Example 2.

[0083] Add soybean oil to an intermittent reactor, fill with nitrogen for protection, raise the temperature to 70°C, add 1% monoglyceride by mass, stir to fully dissolve the monoglyceride in sunflower oil, add glycerol according to a molar ratio of glycerol to soybean oil of 2:1, add lipase, the amount of lipase added is 15wt% of the weight of soybean oil, and start stirring, the stirring speed is 800rpm, and the reaction time is 10h. After the reaction is completed, heat the system temperature to 90°C, stir slowly, the speed is 30rpm, the stirring time is 1h, accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, and recover glycerol as a raw material for new glycerol decomposition reaction.

[0084] The composition of the obtained glycerides is shown in Table 4.

[0085] Table 4. Composition of glycerides in the products of Comparative Examples 1-2

[0086] Comparative Example 1 Comparative Example 2 Monoglyceride (%) 42.2 44.9 Diacylceride (%) 35.0 38.4 Triglyceride (%) 22.7 16.7

[0087] Through comparative analysis, it can be seen that after the reaction, the triglyceride content in the system is much higher than the triglyceride content of the corresponding Example 2, indicating that the system has not reached equilibrium after the reaction, and compared with the comparative example 2 in which monoglyceride is added and the comparative example 1 in which monoglyceride is not added, the triglyceride content in the comparative example 2 is lower than that in the comparative example 1, indicating that the segmented addition of glycerol, homogenization and monoglyceride addition have a strong effect on improving the reaction efficiency. At the same time, the high triglyceride content will affect the subsequent diglyceride yield and the purity of diglyceride in the product.

[0088] Comparative Example 3

[0089] According to the conditions of Example 3, but without using Lipase G50 for esterification, diglyceride was directly produced by conventional methods.

[0090] The first stage molecular distillation is used to remove fatty acids, and the second stage molecular distillation is used to remove monoglycerides.

[0091] Linseed oil is added to an intermittent reactor, nitrogen is filled for protection, the temperature is raised to 50°C, 0.5% monoglyceride is added by mass, and the monoglyceride is fully dissolved in the sunflower seed oil by stirring. Glycerin is added in three stages according to the molar ratio of glycerin to linseed oil of 1.5:1. In the first stage, 30% glycerol is added, homogenized at 40Mpa for 3 minutes, glycerol and linseed oil are fully mixed, lipase is added, and the amount of lipase added is 10wt% of the weight of linseed oil, and stirring is started at 500rpm, the reaction is 1.5h, and the lipase is filtered out. In the second stage, 50% glycerol is added, homogenized at 40Mpa for 3min, lipase is put in again for reaction for 1.5h, the stirring speed is 500rpm, and the lipase is filtered out. In the third stage, 20% glycerol is added, homogenized at 40Mpa for 3min, lipase is put in for reaction for 8h, and the stirring speed is 500rpm. After the reaction, the system temperature was heated to 85°C and slowly stirred at a speed of 10 rpm for 1.5 h to accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, and recover glycerol as a raw material for new glycerol decomposition reaction. The composition of glyceride in the product is as shown in Example 3, with 51.2% monoglyceride, 39.9% diglyceride, 8.8% triglyceride, and a diglyceride yield of 39.9%.

[0092] Fatty acids and monoglycerides are removed by molecular distillation. The conditions for removing fatty acids by primary molecular distillation are: distillation temperature of 170°C, pressure of 5Pa, condenser temperature of 35°C, and the content of diglyceride in the obtained product is 40.4%; monoglyceride is removed by secondary molecular distillation, and the molecular distillation conditions are distillation temperature of 210°C, pressure of 2Pa, condenser temperature of 20°C. After removing monoglyceride, the content of monoglyceride in the product is 1.2%, the content of diglyceride is 81.0%, and the content of triglyceride is 17.8%. Since monoglyceride is removed by molecular distillation, the yield of the obtained product is consistent with the yield in the glycerol hydrolysis reaction, which is 39.9%, the content of glycidyl ester is 6.36mg / Kg, and the content of chloropropanol ester is 1.23mg / Kg.

[0093] Comparative Example 4

[0094] Referring to Example 3, glycerol was added in stages, but homogenization was not performed. Other conditions were the same as in Example 3, and the composition of glycerides in the comparison system was compared.

[0095] Add linseed oil into a batch reactor, fill it with nitrogen for protection, raise the temperature to 50°C, add 0.5% monoglyceride by mass, and stir to fully dissolve the monoglyceride in the sunflower oil;

[0096] Glycerol was added in three stages according to a molar ratio of glycerol to linseed oil of 1.5:1. In the first stage, 30% glycerol was added, lipase NS 40086 was added, and the amount of lipase added was 10wt% of the weight of linseed oil, and stirring was started at a speed of 500rpm. The reaction lasted for 1.5h and the lipase was filtered out. In the second stage, 50% glycerol was added, the lipase was put back in for reaction for 1.5h at a stirring speed of 500rpm, and the lipase was filtered out. In the third stage, 20% glycerol was added, the lipase was put in for reaction for 8h at a stirring speed of 500rpm.

[0097] After the reaction, the system temperature was heated to 85°C and slowly stirred at a speed of 10 rpm for 1.5 h to accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, obtain glycerol hydrolysis product, and recover glycerol as raw material for new glycerol hydrolysis reaction.

[0098] Table 5. Composition of glycerides in the product of Comparative Example 4

[0099] Glyceride composition content(%) Monoglyceride 45.2 Diacylceride 38.3 Triglycerides 15.8

[0100] Comparative Example 5

[0101] Referring to Example 3, different amounts of glycerol were added at different stages, and other conditions were the same as in Example 3, to compare the composition of glyceride in the system.

[0102] Add linseed oil into a batch reactor, fill it with nitrogen for protection, raise the temperature to 50°C, add 0.5% monoglyceride by mass, and stir to fully dissolve the monoglyceride in the sunflower oil;

[0103] Glycerol was added in three stages according to a molar ratio of glycerol to linseed oil of 1.5:1. In the first stage, 5% glycerol was added and homogenized at 40 MPa for 3 minutes to fully mix the glycerol and linseed oil. Lipase NS 40086 was added in an amount of 10 wt% of the weight of the linseed oil, and stirring was started at a speed of 500 rpm. The reaction lasted for 1.5 hours and the lipase was filtered out. In the second stage, 60% glycerol was added and homogenized at 40 MPa for 3 minutes. The lipase was put in again for reaction for 1.5 hours at a stirring speed of 500 rpm and the lipase was filtered out. In the third stage, 35% glycerol was added and homogenized at 40 MPa for 3 minutes. The lipase was put in for reaction for 8 hours at a stirring speed of 500 rpm.

[0104] After the reaction, the system temperature was heated to 85°C and stirred slowly at a speed of 10 rpm for 1.5 h to accelerate the stratification of glycerol and glyceride, separate glycerol from glyceride, and obtain a glycerol hydrolysis product. The glycerol was recovered as a raw material for a new glycerol hydrolysis reaction.

[0105] Table 6. Composition of glycerides in the product of Comparative Example 5

[0106]

[0107]

[0108] The triglyceride content in Comparative Example 5 is higher than that in Example 3, indicating that Comparative Example 5 does not react as thoroughly as Example 3. The reason may be that too little glycerol is added in the first stage, and sufficient emulsifier cannot be formed. Excessive glycerol is added in the second stage, resulting in poor mixing of glycerol and oil, forming a large amount of free glycerol, which covers the surface of lipase to a certain extent, resulting in partial loss of lipase activity, thereby reducing the reaction efficiency.

[0109] Comparative Example 6

[0110] Referring to Example 8, double-imprinted lipase and non-imprinted lipase were used to carry out continuous batch reactions, and the activities of lipases in different batches were compared, and the catalytic stability of lipases was compared.

[0111] Table 7. Activity and enzyme inactivation rate of imprinted and non-imprinted lipases from different batches

[0112] batch Double imprinted lipase Unimprinted lipase 1 33.6％ 15.5％ 2 32.1％ 13.5％ 3 30.7％ 12.2％ 4 30.1％ 11.1％ 5 28.2％ 9.4％ 6 25.4％ 7.1％ Enzyme inactivation rate 24.4％ 54.2％

[0113] The stability of double-imprinted lipase is significantly higher than that of unimprinted lipase. The main reason is that non-ionic surfactant interacts with lipase through hydrophobic force, that is, the polar head of non-ionic surfactant interacts with the hydrophilic group on the surface of lipase, and the non-polar head interacts with the hydrophobic group. Since lipase itself is water-soluble, it shows that the hydrophilic group on the surface of lipase is much higher than the hydrophobic group. Therefore, through the action of non-ionic surfactant, the surface of lipase is changed from hydrophilic to hydrophobic, which can avoid excessive contact between lipase and polar substances in the system to a certain extent, thereby preventing the moisture on the surface of lipase from being robbed and causing the structure of lipase to change and lead to inactivation. Therefore, the addition of non-ionic surfactant is conducive to maintaining good stability of lipase.

[0114] At the same time, the nonionic surfactant interacts with the active center of the lipase, opening the lid of the active center of the lipase. After removing the moisture, the lipase maintains the active catalytic conformation, thereby enhancing the catalytic activity of the lipase.

[0115] Isopropanol has good solubility for nonionic surfactants. At the same time, it contains trace amounts of water, which can keep lipase in a soft conformation. Isopropanol has moderate polarity and will not cause loss of enzyme activity. Therefore, nonionic surfactants soluble in isopropanol can interact well with lipase, thereby improving the imprinting effect and further enhancing the activity of lipase.

[0116] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing high-purity diglyceride oil, characterized in that: include, Add edible oil to a batch reactor, fill it with nitrogen for protection, raise the temperature, add monoglyceride, and stir to fully dissolve the monoglyceride in the edible oil; The method comprises the following steps: using lipase to catalyze glycerol and edible oil for glycerol hydrolysis reaction to obtain a glycerol hydrolysis product; wherein, during the glycerol hydrolysis reaction, glycerol is added in stages for homogenization, and the staged addition of glycerol includes: in the first stage, 10-30% glycerol is added, homogenized at 20-40 MPa for 3-10 minutes, lipase is added, the stirring speed is 500-800 rpm, the reaction is performed for 0.5-1.5 hours, and the lipase is filtered out; in the second stage, 30-50% glycerol is added, homogenized at 20-40 MPa for 3-10 minutes, lipase is added again for reaction for 0.5-1.5 hours, the stirring speed is 500-800 rpm, and the lipase is filtered out; in the third stage, 20-60% glycerol is added, homogenized at 20-40 MPa for 3-10 minutes, lipase is added for reaction for 6-9 hours, and the stirring speed is 600 rpm; Using double-imprinted Lipase G50 as a catalyst and free fatty acids as acyl donors, catalyzing the esterification reaction between monoglyceride and free fatty acids in the glycerol hydrolysis product under vacuum, and removing free fatty acids by molecular distillation, finally obtaining a high-purity diglyceride product, wherein the preparation method of the double-imprinted Lipase G50 includes preparing a buffer solution with a pH value of 5 to 6, adding 15 to 30% of Lipase G50 by mass, stirring at 25° C. for 30 to 45 minutes, filtering and vacuum drying to remove moisture, and obtaining pH-imprinted Lipase G50; A non-ionic surfactant is added to isopropanol at a concentration of 20-100 mg / mL, 15-30% pH imprinted Lipase G50 is added, the mixture is stirred at 25°C for 30-45 minutes, freeze-dried for 12-36 hours, excess imprinted template is eluted with a non-polar solvent, and the solvent is removed by vacuum drying to obtain pH and surfactant dual-imprinted Lipase G50, wherein the non-ionic surfactant is Tween 80, Tween 60 or Tween 40.

2. The preparation method according to claim 1, characterized in that: The conditions of the glycerolysis reaction are as follows: the reaction temperature is 50-70° C., the molar ratio of glycerol to edible oil is 1.5-74:1, and the amount of lipase added is 10-20wt% based on the weight of the edible oil.

3. The preparation method according to claim 1, characterized in that: The glycerol is food grade glycerol, the lipase is commercial lipase, including Novozym 435, Lipozyme RM IM and NS 40086, and the edible oils and fats include sunflower seed oil, soybean oil and linseed oil.

4. The preparation method according to claim 1, characterized in that: The preparation method of the glycerol hydrolysis product further comprises: After the glycerol hydrolysis reaction, the temperature is heated to 85-95°C and stirred slowly at a speed of 10-30 rpm for 0.5-1.5 h to separate the glycerol and glyceride. The glyceride is the glycerol hydrolysis product.

5. The preparation method according to claim 1, characterized in that: The non-polar solvent includes n-hexane and octane.

6. The preparation method according to claim 1, characterized in that: The esterification reaction comprises: The molar ratio of free fatty acids to glycerol backbone of glycerol ester is 2-4:1, the stirring speed is 600-800 rpm, the temperature is 30-50°C, the amount of imprinted lipase Lipase G50 added is 4-8 wt%, the vacuum degree is 10-30 mbar, and the reaction time is 6-10 h; The free fatty acids are fatty acids derived from edible oils and fats, including sunflower oil, soybean oil and linseed oil.

7. The preparation method according to claim 1, characterized in that: The conditions for removing free fatty acids by molecular distillation are: distillation temperature of 150-170°C, pressure of 2-5 Pa, and condenser temperature of 25-35°C.

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