Method for preparing quintuple diglyceride oil for improving blood vessel health through enzymolysis, application and product

Abstract

The invention provides a method for preparing quintuple diglyceride oil for improving blood vessel health through enzymolysis, application and a product, and belongs to the technical field of enzyme engineering. The preparation method comprises the following steps: mixing low-erucic acid rapeseed oil, glycerol, water, fatty acid and lipase, and carrying out enzymolysis reaction, molecular distillation, decoloration and deodorization to obtain diglyceride oil; the lipase is a mixture of Candida antarctica Aspergillus niger lipase, Candida column lipase and bovine pancreas lipase, and the weight ratio of the low-erucic acid rapeseed oil to the glycerin to the water to the fatty acid to the lipase is 500: (80-100): (1-2): (30-60): (10-20). The diglyceride oil prepared by the method can be used for preparing a diglyceride oil composition, and has five functions of reducing blood uric acid, reducing blood total cholesterol, reducing blood triglyceride, reducing blood sugar and increasing blood flow velocity to improve blood vessel health.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and relates to a method, application and product for preparing diglyceride oil that quintuple improves vascular health by enzymatic hydrolysis. Background Art

[0002] Diglyceride is a kind of fat-like substance with a structure in which one acyl group in triglyceride is replaced by hydrogen. The structural formula of diglyceride is: or .

[0003] Among them, R1 and R2 are the same or different saturated or unsaturated aliphatic hydrocarbon group structures.

[0004] Triglyceride is an important energy storage molecule in the human body and an important component of adipose tissue. The synthesis pathways of triglyceride in the human body mainly include the monoglyceride pathway and the phosphatidic acid pathway. Among them, the monoglyceride pathway is: 2-monoacylglycerol is converted into diacylglycerol (i.e., diglyceride) through the acyl transfer action of 2-monoacylglycerol acyltransferase, and diglyceride is converted into triglyceride through the acyl transfer action of diglyceride acyltransferase. It can be seen that the human fat metabolism process involves diglyceride, and diglyceride has a certain effect on regulating lipid metabolism. At the same time, diglyceride is also a new type of food raw material.

[0005] The existing technologies for producing diglyceride mainly include chemical method and enzymatic hydrolysis method. The principle of synthesizing diglyceride by chemical method is to accelerate the esterification reaction of fatty acid with glycerol or monoglyceride by using a catalyst, or to use the transesterification reaction of oil and fat. However, the synthesis of diglyceride by chemical method is limited by problems such as low reaction specificity, consumption of solvents and possible side reactions that are not conducive to food safety. The production of diglyceride by enzymatic hydrolysis method has mild conditions and high selectivity, and is suitable for the production of food-grade diglyceride. However, in the existing process of producing diglyceride by enzymatic hydrolysis method, the production efficiency of enzyme preparations is limited, and the types of substances that can be used in food production to improve the enzymatic hydrolysis rate are also limited. In addition, the post-treatment process of enzymatic hydrolysis is relatively cumbersome, which may cause the carbon-carbon double bonds in unsaturated fatty acids in the product during the process to be oxidized, resulting in a decrease in iodine value. Therefore, there is an urgent need for a production process that reasonably matches the existing enzymes and auxiliaries used in food production and their ratios to improve the yield, purity and iodine value of diglyceride. Summary of the Invention

[0006] In view of this, in response to the above problems of the prior art, the object of the present invention is to provide a method, application and product for preparing diglyceride oil that quintuply improves vascular health through enzymatic hydrolysis. The present invention selects a mixture of three enzymes, namely Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase, as the enzyme for the medium reaction. At the same time, by optimizing the ratio of low-erucic rapeseed oil, glycerol, water, fatty acids and lipase, the yield, purity and iodine value of the enzymatic hydrolysis reaction for preparing low-erucic rapeseed diglyceride from low-erucic rapeseed oil are successfully improved, while reducing the overall production cost and improving the product quality.

[0007] To achieve the above object of the invention, on the one hand, the present invention provides a method for preparing diglyceride oil that quintuply improves vascular health through enzymatic hydrolysis, comprising the following steps: Mix low-erucic rapeseed oil, glycerol, water, fatty acids and lipase, carry out enzymatic hydrolysis reaction, molecular distillation, decolorization and deodorization to obtain diglyceride oil; Wherein, the mass content of erucic acid in the low-erucic rapeseed oil is less than or equal to 3%; The lipase is a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase, and the weight ratio of the low-erucic rapeseed oil, glycerol, water, fatty acids and lipase is 500: 80-100: 1-2: 30-60: 10-20.

[0008] Preferably, the fatty acid is selected from at least one of linoleic acid, α-linolenic acid, oleic acid and eicosapentaenoic acid, and more preferably a mixture of linoleic acid and α-linolenic acid.

[0009] As an example of the present invention, the fatty acid is a mixture of linoleic acid and α-linolenic acid with a weight ratio of 1:1.

[0010] Preferably, the lipase is a mixture composed of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase mixed in a weight ratio of 7: 1-2: 1-2.

[0011] More preferably, and as an example of the present invention, the lipase is a mixture composed of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase mixed in a weight ratio of 7: 1.5: 1.5.

[0012] Preferably, the weight ratio of the low-erucic rapeseed oil, glycerol, water, fatty acids and lipase is 500: 90: 1.5: 45: 15.

[0013] Preferably, the temperature of the enzymatic hydrolysis reaction is 50-65 °C, and the time of the enzymatic hydrolysis is 1-9 h.

[0014] As a specific example of the present invention, the temperature of the enzymatic hydrolysis reaction is 60 °C, and the time of the enzymatic hydrolysis reaction is 8 h.

[0015] Preferably, the molecular distillation includes first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation; The temperature of the first-stage molecular distillation is 100-160 °C, and the vacuum degree is less than 500 Pa; The temperature of the second-stage molecular distillation is 170-200 °C, and the vacuum degree is less than 10 Pa; The temperature of the third-stage molecular distillation is 220-280 °C, and the vacuum degree is less than 10 Pa; The temperature of the condensation surface of the molecular distillation is 35-55 °C, more preferably 40 °C; the molecular distillation is repeated 3-5 times, more preferably 4 times.

[0016] On the other hand, the present invention provides a diglyceride oil prepared by the above method.

[0017] On yet another aspect, the present invention provides the application of the diglyceride oil prepared by the above method in the production of oil products.

[0018] The oil products include, but are not limited to, liquid edible oils, oil microcapsule powders, and gels.

[0019] Preferably, the oil product is a liquid edible oil.

[0020] Finally, the present invention provides a diglyceride oil composition, comprising low-erucic rapeseed diglyceride oil, corn diglyceride oil, edible vegetable oil, and food additives; The low-erucic rapeseed diglyceride oil is the diglyceride oil prepared by the above method.

[0021] The term "edible vegetable oil" refers to edible oils made from edible vegetable oil materials or crude vegetable oils. It includes, but is not limited to, soybean oil, sunflower oil, linseed oil, sesame oil, grape seed oil, pumpkin seed oil, peanut oil, walnut oil, almond oil, hazelnut oil, macadamia nut oil, olive oil, coconut oil, palm oil, avocado oil, rice bran oil, corn oil, wheat germ oil, tea oil, peony seed oil, perilla seed oil, hemp oil, seabuckthorn fruit oil, cyperus esculentus oil, xanthoceras sorbifolia oil, etc.

[0022] Preferably, the edible vegetable oil is selected from one or more of soybean oil, sunflower oil, linseed oil, grape seed oil, peanut oil, olive oil, corn oil, wheat germ oil, tea oil, peony seed oil, cyperus esculentus oil, and xanthoceras sorbifolia oil.

[0023] More preferably, the edible vegetable oil is selected from peanut oil and / or corn oil.

[0024] More preferably, and as a specific example of the present invention, the edible vegetable oil is high-oleic acid peanut oil.

[0025] Further preferably, the oleic acid content in the high-oleic acid peanut oil > 75%.

[0026] The term "food additive" refers to a substance applied to food that can improve or help improve the sensory requirements, physical and chemical indexes, nutrition and other properties of food. The food additives include, but are not limited to, the additives that can be added to edible oil products as stipulated in GB 2760-2024 "National Food Safety Standard for the Use of Food Additives".

[0027] The food additives include antioxidants, emulsifiers, defoamers, anticaking agents, acidity regulators, color protectants, metal ion chelating agents, natural extracts, etc.

[0028] Preferably, the food additives are selected from one or more of the following: tert-Butylhydroquinone, butylated hydroxyanisole, dibutylhydroxytoluene, ascorbyl palmitate, vitamin E, rosemary extract, tea polyphenols, glycyrrhizin antioxidant, soy lecithin, sunflower lecithin, polyglycerol fatty acid ester, citric acid, sodium citrate, curcumin, β-carotene.

[0029] More preferably, the food additive is vitamin E.

[0030] Furthermore, the diglyceride oil composition includes: Low erucic acid rapeseed diglyceride oil, corn diglyceride oil, high-oleic acid peanut oil and vitamin E.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a technology for enzymatically producing diglyceride using a specific enzyme combination and the ratio of enzymatic hydrolysis reactants, effectively improving the mass percentage content of diglyceride, the yield of diglyceride and the iodine value. Specifically, it is reflected in the following aspects: (1) Three enzymes, namely Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase, are compounded. Through the synergistic effect among the three enzymes, the mass percentage content of diglyceride and the yield of diglyceride are improved.

[0032] (2) The present invention establishes a suitable microenvironment for enzymatically producing diglyceride by adjusting the ratios of low erucic acid rapeseed oil, glycerol, water, fatty acid and lipase, which is conducive to maximizing the enzymatic hydrolysis effect after the compounding of the three enzymes.

[0033] (3)The inventors unexpectedly found that during the enzymatic production of diglycerides using the above enzyme preparation and enzymatic reaction raw materials, the entire enzyme reaction system (specific component types and weight ratios of the vegetable oil, glycerol, fatty acids, water, and lipase used) has a certain selective effect on different types of fatty acids in the fatty acid exchange reaction, which is manifested as an increase in the iodine value of the final diglycerides. Description of the Drawings

[0034] Figure 1 It is a bar chart of the uric acid level in the blood of mice on the 42nd day of the experiment in Effect Example 2.

[0035] Figure 2 It is a bar chart of the difference in the uric acid level in the blood of mice between the 42nd day and the 0th day of the experiment in Effect Example 2.

[0036] Figure 3 It is a bar chart of the total cholesterol level in the blood of mice on the 42nd day of the experiment in Effect Example 2.

[0037] Figure 4 It is a bar chart of the difference in the total cholesterol level in the blood of mice between the 42nd day and the 0th day of the experiment in Effect Example 2.

[0038] Figure 5 It is a bar chart of the triglyceride level in the blood of mice on the 42nd day of the experiment in Effect Example 2.

[0039] Figure 6 It is a bar chart of the difference in the triglyceride level in the blood of mice between the 42nd day and the 0th day of the experiment in Effect Example 2.

[0040] Figure 7 It is a bar chart of the blood glucose level of zebrafish in Effect Example 3. Detailed Embodiments

[0041] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary illustration of the scope claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes and modifications should also fall within the scope claimed by the present invention.

[0042] The present invention will be further described below by way of specific examples. All the various chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels unless otherwise specified. The same chemical reagents of different brands have no significant impact on the technical solutions and technical effects of the present invention. Unless otherwise specified, the contents described below are mass contents. Unless otherwise specified, it is understood that the operations are carried out at room temperature.

[0043] In the following examples and comparative examples, the sources of some reagents are shown in Table 1: Table 1

[0044] In the following examples and comparative examples, the bovine pancreatic lipase used refers to the prior art "Bovine pancreatic lipase. I. Isolation, homogeneity, and characterization" (KM Shahani et al., Journal of Dairy Science, 1976, 59(3): 369-375). The specific steps are as follows: (1) Place fresh bovine pancreas in a 0.01 mol / L sucrose solution at 0 °C, remove fat and non-glandular tissues, cut into small pieces, and quick-freeze at -20 °C for later use.

[0045] (2) Thaw 500 g of the small pieces of bovine pancreas prepared in (1), mix with 1000 mL of 0.01 mol / L sucrose aqueous solution at 0 - 5 °C, and stir for 90 s. Filter using five layers of gauze to obtain 1400 mL of clear and homogeneous liquid. Centrifuge (15000 g, 30 min, -4 °C). The liquid is divided into three layers, with fat on the upper layer, precipitate on the lower layer, and supernatant on the middle layer. Use a dropper to suck the middle-layer liquid and combine them, totaling 1200 mL. Add 0.22 g of diisopropyl fluorophosphate to inactivate proteases to protect the esterase. Lyophilize to obtain 24 g of dry powder, and store it frozen at -20 °C for later use.

[0046] (3) Take 5 g of the dry powder prepared in (2), mix with 75 mL of 0.01 mol / L cold sucrose solution, and stir magnetically (200 rpm, 15 min) at 4 °C. Centrifuge the mixture at 4 °C (2000 g, 15 min), and collect the supernatant into a brown bottle. Resuspend the precipitate with 75 mL of 0.01 mol / L cold sucrose solution, and repeat the above stirring and centrifugation steps. Combine the two supernatants to obtain a crude extract of bovine pancreatic lipase.

[0047] (4) Place the crude extract of bovine pancreatic lipase obtained in (3) (about 150 mL in total) in a container, immerse the container in an acetone-dry ice bath, cool it to -10 °C, and add cold acetone to the crude extract while stirring (a total of 16.6 mL of cold acetone is added). After stirring for 25 min, centrifuge at -4 °C (10000 g, 15 min). Collect the supernatant and pour it into a beaker, then place it back into the acetone-dry ice bath and discard the precipitate. Add cold acetone again to increase the acetone concentration in the supernatant to 45%. At this time, the precipitate contains enriched lipase and some impurities. Collect the precipitate and dissolve it in 20 mL of 0.01 mol / L sucrose solution. Adjust the pH to 8.5 using 1 mol / L sodium hydroxide aqueous solution for the next column chromatography separation.

[0048] (5) Column chromatography separation.

[0049] Column separation was carried out using Sephadex G-100 dextran gel. As an example of the present invention, the gel column used had a diameter of 2 cm, a length of 32 cm, and an empty column volume of 35 mL. During separation, 5 mL of the crude enzyme solution obtained in (4) was loaded onto the column, and eluted with a 0.01 mol / L sucrose solution at a flow rate of 12 mL / h. The fractions were collected and freeze-dried to obtain bovine pancreatic lipase.

[0050] Example 1 A method for preparing a diglyceride oil.

[0051] (1) 500 parts by weight of vegetable oil, 90 parts by weight of glycerol, 1.5 parts by weight of water, 22.5 parts by weight of linoleic acid, 22.5 parts by weight of α-linolenic acid, and 15 parts by weight of lipase were stirred and thoroughly mixed. The lipase was a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase, and bovine pancreatic lipase in a weight ratio of 7:1.5:1.5.

[0052] (2) The mixture obtained in step (1) was enzymatically hydrolyzed in an environment at 60 °C for 8 hours. After the enzymatic hydrolysis was completed, it was allowed to stand, and the supernatant was retained for molecular distillation. The process of molecular distillation included first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation. The temperature of the first-stage molecular distillation was 100 - 160 °C, and the vacuum degree was less than 500 Pa; the temperature of the second-stage molecular distillation was 170 - 200 °C, and the vacuum degree was less than 10 Pa; the temperature of the third-stage molecular distillation was 220 - 280 °C, and the vacuum degree was less than 10 Pa; the temperature of the condensation surface of the molecular distillation was 50 °C. The molecular distillation was repeated 4 times. The fractions at 170 - 200 °C were collected and combined, and cooled to room temperature. 1% by weight of activated carbon was added, stirred for 0.5 h for decolorization and deodorization, and filtered using a filter element to remove the activated carbon, obtaining low-erucic rapeseed diglyceride oil.

[0053] Example 2 Compared with Example 1, the difference was that the lipase used in step (1) was a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase, and bovine pancreatic lipase in a weight ratio of 7:2:1, and the rest were the same.

[0054] Example 3 Compared with Example 1, the difference was that the lipase used in step (1) was a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase, and bovine pancreatic lipase in a weight ratio of 7:1:2, and the rest were the same.

[0055] Example 4 Compared with Example 1, the difference lies in that in step (1), glycerol is changed to 80 parts by weight, water is changed to 2 parts by weight, linoleic acid is changed to 15 parts by weight, α-linolenic acid is changed to 15 parts by mass, and lipase is changed to 20 parts by weight, and the rest are the same.

[0056] Example 5 Compared with Example 1, the difference lies in that in step (1), glycerol is changed to 100 parts by weight, water is changed to 1 part by weight, linoleic acid is changed to 30 parts by weight, α-linolenic acid is changed to 30 parts by weight, and lipase is changed to 10 parts by weight, and the rest are the same.

[0057] Comparative Example 1 Compared with Example 1, the difference lies in that in step (1), the lipase used is a mixture of Candida cylindracea lipase and bovine pancreatic lipase with a weight ratio of 8:2, and the rest are the same.

[0058] Comparative Example 2 Compared with Example 1, the difference lies in that in step (1), the lipase used is a mixture of Candida antarctica Aspergillus niger lipase and Candida cylindracea lipase with a weight ratio of 7:3, and the rest are the same.

[0059] Comparative Example 3 Compared with Example 1, the difference lies in that in step (1), the lipase used is a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and Novozym 435 with a weight ratio of 7:2:1, and the rest are the same.

[0060] Comparative Example 4 Compared with Example 1, the difference lies in that in step (1), 1.5 parts by weight of water is not added, and glycerol is changed to 91.5 parts by weight, and the rest are the same.

[0061] Comparative Example 5 Compared with Example 1, the difference lies in that in step (1), glycerol is changed to 130 parts by weight, α-linolenic acid is changed to 35 parts by weight, linoleic acid is not used, and lipase is changed to 5 parts by weight, and the rest are the same.

[0062] In Examples 1 - 5 and Comparative Examples 1 - 5, when the vegetable oil in step S1 is low-erucic rapeseed oil with an erucic acid weight content ≤ 3%, the diglyceride oil prepared is low-erucic rapeseed diglyceride oil; when the vegetable oil in step S1 is corn oil, the diglyceride oil prepared is corn diglyceride oil.

[0063] The yields of the low-erucic rapeseed diglyceride oil and corn diglyceride oil prepared by the above method are calculated. The calculation formula is as follows: Diglyceride oil yield = Diglyceride oil output × Mass percentage of diglyceride in diglyceride oil ÷ Vegetable oil input amount × 100%.

[0064] The weight content of diglyceride in diglyceride oil was detected by the method provided by the existing technology (Wang Yong et al., "Study on the Determination of Diglyceride Content by Reversed-Phase High Performance Liquid Chromatography", Journal of the Chinese Cereals and Oils Association, 3(2010):5).

[0065] The iodine value of diglyceride oil was determined by the method described in Standard GB / T5532-2022.

[0066] The weight content of diglyceride, the yield, and the iodine value of diglyceride oil in the low erucic acid rapeseed diglyceride oil and corn diglyceride oil prepared in Examples 1-5 and Comparative Examples 1-5 were determined. The results are shown in Table 2 and Table 3.

[0067] Table 2:

[0068] Table 3:

[0069] It can be seen that the preparation methods provided in Examples 1-5 can prepare low erucic acid rapeseed diglyceride oil and corn diglyceride oil with higher diglyceride content and higher iodine value.

[0070] Examples 6-9 provide the formulations of diglyceride oil compositions, as shown in Table 4.

[0071] Table 4:

[0072] Among them, in the diglyceride oil compositions provided in Examples 6-9, the preparation methods of the low erucic acid rapeseed diglyceride oil and corn diglyceride oil are as follows: In Example 1, when the vegetable oil in step S1 is corn oil, the diglyceride oil prepared is the corn diglyceride oil in Examples 6-9.

[0073] In Example 1, when the vegetable oil in step S1 is rapeseed oil with erucic acid content less than or equal to 3%, the diglyceride oil prepared in Example 1 is the low erucic acid rapeseed diglyceride oil in Examples 6-9.

[0074] The preparation method of the diglyceride oil compositions provided in Examples 6-9 is: mix each component according to the formulated amount.

[0075] Effect Example 1 Effect of diglyceride oil composition on blood flow velocity in zebrafish with thrombosis model.

[0076] 1. Experimental conditions 1.1. Experimental animals Zebrafish were all raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L in terms of CaCO3). They were provided by the fish-raising center of the Innovation Experiment Center of Huante Biotechnology. The experimental animal use license number is: SYXK(Zhe)2022 - 0004. The feeding management conforms to the requirements of international AAALAC accreditation (accreditation number: 001458). IACUC ethical review number: IACUC - 2023 - 8002 - 01.

[0077] 1.2 Samples, Instruments and Consumables The sample detected in this effect example was the diglyceride oil composition provided in Example 6.

[0078] Dissecting microscope (SZX7, OLYMPUS); precision electronic balance (CP214, OHAUS); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd.); 6-well plate (Zhejiang Beilambo Biotechnology Co., Ltd.); heartbeat blood flow analysis system (Zebra Blood 3.4, ViewPoint Life Sciences). Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd.); isoproterenol hydrochloride (batch number VNVFK-QF, Tokyo Chemical Industry Co., Ltd. (Shanghai)).

[0079] 2 Evaluation Methods 2.1 Determination of the Maximum Test Concentration (MTC) Randomly select 3-day post-fertilization (3 dpf) melanin allele mutant Albino strain zebrafish into 6-well plates, with 30 zebrafish in each well. Different concentrations of the diglyceride oil provided in Example 6 were given respectively, and a normal control group and a model control group were set up simultaneously. The volume of each well was 3 mL. Except for the normal control group, isoproterenol hydrochloride was dissolved in water in the remaining experimental groups to establish a zebrafish qi-stagnation and blood-stasis thrombosis model. After treatment at 28 °C for 2 days, the MTC of the sample on the model zebrafish was measured.

[0080] 2.2 Evaluation of the Efficacy against Qi-Stagnation and Blood-Stasis Thrombosis Randomly select 3-day post-fertilization (3dpf) zebrafish of the melanin allele mutant Albino strain and place 30 zebrafish in each well of a 6-well plate. Treat them with a certain concentration of the sample (in Example 6, give the diglyceride oil composition provided in Example 6 at 2000 μg / mL, the peanut oil group is given peanut oil at 2000 μg / mL, and the model control group is given the same volume of zebrafish culture medium). At the same time, set up a normal control group, with a volume of 3 mL per well. Except for the normal control group, the other experimental groups are given isoproterenol hydrochloride in water to establish a zebrafish model of qi stagnation and blood stasis thrombosis. After treatment at 28 °C for 2 days, randomly select 10 zebrafish from each experimental group and place them under a heartbeat blood flow analysis system to record the zebrafish blood flow video, and analyze and statistically calculate the zebrafish blood flow velocity. The anti-qi stagnation and blood stasis thrombosis efficacy of the sample is evaluated based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis, and P < 0.05 indicates that the difference is statistically significant.

[0081] 3. Detection Results 3.1 Maximum Detection Concentration The experimental results for determining the maximum detection concentration are shown in Table 5 below.

[0082] Table 5:

[0083] It can be seen that the maximum detection concentration of the diglyceride oil composition in Example 6 for improving the efficacy of qi stagnation and blood stasis is 2000 μg / mL.

[0084] 3.2 Evaluation of the Efficacy against Qi Stagnation and Blood Stasis Thrombosis The evaluation results of the efficacy against qi stagnation and blood stasis thrombosis are shown in Table 6 below.

[0085] Table 6:

[0086] Among them, * represents p < 0.05 compared with the model control group, and *** represents p < 0.001 compared with the model control group. Compared with the model control group, after using the diglyceride oil composition in Example 6, the blood flow velocity of zebrafish increased by 17%, showing an obvious effect in improving high thrombosis, while there was no significant difference in the peanut oil group.

[0087] It can be seen that using the diglyceride oil provided by the preparation process of the present invention, a diglyceride oil composition can be obtained, which has the effect of increasing blood flow velocity and improving the efficacy of qi stagnation and blood stasis thrombosis.

[0088] Effect Example 2 Evaluation of the effects of the diglyceride oil composition on the blood uric acid level, blood triglyceride level, and blood cholesterol level in Uox gene knockout mice.

[0089] SPF - level C57BL / 6J mice were selected for the experiment. The hyperuricemia mouse model was a male mouse model with Uox gene knockout, provided by the Medical Research Center of Qingdao University Affiliated Hospital. Uox, also known as urate oxidase, is responsible for oxidatively degrading uric acid small molecules in animals, thereby maintaining the blood uric acid concentration in animals at a normal level. In male C57BL / 6J mice with Uox gene knockout, the decreased Uox expression leads to disordered uric acid metabolism. The blood uric acid level of the successfully modeled mice is stably between 400 - 600 μmol / L and they can survive for a long time. When grouping, the body weight and age of the experimental animals were: Uox - gene - knockout male mice: 18 - 22 g, 8 - 12 weeks old; wild - type male mice: 20 - 25 g, 8 - 12 weeks old.

[0090] Animal feeding adaptation period: 7 days. The experimenters observed beside the cages once a day during the adaptation period.

[0091] Mice were housed in individually ventilated cages (IVC), with no more than 5 mice per cage. During the feeding period, the environmental parameters of the animal room were recorded, and no other types of animals were housed in the same room during the experiment. The feeding environment was: temperature 20 - 26 °C, relative humidity 40% - 70%, ventilation was carried out using an independent ventilation cage system for ventilation and air exchange, the light was alternated between light and dark every 12 hours, the lights were turned off at 7 pm every day and turned on at 7 am the next day.

[0092] The SPF mouse maintenance feed sterilized by cobalt - 60 irradiation was provided by Shanghai Model Organisms Center, Inc.; the lipid - substituted feed was provided by Beijing Keao Xieli Feed Co., Ltd., and the diglyceride oil composition of Example 7 was used to replace the vegetable oil added in the feed. Each batch of feed was provided with a quality inspection report including detection indexes such as conventional nutritional components, heavy metals, pesticides, and microbiology. In the qualified feed, there were known to be no pollutants affecting the quality of this experiment. The animals freely drank self - made deionized water. Water quality analysis including pH value, conductivity, microorganisms, and heavy metals was carried out once per quarter; a comprehensive water quality test including sensory indexes, physical and chemical indexes, and microorganisms was carried out once a year. There were known to be no pollutants affecting the quality of this experiment. The corncob bedding used in this experiment was provided by Shanghai Model Organisms Center, Inc., and each batch of bedding was provided with a quality inspection report including detection indexes such as heavy metals and pesticides. In the qualified bedding, there were known to be no pollutants affecting the quality of this experiment.

[0093] The Provantis - built grouping module was used to randomly group the animals. When randomly grouping, the selected Uox - gene - knockout mice were assigned to 4 groups. The weight differences of the animals used for grouping were all within ±20% of the average weight. After grouping, the average weight of each group of animals had no statistical difference at the 5% test level.

[0094] The experimental groups and administration doses are shown in Table 7.

[0095] Table 7:

[0096] Among them, the normal uric acid control group was wild-type C57BL / 6J male mice, with the genotype denoted as HO-; the hyperuricemia model group and the group in Example 7 were C57BL / 6J male mice with the Uox gene knocked out, with the genotype denoted as HO+. The mice in the normal uric acid control group, hyperuricemia model group, and the group in Example 7 were all 6 - 8 weeks old and weighed 20 g ± 2 g. The normal uric acid control group and the hyperuricemia model group were gavaged with distilled water once a day (200 μL) and fed with maintenance feed for mice, while the group in Example 7 was gavaged with the diglyceride oil composition provided in Example 7 once a day (200 μL) and fed with oil replacement feed.

[0097] All animals were observed once a day (observed for 3 days before grouping, including the day of grouping). Before the first gavage and after administration, the respiratory, motor and other behavioral changes of the animals were closely observed, and then observed every day. All animals were weighed on the day of grouping (day 0), day 14, 28, and 42 after grouping. Blood was collected from the medial canthus, serum was separated, and blood biochemical indexes were detected once.

[0098] Body weight and blood biochemistry were expressed as mean ± standard deviation. GraphPad Prism 9.5 software was used for data statistical analysis and chart making. One-way ANOVA was used for comparison, and P < 0.05 was considered statistically significant. If there were differences in the results of one-way ANOVA, Dunnett or Tukey correction was used for pairwise comparison, and corrected P < 0.05 was considered statistically significant. For comparisons involving gavage time and group as two factors, two-way ANOVA was used. If there were differences in the results of two-way ANOVA, Tukey's HSD correction was used for pairwise comparison, and corrected P less than 0.05 was considered statistically significant.

[0099] The experimental results are as follows: 1. General observation: There were no obvious changes in the respiration, defecation, movement, mental state, etc. of all animals before and after gavage.

[0100] 2. Changes in serum uric acid, triglyceride and total cholesterol in mice The results of the mean ± standard deviation of the three biochemical indexes of serum uric acid, triglyceride and total cholesterol concentrations on days 0, 14, 28, and 42 of the experiment are shown in Table 8.

[0101] Table 8:

[0102] On the 42nd day of the experiment, the detection results of blood uric acid in mice were as Figure 1 shown, and the difference in the blood uric acid detection results from those on the 0th day of the experiment was as Figure 2 shown; the detection results of the total blood cholesterol concentration in mice were as Figure 3 shown, and the difference in the total blood cholesterol concentration detection results from those on the 0th day of the experiment was as Figure 4 shown; the detection results of the blood triglyceride concentration in mice were as Figure 5 shown, and the difference in the blood triglyceride concentration detection results from those on the 0th day of the experiment was as Figure 6 shown.

[0103] It can be seen that compared with the model control group, the diglyceride oil composition provided in Example 7 has the effects of reducing the blood uric acid level in mice (p < 0.05), and significantly reducing the blood triglyceride and cholesterol levels in mice (p < 0.001).

[0104] Effect Example 3 Effect of the diglyceride oil composition on blood glucose in hyperglycemic zebrafish with thrombosis models.

[0105] 1. Experimental conditions 1.1. 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), provided by the fish culture center of the Innovative Experiment Center of Huante Biotechnology. 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). IACUC ethical review number: IACUC - 2024 - 10165 - 01.

[0106] 1.2. Samples, instruments and consumables The samples detected in this effect example were the diglyceride oil compositions provided in Example 6, Example 8, and Example 9.

[0107] Dissecting microscope (SZX7, OLYMPUS); precision electronic balance (CP214, OHAUS); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd.); blood glucose meter (ACCU - CHEK Performa, Roche Diagnostic Products (Shanghai) Co., Ltd.); blood glucose meter test strips (batch number 670452, Roche Diagnostic Products (Shanghai) Co., Ltd.). Egg yolk powder (batch number 20230203, Zhejiang Aige Biotechnology Co., Ltd.); anhydrous glucose (batch number C15778026, Shanghai Macklin Biochemical Technology Co., Ltd.); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma).

[0108] 2. Evaluation Method 2.1 Determination of Maximum Test Concentration (MTC) Randomly select wild-type AB strain zebrafish at 5 days post-fertilization (5 dpf) and place 30 zebrafish in each beaker. Different concentrations of diglyceride oil composition are given respectively, and a normal control group and a model control group are set up at the same time. The volume of each beaker is 25 mL. Except for the normal control group, high-sugar and high-fat feed is given by water solution to the rest of the experimental groups to establish a hyperglycemic model of zebrafish. After treatment at 28 °C for 2 days, the MTC of the samples on the model zebrafish is measured.

[0109] 2.2 Evaluation of Hypoglycemic Efficacy Randomly select wild-type AB strain zebrafish at 5 dpf and place 30 zebrafish in each beaker. Different concentrations of diglyceride oil composition (dissolved with DMSO) are given respectively, and the positive control group is given metformin solution at a concentration of 400 μg / mL. A normal control group and a model control group are set up at the same time. Except for the normal control group, high-sugar and high-fat feed is given by water solution to the rest of the experimental groups to establish a qi stagnation and blood stasis thrombosis model of zebrafish. After treatment at 28 °C for 2 days, data are collected with a blood glucose meter, and the glucose levels of zebrafish are analyzed and statistically analyzed. The hypoglycemic efficacy of the samples is evaluated based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Statistical analysis is performed using SPSS 26.0 software. P < 0.05 indicates that the difference is statistically significant.

[0110] 3. Detection Results 3.1 Maximum Test Concentration The experimental results for determining the maximum test concentration are shown in Table 9 below.

[0111] Table 9

[0112] It can be seen that the maximum test concentration of the diglyceride oil composition in the hypoglycemic efficacy test of Examples 6, 8, and 9 is 2000 μg / mL.

[0113] 3.2 Evaluation of Hypoglycemic Efficacy Under the evaluation conditions of 2.2, the hypoglycemic efficacy evaluation results of Examples 6, 8, and 9 are shown in Table 10 and Figure 7 as follows.

[0114] Table 10

[0115] Among them, *** represents p < 0.001 compared with the model control group.

[0116] It can be seen that the diglyceride oil composition provided by the preparation process of the present invention has the effect of reducing blood sugar.

[0117] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting 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 shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride oil that quintuply improves vascular health by enzymatic hydrolysis, characterized in that, It includes the following steps: Mix rapeseed oil with low erucic acid, glycerol, water, fatty acid and lipase, carry out enzymatic hydrolysis reaction, molecular distillation, decolorization and deodorization to obtain diglyceride oil; The lipase is a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase. The weight ratio of the rapeseed oil with low erucic acid, glycerol, water, fatty acid and lipase is 500:80 - 100:1 - 2:30 - 60:10 - 20.

2. The method according to claim 1, wherein The fatty acid is selected from at least one of linoleic acid, oleic acid and eicosapentaenoic acid; the lipase is a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase mixed in a weight ratio of 7:1 - 2:1 - 2.

3. The method according to claim 2, wherein The lipase is a mixture of Candida antarctica Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreatic lipase mixed in a weight ratio of 7:1.5:1.

5.

4. The method according to claim 1, wherein The temperature of the enzymatic hydrolysis reaction is 50 - 65 °C, and the time of the enzymatic hydrolysis reaction is 1 - 9 h.

5. The method according to claim 1, wherein The molecular distillation includes the first - stage molecular distillation, the second - stage molecular distillation and the third - stage molecular distillation; The temperature of the first - stage molecular distillation is 100 - 160 °C, and the vacuum degree is less than 500 Pa; The temperature of the second - stage molecular distillation is 170 - 200 °C, and the vacuum degree is less than 10 Pa; The temperature of the third - stage molecular distillation is 220 - 280 °C, and the vacuum degree is less than 10 Pa; The temperature of the condensation surface of the molecular distillation is 35 - 55 °C, and the molecular distillation is repeated 3 - 5 times.

6. The method according to claim 1, wherein The weight ratio of the rapeseed oil with low erucic acid, glycerol, water, fatty acid and lipase is 500:90:1.5:45:

15.

7. The diglyceride oil prepared by the method according to any one of claims 1 - 6.

8. The application of the diglyceride oil prepared by the method according to any one of claims 1 - 6 in the production of oil products.

9. A diglyceride oil composition, characterized in that, It includes rapeseed diglyceride oil with low erucic acid, corn diglyceride oil, edible vegetable oil and food additive; Among them, the preparation method of the rapeseed diglyceride oil with low erucic acid is the method according to any one of claims 1 - 6.

10. The diglyceride oil composition according to claim 9, wherein The edible vegetable oil is selected from corn oil and / or peanut oil, and the food additive is vitamin E.

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