Process for the preparation of diglyceride oils with improved high thrombotic efficacy by enzymatic hydrolysis and products and uses thereof

Abstract

The application provides a method for preparing diglyceride oil with improved high thrombus efficacy by enzymolysis, and products and applications thereof, and relates to the technical field of enzymatic processing. Low rapeseed diester oil and corn diester oil in the diglyceride oil are obtained by twice enzymolysis and twice molecular distillation, and the enzymes used in the twice enzymolysis include lipase and partial glyceride lipase; the lipase is a composite lipase of three enzymes, and the mass ratio of the composite lipase to the partial glyceride lipase is 1:2-4.5; the low rapeseed diester oil, the corn diester oil, the maple seed oil and the peanut oil are mixed to obtain the diglyceride oil. The preparation method improves the yield and activity of the diglyceride oil; and the obtained diglyceride oil has the effect of significantly improving blood flow speed and has good improvement effect on high thrombus.

Description

Methods for preparing diglyceride oil with anti-thrombotic effects via enzymatic hydrolysis, its products, and applications. Technical Field

[0001] This invention belongs to the field of enzymatic processing technology, specifically relating to a method for preparing diglyceride oil with the effect of improving high blood thrombosis through enzymatic hydrolysis, as well as its products and applications. Background Technology

[0002] Diacylglycerol (DAG), also known as diglyceride, is an ester formed when a fatty acid in the triglyceride backbone is replaced by a hydroxyl group. It is also an intermediate product of the metabolism of natural edible oils in the body. Diacylglycerol has the effects of inhibiting the rise of serum triglycerides, preventing the accumulation of fat in the body, increasing β-oxidation, reducing weight, lowering blood lipids, and preventing the formation of arterial thrombosis.

[0003] Diacylglycerols are trace components in natural oils, typically comprising less than 10% of the total composition, with their relative content varying depending on the source of the edible oil. Diacylglycerols are scarce in nature and are primarily obtained through further processing using oil modification techniques. Currently, the main methods for preparing diglycerides include chemical synthesis, enzymatic methods, and microbial methods. While chemical synthesis offers a simple reaction route, shorter processing time, and relatively lower cost, making it easy to scale up, the resulting products are usually a mixture of monoglycerides, diglycerides, and glycerol, resulting in lower purity and yield of diglycerides. Furthermore, the flavor and color of the product obtained through chemical methods are often inferior, limiting its application. Microbial methods are technically immature and require strict cultivation environments, hindering large-scale production. Enzymatic methods primarily utilize lipases, which catalyze the hydrolysis, alcoholysis, esterification, transesterification, and reverse synthesis of esters under specific conditions. Due to their specificity, high efficiency, and environmental friendliness, enzymatic methods are widely used in the preparation of diglycerides.

[0004] Currently, there are some reports on the enzymatic preparation of diglyceride oil: Chinese invention patent CN114058649A discloses a solvent-free enzymatic preparation process for diglyceride oil, including the following steps: (1) Glycerol pre-adsorption method ultrasonic enzyme reaction: weigh glycerol, pre-adsorb glycerol to obtain pre-adsorbed glycerol; weigh vegetable oil, first add pre-adsorbed glycerol and immobilized lipase to vegetable oil for enzymatic reaction I, then add metaglyceryl lipase for enzymatic reaction II, and use ultrasound to promote the reaction throughout the reaction process; (2) centrifugation; (3) quadruple molecular distillation; (4) white clay activated carbon decolorization; (5) double low temperature deodorization; this technical solution first uses immobilized lipase for enzymatic reaction I, and then uses metaglyceryl lipase for enzymatic reaction II, which greatly improves the yield of diglycerides. However, this method does not explain the specific vegetable oil components, enzyme types and diglyceride preparation methods on the effect of improving the efficacy of high thrombosis, nor does it record whether the type of enzyme has a synergistic effect on thrombolysis.

[0005] Chinese invention patent CN115678676A discloses a flavored diglyceride oil and its preparation method, comprising the following steps: mixing crude oil, enzyme preparation, and chelating agent; adjusting the water content; high-speed shearing; placing in a shaker for incubation; filtering to obtain a degummed solution; adding glycerol and lipase to the degummed solution; reacting in a vacuum reactor; collecting the reaction gas simultaneously using a gas adsorbent; centrifuging to obtain the oil layer to obtain the reaction product; subjecting the reaction product to molecular distillation to obtain a mixture with a heavy phase of diglycerides and triglycerides; separating to obtain diglyceride oil; and passing the collected reaction gas into the diglyceride oil for desorption to obtain the flavored diglyceride oil. This invention uses an enzymatic degumming method, which does not cause loss of aroma substances, and the flavor substance retention rate of the oil reaches over 90%. However, the products obtained by the prior art still have a low content of diglycerides and contain a large number of other components. Summary of the Invention

[0006] This invention addresses the problems of existing technologies by providing a method for preparing diglyceride oil with improved thrombotic efficacy through enzymatic hydrolysis, along with its products and applications. The invention first employs two enzymatic hydrolysis processes and two molecular distillations to obtain low-erucic acid rapeseed diglyceride oil and corn diglyceride oil; then, these are mixed with Acer truncatum seed oil and peanut oil to obtain diglyceride oil. The enzymes used in the enzymatic hydrolysis include lipase and metaglyceryl lipase. By controlling the types and ratios of enzymes and the reaction conditions of each enzymatic hydrolysis reaction, this invention not only improves the yield and activity of the diglyceride oil but also enhances the thrombolytic opening rate and blood flow velocity in arteries / venous vessels. The diglyceride oil has a significant effect on improving thrombotic efficacy when applied in food preparation.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] First, this invention provides a method for preparing diglyceride oil with the effect of improving hyperthrombosis through enzymatic hydrolysis, comprising the following steps:

[0009] (1) Preparation of low-erucic acid rapeseed diester oil:

[0010] (1.1) Mix rapeseed oil with 0.15-0.3 times its weight of water and heat to 45-60℃ to obtain rapeseed oil mixture;

[0011] (1.2) Under heat preservation conditions, the rapeseed oil mixture from step (1.1) and the compound lipase are mixed and stirred for 1-6 hours to obtain the first enzymatic hydrolysis mixture.

[0012] (1.3) Centrifuge the first enzymatic hydrolysis mixture from step (1.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0013] The two-stage molecular distillation is as follows: first-stage molecular distillation at 100-160℃; second-stage molecular distillation at 190-230℃.

[0014] (1.4) The distillation product of step (1.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 50-70℃ and stirred for 1-8 hours to obtain the second enzymatic hydrolysis mixture.

[0015] The mass of the glycerol is 1-1.5 times the mass of the rapeseed oil;

[0016] (1.5) Centrifuge the second enzymatic hydrolysis mixture from step (1.4), and perform molecular distillation of the supernatant by gradient heating to obtain low erucic acid rapeseed diester oil.

[0017] (2) Preparation of corn diester oil: The method is the same as in step (1) to prepare low erucic acid rapeseed diester oil, except that the rapeseed oil in step (1) is replaced with corn oil to obtain corn diester oil;

[0018] (3) Low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil are mixed to obtain glycerol oil.

[0019] Preferably, in step (1.1), the temperature for heating is 50°C.

[0020] Preferably, in step (1.1), rapeseed oil is mixed with 0.22 times its weight of water.

[0021] Preferably, in step (1.2), the temperature of the heat preservation condition is the same as the temperature of the heating in step (1.1), which is 45-60℃.

[0022] Preferably, in step (1.2), the enzymatic hydrolysis reaction time is 3.5-5 hours; more preferably, in step (1.2), the enzymatic hydrolysis reaction time is 4 hours.

[0023] Preferably, in step (1.2), the complex lipase is a mixture of three lipases.

[0024] More preferably, in step (1.2), the lipase is derived from Rhizopus niveus, salivary glands or forestomach of calf, kid or lamb, goat gullets, hog or bovine pancreas, Candida cylindracea, Aspergillus niger, Rhizopus oryzae, Rhizomucor miehei, Aspergillus oryzae, Candida antarctica lipase, or Thermomyces lanuginosus.

[0025] More preferably, in step (1.2), the lipase is derived from Aspergillus niger, Rhizopus oryzae, Rhizomucor miehei, Aspergillus oryzae, Candida antarctica lipase, or Thermomyces lanuginosus.

[0026] More preferably, in step (1.2), the complex lipase is a complex lipase composed of lipase (Lipozyme) TL 100L, lipase 435 (Novozym 435) and lipase (Lipozyme) CALB in a mass ratio of 7-11:1-3:4.5-7.5; the lipase TL 100L is derived from *Thermophilic Mycospora spp.*, the lipase 435 is derived from *Aspergillus oryzae*, and the lipase CALB is derived from *Candida antarcticis*.

[0027] More preferably, in step (1.2), the complex lipase is a complex lipase composed of lipase TL100L, Novozym 435 and lipase CALB in a mass ratio of 8-10:2:5-7.

[0028] More preferably, in step (1.2), the complex lipase is a complex lipase composed of lipase TL100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0029] Preferably, in step (1.3), the secondary molecular distillation is: first-stage molecular distillation at 130°C; second-stage molecular distillation at 220°C.

[0030] Preferably, in step (1.4), the mass of the glycerol is 1.2-1.3 times the mass of the rapeseed oil; more preferably, in step (1.4), the mass of the glycerol is 1.28 times the mass of the rapeseed oil.

[0031] Preferably, in step (1.4), the metaglycerol lipase is a metaglycerol lipase (AOL) derived from Aspergillus oryzae.

[0032] Preferably, in step (1.4), the temperature of the enzymatic hydrolysis reaction is 60-65℃ and the hydrolysis time is 5-7h.

[0033] More preferably, in step (1.4), the temperature of the enzymatic hydrolysis reaction is 60°C and the hydrolysis time is 6h.

[0034] Preferably, the mass ratio of the compound lipase in step (1.2) to the glycerol lipase in step (1.4) is 1:2-4.5; more preferably, the mass ratio is 1:2.5-3.6; and even more preferably, the mass ratio is 1:3.

[0035] Preferably, in step (1.4), the mass ratio of glycerol to glyceryl lipase is 100:4-7.5.

[0036] More preferably, in step (1.4), the mass ratio of glycerol to glyceryl lipase is 100:5-7.

[0037] More preferably, in step (1.4), the mass ratio of glycerol to glyceryl lipase is 100:6.

[0038] Preferably, in step (1.5), the gradient temperature molecular distillation specifically includes: ① 110-120℃, ② 140-150℃, ③ 160-180℃, ④ 210-230℃; after molecular distillation processes ①-④, diglyceride oil is obtained.

[0039] More preferably, in step (1.5), the parameters of the gradient temperature molecular distillation are: ①115℃, ②145℃, ③170℃, ④220℃; after the molecular distillation process of ①-④, diglyceride oil is obtained.

[0040] Preferably, the diglyceride oil obtained in step (3) includes the following components: 0.1-20 parts of Acer truncatum seed oil, 45-90 parts of low erucic acid rapeseed diglyceride oil, 15-30 parts of corn diglyceride oil and 4-12 parts of peanut oil.

[0041] More preferably, the diglyceride oil obtained in step (3) includes the following components: 0.1-5 parts of Acer truncatum seed oil, 45-90 parts of low erucic acid rapeseed diglyceride oil, 15-30 parts of corn diglyceride oil and 4-12 parts of peanut oil.

[0042] More preferably, the diglyceride oil obtained in step (3) includes the following components: 0.5-2 parts of Acer truncatum seed oil, 65-75 parts of low erucic acid rapeseed diglyceride oil, 20-25 parts of corn diglyceride oil and 6-10 parts of peanut oil.

[0043] More preferably, the diglyceride oil obtained in step (3) includes the following components: 1 part of Acer truncatum seed oil, 70 parts of low erucic acid rapeseed diglyceride oil, 23 parts of corn diglyceride oil and 8 parts of peanut oil.

[0044] Then, the present invention provides a diglyceride oil prepared by the above method.

[0045] Preferably, the diglyceride oil comprises the following components: 0.1-20 parts of Acer truncatum seed oil, 45-90 parts of low-erucic acid rapeseed diglyceride oil, 15-30 parts of corn diglyceride oil, and 4-12 parts of peanut oil.

[0046] More preferably, the diglyceride oil comprises, by weight, the following components: 0.1-5 parts of Acer truncatum seed oil, 45-90 parts of low-erucic acid rapeseed diglyceride oil, 15-30 parts of corn diglyceride oil, and 4-12 parts of peanut oil.

[0047] More preferably, the diglyceride oil comprises, by weight, the following components: 0.5-2 parts of Acer truncatum seed oil, 65-75 parts of low-erucic acid rapeseed diglyceride oil, 20-25 parts of corn diglyceride oil, and 6-10 parts of peanut oil.

[0048] More preferably, the diglyceride oil comprises, by weight, the following components: 1 part of Acer truncatum seed oil, 70 parts of low-erucic acid rapeseed diglyceride oil, 23 parts of corn diglyceride oil and 8 parts of peanut oil.

[0049] Finally, this invention provides the application of diglyceride oil in the preparation of foods that improve hyperthrombosis.

[0050] Preferably, the diglyceride oil is the diglyceride oil described above or the diglyceride oil prepared by the above preparation method.

[0051] Preferably, the food that improves high blood clots is a food that can increase the thrombolysis rate and increase blood flow velocity.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] 1. The diglyceride oil of the present invention is prepared by enzymatic hydrolysis and subsequent enzymatic esterification. Specifically, a complex lipase with specific components is used for enzymatic hydrolysis, followed by enzymatic esterification using a glyceryl ester lipase. By controlling the type and ratio of enzymes and the reaction conditions of the two enzymatic hydrolysis reactions, the present invention improves the yield and activity of diglyceride oil. The resulting diglyceride oil can significantly improve blood flow velocity and has a good effect on improving high thrombosis.

[0054] 2. The raw materials for preparing diglyceride oil according to the present invention include the following components: 0.1-20 parts of Acer truncatum seed oil, 45-90 parts of low erucic acid rapeseed diglyceride oil, 15-30 parts of corn diglyceride oil and 4-12 parts of peanut oil. The components complement each other and are reasonably combined through a specific ratio. The resulting diglyceride oil has a high thrombolysis rate and blood flow velocity. Detailed Implementation

[0055] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0056] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0057] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels. In the following embodiments, products from different manufacturers do not have a significant impact on the efficacy. The metaglycerol lipase used is a metaglycerol lipase (AOL) derived from Aspergillus oryzae.

[0058] Example 1

[0059] A diglyceride oil that improves high blood clots, comprising, by weight: 1 part Acer truncatum seed oil, 70 parts low erucic acid rapeseed diglyceride oil, 23 parts corn diglyceride oil and 8 parts peanut oil.

[0060] The preparation method of diglyceride oil is as follows:

[0061] (1) Preparation of low-erucic acid rapeseed diester oil:

[0062] (1.1) Mix rapeseed oil with 0.22 times its weight of water and heat to 50°C to obtain rapeseed oil mixture;

[0063] (1.2) Under the condition of keeping warm at 50℃, the rapeseed oil mixture from step (1.1) and the compound lipase are mixed and stirred for 4 hours to obtain the first enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0064] (1.3) Centrifuge the first enzymatic hydrolysis mixture from step (1.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0065] The temperature for the first stage of molecular distillation is 130℃; the temperature for the second stage of molecular distillation is 220℃.

[0066] (1.4) The distillation product of step (1.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 60°C and stirred for 6 hours to obtain the second enzymatic hydrolysis mixture.

[0067] The mass of the glycerol is 1.28 times the mass of the rapeseed oil; the mass ratio of glycerol to glyceryl ester lipase is 100:6; the mass ratio of compound lipase to glyceryl ester lipase is 1:3.

[0068] (1.5) Centrifuge the second enzymatic hydrolysis mixture from step (1.4), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①115℃; ②145℃; ③170℃; ④220℃ to obtain low erucic acid rapeseed diester oil.

[0069] (2) Preparation of corn diester oil:

[0070] (2.1) Mix corn oil with 0.22 times its weight of water and heat to 50°C to obtain corn oil mixture;

[0071] (2.2) Under the condition of keeping warm at 50℃, the corn oil mixture from step (2.1) and the compound lipase are mixed and stirred for 4 hours to obtain the first enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0072] (2.3) Centrifuge the first enzymatic hydrolysis mixture from step (2.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0073] The temperature for the first stage of molecular distillation is 130℃; the temperature for the second stage of molecular distillation is 220℃.

[0074] (2.4) The distillation product of step (2.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 60°C and stirred for 6 hours to obtain the second enzymatic hydrolysis mixture.

[0075] The mass of the glycerol is 1.28 times the mass of the corn oil; the mass ratio of glycerol to glyceryl ester lipase is 100:6; the mass ratio of compound lipase to glyceryl ester lipase is 1:3.

[0076] (2.5) Centrifuge the second enzymatic hydrolysis mixture from step (2.4), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①115℃; ②145℃; ③170℃; ④220℃ to obtain corn diester oil;

[0077] (3) Mix low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil to obtain glyceryl diester oil.

[0078] Example 2

[0079] A diglyceride oil for improving high blood clots, comprising, by weight: 0.5 parts of Acer truncatum seed oil, 65 parts of low erucic acid rapeseed diglyceride oil, 20 parts of corn diglyceride oil and 6 parts of peanut oil.

[0080] The preparation method of diglyceride oil is as follows:

[0081] (1) Preparation of low-erucic acid rapeseed diester oil:

[0082] (1.1) Mix rapeseed oil with 0.15 times its weight of water and heat to 60°C to obtain rapeseed oil mixture;

[0083] (1.2) Under the condition of keeping warm at 60℃, the rapeseed oil mixture from step (1.1) and the compound lipase are mixed and stirred for 1 hour to obtain the first enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 8:2:5.

[0084] (1.3) Centrifuge the first enzymatic hydrolysis mixture from step (1.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0085] The temperature for the first stage of molecular distillation is 100℃; the temperature for the second stage of molecular distillation is 230℃.

[0086] (1.4) The distillation product of step (1.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 50°C and stirred for 8 hours to obtain the second enzymatic hydrolysis mixture.

[0087] The mass of glycerol is 1.2 times the mass of rapeseed oil; the mass ratio of glycerol to glyceryl ester lipase is 100:7; the mass ratio of compound lipase to glyceryl ester lipase is 1:2.

[0088] (1.5) Centrifuge the second enzymatic hydrolysis mixture from step (1.4), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①110℃; ②140℃; ③180℃; ④230℃ to obtain low erucic acid rapeseed diester oil.

[0089] (2) Preparation of corn diester oil:

[0090] (2.1) Mix corn oil with 0.15 times its weight of water and heat to 60°C to obtain corn oil mixture;

[0091] (2.2) Under the condition of keeping warm at 60℃, the corn mixed oil from step (2.1) and the compound lipase are mixed and stirred for 1 hour to obtain the first enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 8:2:5.

[0092] (2.3) Centrifuge the first enzymatic hydrolysis mixture from step (2.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0093] The temperature for the first stage of molecular distillation is 100℃; the temperature for the second stage of molecular distillation is 230℃.

[0094] (2.4) The distillation product of step (2.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 50°C and stirred for 8 hours to obtain the second enzymatic hydrolysis mixture.

[0095] The mass of glycerol is 1.2 times the mass of corn oil; the mass ratio of glycerol to glyceryl ester lipase is 100:7; the mass ratio of complex lipase to glyceryl ester lipase is 1:2.

[0096] (2.5) Centrifuge the second enzymatic hydrolysis mixture from step (2.4), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①110℃; ②140℃; ③180℃; ④230℃ to obtain corn diester oil;

[0097] (3) Mix low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil to obtain glyceryl diester oil.

[0098] Example 3

[0099] A diglyceride oil that improves high blood clots, comprising, by weight: 2 parts Acer truncatum seed oil, 75 parts low erucic acid rapeseed diglyceride oil, 25 parts corn diglyceride oil and 10 parts peanut oil.

[0100] The preparation method of diglyceride oil is as follows:

[0101] (1) Preparation of low-erucic acid rapeseed diester oil:

[0102] (1.1) Mix rapeseed oil with 0.3 times its weight of water and heat to 45°C to obtain rapeseed oil mixture;

[0103] (1.2) Under the condition of keeping warm at 45℃, the rapeseed oil mixture from step (1.1) and the compound lipase are mixed and stirred for 6 hours to obtain the first enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB with a mass ratio of 10:2:7.

[0104] (1.3) Centrifuge the first enzymatic hydrolysis mixture from step (1.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0105] The temperature for the first-stage molecular distillation is 160℃; the temperature for the second-stage molecular distillation is 190℃.

[0106] (1.4) The distillation product of step (1.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 70°C and stirred for 4 hours to obtain the second enzymatic hydrolysis mixture.

[0107] The mass of glycerol is 1.3 times the mass of rapeseed oil; the mass ratio of glycerol to metaglycerol lipase is 100:5; the mass ratio of compound lipase to metaglycerol lipase is 1:4.5.

[0108] (1.5) Centrifuge the second enzymatic hydrolysis mixture from step (1.4), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①120℃; ②150℃; ③160℃; ④210℃ to obtain low erucic acid rapeseed diester oil.

[0109] (2) Preparation of corn diester oil:

[0110] (2.1) Mix corn oil with 0.3 times its weight of water and heat to 45°C to obtain corn oil mixture;

[0111] (2.2) Under the condition of keeping warm at 45℃, the corn oil mixture from step (2.1) and the compound lipase are mixed and stirred for 6 hours to obtain the first enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB with a mass ratio of 10:2:7.

[0112] (2.3) Centrifuge the first enzymatic hydrolysis mixture from step (2.2), and obtain the distilled product by secondary molecular distillation of the supernatant;

[0113] The temperature for the first-stage molecular distillation is 160℃; the temperature for the second-stage molecular distillation is 190℃.

[0114] (2.4) The distillation product of step (2.3) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 70°C and stirred for 4 hours to obtain the second enzymatic hydrolysis mixture.

[0115] The mass of glycerol is 1.3 times the mass of corn oil; the mass ratio of glycerol to glyceryl ester lipase is 100:5; the mass ratio of compound lipase to glyceryl ester lipase is 1:4.5.

[0116] (2.5) Centrifuge the second enzymatic hydrolysis mixture from step (2.4), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①120℃; ②150℃; ③160℃; ④210℃ to obtain corn diester oil;

[0117] (3) Mix low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil to obtain glyceryl diester oil.

[0118] Example 4

[0119] Unlike Example 1, in the composite lipase described in steps (1.2) and (2.2), the mass ratio of lipase TL100L, Novozym 435 and lipase CALB is 7:1:4.5.

[0120] Everything else is the same as in Example 1.

[0121] Example 5

[0122] Unlike Example 1, in the complex lipase described in steps (1.2) and (2.2), the mass ratio of lipase TL100L, Novozym 435 and lipase CALB is 11:3:7.5.

[0123] Everything else is the same as in Example 1.

[0124] Comparative Example 1

[0125] Unlike Example 1, the compound lipase used to prepare low erucic acid rapeseed ester oil and corn ester oil was replaced with a single lipase TL 100L.

[0126] Everything else is the same as in Example 1.

[0127] Comparative Example 2

[0128] Unlike Example 1, the compound lipase used to prepare low erucic acid rapeseed ester oil and corn ester oil was replaced with a mixture of Novozym 435 and CALB lipase in a mass ratio of 2:6.

[0129] Everything else is the same as in Example 1.

[0130] Comparative Example 3

[0131] Unlike Example 1, the preparation methods of low erucic acid rapeseed ester oil and corn ester oil did not use complex lipase.

[0132] The preparation method of diglyceride oil is as follows:

[0133] (1) Preparation of low-erucic acid rapeseed diester oil:

[0134] (1.1) Mix rapeseed oil with 0.22 times its weight of water and heat to 50°C to obtain rapeseed oil mixture;

[0135] (1.2) The rapeseed oil mixture was subjected to two-stage molecular distillation to obtain the distilled product; the temperature of the first-stage molecular distillation was 130℃; the temperature of the second-stage molecular distillation was 220℃;

[0136] (1.3) The distillation product of step (1.2) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 60°C and stirred for 6 hours to obtain the enzymatic hydrolysis mixture.

[0137] The mass of the glycerol is 1.28 times the mass of the rapeseed oil; the mass ratio of glycerol to metaglycerol lipase is 100:6;

[0138] (1.4) The enzymatic hydrolysis mixture was centrifuged, and the supernatant was subjected to molecular distillation with gradient temperature increases: ①115℃; ②145℃; ③170℃; ④220℃ to obtain low erucic acid rapeseed diester oil.

[0139] (2) Preparation of corn diester oil:

[0140] (2.1) Mix corn oil with 0.22 times its weight of water and heat to 50°C to obtain corn oil mixture;

[0141] (2.2) The corn oil mixture was subjected to two-stage molecular distillation to obtain the distilled product; the temperature of the first-stage molecular distillation was 130℃; the temperature of the second-stage molecular distillation was 220℃;

[0142] (2.3) The distillation product of step (2.2) is mixed with glyceryl lipase and glycerol, and the mixture is heated to 60°C and stirred for 6 hours to obtain the enzymatic hydrolysis mixture.

[0143] The mass of the glycerol is 1.28 times the mass of the corn oil; the mass ratio of glycerol to glyceryl lipase is 100:6;

[0144] (2.4) The enzymatic hydrolysis mixture was centrifuged, and the supernatant was subjected to molecular distillation with gradient temperature increases: ①115℃; ②145℃; ③170℃; ④220℃ to obtain corn diester oil;

[0145] (3) Mix low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil to obtain glyceryl diester oil.

[0146] Everything else is the same as in Example 1.

[0147] Comparative Example 4

[0148] Unlike Example 1, the preparation methods of low erucic acid rapeseed ester oil and corn ester oil did not use metaglycerol lipase.

[0149] The preparation method of diglyceride oil is as follows:

[0150] (1) Preparation of low-erucic acid rapeseed diester oil:

[0151] (1.1) Mix rapeseed oil with 0.22 times its weight of water and heat to 50°C to obtain rapeseed oil mixture;

[0152] (1.2) Under the condition of keeping warm at 50℃, the rapeseed oil mixture from step (1.1) is mixed with compound lipase and glycerol, and the mixture is stirred and enzymatically hydrolyzed for 4 hours to obtain the enzymatic hydrolysate; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0153] The mass of glycerol is 1.28 times that of rapeseed oil; the mass ratio of glycerol to complex lipase is 100:2.

[0154] (1.3) Centrifuge the enzymatic hydrolysis mixture from step (1.2), and obtain the distilled product by two-stage molecular distillation of the supernatant; the temperature of the first-stage molecular distillation is 130℃; the temperature of the second-stage molecular distillation is 220℃.

[0155] (1.4) The distilled product was subjected to molecular distillation with gradient temperature increases: ①115℃; ②145℃; ③170℃; ④220℃ to obtain low erucic acid rapeseed diester oil.

[0156] (2) Preparation of corn diester oil:

[0157] (2.1) Mix corn oil with 0.22 times its weight of water and heat to 50°C to obtain corn oil mixture;

[0158] (2.2) Under the condition of keeping warm at 50℃, the corn oil mixture from step (2.1) is mixed with compound lipase and glycerol, and the enzymatic hydrolysis reaction is stirred for 4 hours to obtain the enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0159] The mass of glycerol is 1.28 times that of corn oil; the mass ratio of glycerol to complex lipase is 100:2.

[0160] (2.3) Centrifuge the enzymatic hydrolysis mixture from step (2.2), and obtain the distilled product by two-stage molecular distillation of the supernatant; the temperature of the first-stage molecular distillation is 130℃; the temperature of the second-stage molecular distillation is 220℃;

[0161] (2.4) The distilled product was subjected to molecular distillation with gradient temperature increases: ①115℃; ②145℃; ③170℃; ④220℃ to obtain corn diester oil;

[0162] (3) Mix low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil to obtain glyceryl diester oil.

[0163] Everything else is the same as in Example 1.

[0164] Comparative Example 5

[0165] Unlike Example 1, in steps (1.4) and (2.4), the mass ratio of glycerol to glyceryl lipase is 100:2. All other steps are the same as in Example 1.

[0166] Comparative Example 6

[0167] Unlike Example 1, the mass ratio of compound lipase to glycerol lipase is 1:10, and the mass ratio of glycerol to glycerol lipase is 100:6. All other aspects are the same as in Example 1.

[0168] Comparative Example 7

[0169] Unlike Example 1, the preparation methods for low-erucic acid rapeseed diester oil and corn diester oil do not involve two enzymatic hydrolysis and two molecular distillations; instead, they involve only one enzymatic hydrolysis and one molecular distillation operation. The steps are as follows:

[0170] (1) Preparation of low-erucic acid rapeseed diester oil:

[0171] (1.1) Mix rapeseed oil with 0.22 times its weight of water and heat to 50°C to obtain rapeseed oil mixture;

[0172] (1.2) Under the condition of keeping warm at 50℃, the rapeseed oil mixture from step (1.1) is mixed with compound lipase, glyceryl ester lipase and glycerol, and the mixture is stirred and enzymatically hydrolyzed for 4 hours to obtain the enzymatic hydrolysate; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0173] The mass of the glycerol is 1.28 times the mass of the rapeseed oil; the mass ratio of glycerol to glyceryl ester lipase is 100:6; the mass ratio of compound lipase to glyceryl ester lipase is 1:3.

[0174] (1.3) Centrifuge the enzymatic hydrolysis mixture from step (1.2), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①115℃; ②145℃; ③170℃; ④220℃ to obtain low erucic acid rapeseed diester oil.

[0175] (2) Preparation of corn diester oil:

[0176] (2.1) Mix corn oil with 0.22 times its weight of water and heat to 50°C to obtain corn oil mixture;

[0177] (2.2) Under the condition of keeping warm at 50℃, the corn oil mixture from step (2.1) is mixed with compound lipase, glyceryl ester lipase and glycerol, and stirred for 4 hours to obtain enzymatic hydrolysis mixture; the compound lipase is a mixture of lipase TL 100L, Novozym 435 and lipase CALB in a mass ratio of 9:2:6.

[0178] The mass of the glycerol is 1.28 times the mass of the corn oil; the mass ratio of glycerol to glyceryl ester lipase is 100:6; the mass ratio of compound lipase to glyceryl ester lipase is 1:3.

[0179] (2.3) Centrifuge the enzymatic hydrolysis mixture from step (2.2), and perform molecular distillation with gradient temperature increases on the supernatant obtained: ①115℃; ②145℃; ③170℃; ④220℃ to obtain corn diester oil;

[0180] (3) Mix low erucic acid rapeseed oil, corn oil, maple seed oil and peanut oil to obtain glyceryl diester oil.

[0181] Comparative Example 8

[0182] Unlike Example 1, no water was added in steps (1.1) and (2.1).

[0183] Rapeseed oil and corn oil are heated directly to 50°C to obtain rapeseed oil blend and corn oil blend, respectively.

[0184] Everything else is the same as in Example 1.

[0185] Comparative Example 9

[0186] Unlike Example 1, the enzymatic hydrolysis temperatures in steps (1.4) and (2.4) are different; the hydrolysis temperature is set to 80°C. All other steps are the same as in Example 1.

[0187] Experiment 1 Content Detection

[0188] The content of diglycerides in low-erucic acid rapeseed oil and corn oil was determined by gas chromatography, with the experiment repeated three times. The results are shown in Table 1.

[0189] Table 1

[0190]

[0191] The data in Table 1, compared to Example 1, are... △ P≤0.05, △△ P≤0.01, indicating a significant difference.

[0192] As can be seen from the data in Table 1, the present invention uses two enzymatic hydrolysis and two molecular distillation methods to prepare low erucic acid rapeseed diester oil and corn diester oil, respectively, which significantly increases the content of diglycerides in the diester oil.

[0193] As can be seen from the data of the examples and comparative examples 1-6, the content of diglycerides can be significantly increased by combining a compound lipase with a glyceryl lipase of a specific composition in a specific ratio.

[0194] Comparison of data from the examples and Comparative Examples 7-9 shows that the preparation of low-erucic acid rapeseed diglyceride oil and corn diglyceride oil using a two-stage enzymatic hydrolysis and two-stage molecular distillation method is beneficial to the completeness of subsequent enzymatic hydrolysis and the stability of molecular distillation. Controlling the temperature of enzymatic hydrolysis to increase the content of diglycerides results in diglyceride oils that are rich in diglycerides and have stable properties.

[0195] Experiment 2: Detection of efficacy against qi stagnation and blood stasis type thrombosis

[0196] Experimental animals: Zebrafish were all raised in fish culture water at 28°C (Water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L calculated as CaCO3). The license number for the use of experimental animals was: SYXK(Zhe)2022 - 0004, and the feeding management met the requirements of international AAALAC certification (certification number: 001458).

[0197] Evaluation of the efficacy against qi stagnation and blood stasis type thrombosis: Randomly select zebrafish of the Albino strain in a 6 - well plate, with 30 zebrafish in each well. The sample was administered by dissolving in water at a concentration of 2000 μg / mL. At the same time, a normal control group and a model control group were set up, and the volume of each well was 3 mL. Except for the normal control group, the other experimental groups were all given isoproterenol hydrochloride by dissolving in water to establish a zebrafish qi stagnation and blood stasis thrombosis model. After being treated at 28°C for 2 days, the zebrafish were placed under a heartbeat blood flow analysis system to record the blood flow video of the zebrafish, and the blood flow velocity of the zebrafish was analyzed and statistically calculated. The statistical analysis results of this index were used to evaluate the efficacy of diglyceride oil against qi stagnation and blood stasis type thrombosis. SPSS 26.0 software was used for statistical analysis, and P < 0.05 indicated that the difference was statistically significant.

[0198] The results of zebrafish blood flow velocity are shown in Table 2.

[0199] Table 2

[0200]

[0201] In the data of Table 2, compared with Example 1, △ P ≤ 0.05, △△ P ≤ 0.01, there were significant differences; compared with the model control group, for each experimental group, # P ≤ 0.05, ## P ≤ 0.01.

[0202] It can be seen from Table 2 that the diglyceride oil of the present invention can significantly increase the blood flow velocity in zebrafish with thrombosis models, indicating that it has the efficacy of improving qi stagnation and blood stasis. The diglyceride oil of the present invention has a better ability to improve high thrombosis compared with the products of the comparative examples.

[0203] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride oil with the effect of improving hyperthrombosis by enzymatic hydrolysis, characterized in that, The steps include: (1) Preparation of low erucic acid rapeseed diester oil: (1.1) Rapeseed oil is mixed with 0.15-0.3 times its weight of water and heated to 45-60℃ to obtain rapeseed oil mixture; (1.2) Under heat preservation conditions, the rapeseed oil mixture from step (1.1) is mixed with a complex lipase and stirred for 1-6 hours to obtain the first enzymatic hydrolysis mixture; the complex lipase is a complex lipase composed of lipase TL100L, lipase 435 and lipase CALB in a mass ratio of 7-11:1-3:4.5-7.5; (1.3) The first enzymatic hydrolysis mixture from step (1.2) is centrifuged, and the supernatant is subjected to secondary molecular distillation to obtain the distilled product; the secondary molecular distillation is: first molecular distillation at 100-160℃; second molecular distillation at 190-230℃; (1.4) The distillation product of step (1.3) is mixed with glyceryl ester lipase and glycerol, and heated to 50-70℃ for stirring and enzymatic hydrolysis for 1-8h to obtain the second enzymatic hydrolysis mixture; the mass ratio of compound lipase to glyceryl ester lipase is 1:3; the mass of glycerol is 1-1.5 times the mass of rapeseed oil; the mass ratio of glycerol to glyceryl ester lipase is 100:6; (1.5) the second enzymatic hydrolysis mixture of step (1.4) is centrifuged, and the supernatant is subjected to gradient heating molecular distillation to obtain low erucic acid rapeseed diester oil; (2) corn diester oil is prepared: the method is the same as that of step (1) to prepare low erucic acid rapeseed diester oil, and the rapeseed oil in step (1) is replaced with corn oil to obtain corn diester oil; (3) low erucic acid rapeseed diester oil, corn diester oil, Acer truncatum seed oil and peanut oil are mixed to obtain diglyceride oil.

2. The method according to claim 1, characterized in that, In step (1.1), rapeseed oil is mixed with 0.22 times its weight of water and heated to 50°C; in step (1.2), the temperature of the heat preservation condition is the same as the temperature of the heating in step (1.1), which is 45-60°C; in step (1.2), the enzymatic hydrolysis reaction time is 3.5-5h; in step (1.3), the secondary molecular distillation is: first-stage molecular distillation at 130°C; second-stage molecular distillation at 220°C.

3. The method according to claim 1, characterized in that, In step (1.4), the metaglycerol lipase is a metaglycerol lipase derived from Aspergillus oryzae.

4. The method according to claim 1, characterized in that, In step (1.4), the mass of the glycerol is 1.2-1.3 times the mass of the rapeseed oil; in step (1.4), the temperature of the enzymatic hydrolysis reaction is 60-65℃ and the enzymatic hydrolysis time is 5-7h.

5. The method according to claim 1, characterized in that, In step (1.5), the gradient temperature molecular distillation specifically involves: ① 110-120℃, ② 140-150℃, ③ 160-180℃, ④ 210-230℃; after molecular distillation processes ①-④, diglyceride oil is obtained.

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