Method for preparing diglyceride with effects of reducing blood lipid, total cholesterol, blood glucose and uric acid by enzymatic hydrolysis, product thereof and application

Abstract

The present invention discloses a method for preparing diglycerides with the effects of reducing blood lipid, total cholesterol, blood glucose and uric acid through enzymatic hydrolysis, as well as products and applications thereof, belonging to the technical field of diglyceride production. The method comprises the following steps: (1) mixing vegetable oil, glycerol, complex lipase and water for glycerolysis reaction to obtain a reaction product; (2) separating the reaction product and collecting the upper layer liquid; (3) subjecting the upper layer liquid to distillation separation, decolorization and deodorization to obtain diglycerides; the vegetable oil in step (1) is low-erucic rapeseed oil and corn oil; the complex lipase in step (1) is Rhizopus javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 2-4:1:5-8. The present invention can reduce the dosage of enzymes, lower the cost, and the prepared diglycerides have better activities of reducing blood lipid, total cholesterol, blood glucose and uric acid, and have more advantages in pharmaceutical preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of diglyceride production, and particularly relates to a method for preparing diglyceride with the effects of reducing blood lipid, total cholesterol, blood glucose and uric acid by enzymatic hydrolysis, and its products and applications. Background Art

[0002] Diglyceride (DAG) is a natural component of oils and fats and an intermediate product of oil and fat metabolism. It has the flavor of ordinary oils and fats and high safety. The absorption and metabolism modes of diglyceride and triglyceride in the body are different. It has the effects of reducing visceral fat, inhibiting weight gain and reducing blood lipid, so it has received extensive attention.

[0003] In recent years, preparing DAG oil with nutritional and health functions from ordinary oils and fats has become one of the main directions of oil and fat development. For example, CN101270375A discloses a process for preparing 1,3-diglyceride by enzymatic method in a tert-butanol system. The process steps are as follows: acyl acceptor monoglyceride and acyl donor with a molar ratio of 1:0.5 - 1:3 are added to tert-butanol based on 20 - 200% of the mass of acyl acceptor monoglyceride and lipase based on 2 - 20% of the mass of acyl acceptor monoglyceride, and then they are loaded into any biochemical reactor suitable for enzymatic reaction and mixed evenly. The temperature is controlled at 30 - 65°C and the reaction is carried out for 1 - 12 hours. The conversion rate of acyl acceptor monoglyceride to diglyceride reaches 70 - 90%, and the content of 1,3-diglyceride in the generated diglyceride reaches more than 75%. The acyl donor is fatty acid, short-chain fatty acid ester, monoglyceride, triglyceride, animal and vegetable oils or incomplete hydrolysis products of oils and fats.

[0004] Hua Di et al. disclosed a method for continuously preparing diglyceride by enzymatic glycerolysis in the research on continuously preparing diglyceride by enzymatic method. By comparing the reaction effects of different lipases and different solvent systems, it was selected to continuously carry out the reaction in a solvent-free system using a Novozyme 435 immobilized lipase packed column reactor. Further optimizing the reaction conditions, the optimal process conditions were determined as follows: the molar ratio of soybean oil to glycerol was 1:2, the reaction temperature was 60°C, and the flow rate was 0.05 g / min. Under these conditions, the content of diglyceride in the product could reach 58.17%.

[0005] CN103243126A discloses a method for preparing diglyceride, comprising the following steps: 1) Selection of raw materials: Select rapeseed oil, glycerol, bio-enzyme and water according to the mass parts of each raw material as follows: 100 parts of rapeseed oil, 30 parts of glycerol, 1-8 parts of bio-enzyme, 0-2 parts of water, with the water content of glycerol being 3-5wt%; 2) Mixing: Mix rapeseed oil, glycerol, bio-enzyme and water, stir and heat to 60-80°C, with the stirring speed being 120-130 revolutions per minute, and react for 2-6 hours to obtain a mixture; 3) Esterification and dehydration of the mixture to obtain an esterification and dehydration product; 4) Settling and deglycerolization of the esterification and dehydration product to obtain a sediment; 5) Further screening by molecular distillation: ① Vacuum dehydration; ② Removal of fatty acids; ③ Removal of monoglyceride; ④ Removal of triglyceride: Then at 180-240°C, with a vacuum degree of 1-5 Pa, vacuum remove triglyceride for 10-20 minutes to obtain diglyceride, and the content of diglyceride in this invention reaches more than 85wt%.

[0006] Although there are many methods for preparing triglyceride, it is still a very challenging task to prepare a diglyceride with more abundant functions at present. Summary of the Invention

[0007] The present invention aims to provide a method for preparing diglyceride with functions of reducing blood lipid, total cholesterol, blood glucose and uric acid through enzymatic hydrolysis, as well as its products and applications. The diglyceride prepared by the enzymatic hydrolysis process of the present invention has multiple functions such as reducing blood lipid, total cholesterol, blood glucose and uric acid. Compared with the prior art, the drugs prepared by using this diglyceride have better effects of reducing blood lipid, total cholesterol, blood glucose and uric acid, with obvious advantages.

[0008] In order to achieve the above technical solutions, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a method for preparing diglyceride with functions of reducing blood lipid, total cholesterol, blood glucose and uric acid through enzymatic hydrolysis, comprising the following steps:

[0010] (1) Mix vegetable oil, glycerol, compound lipase and water to carry out glycerolysis reaction to obtain a reactant;

[0011] (2) Separate the reactant obtained in step (1) and collect the upper layer liquid;

[0012] (3) Distill, separate, decolorize and deodorize the upper layer liquid obtained in step (2) to obtain diglyceride.

[0013] In some embodiments, the mass ratio of the vegetable oil to the glycerol in step (1) is 0.6-1.1:1; preferably 0.7-1:1.

[0014] In some embodiments, the vegetable oil in step (1) is at least one of rapeseed oil with low erucic acid content, corn oil, sunflower oil, and peanut oil. Preferably, it is rapeseed oil with low erucic acid content and corn oil; further preferably, the mass ratio of the rapeseed oil with low erucic acid content to the corn oil is 1.5 - 5:1; even more preferably, it is 3 - 3.5:1.

[0015] In some embodiments, the addition amount of the composite lipase in step (1) is 0.05 - 5% of the total mass of the vegetable oil and glycerol; further preferably, it is 0.1 - 2%.

[0016] In some embodiments, the composite lipase in step (1) is at least one of Hansenula polymorpha lipase, Aspergillus niger lipase, Mucor javanicus lipase, Aspergillus oryzae lipase, Candida cylindracea lipase, pancreatic lipase, Rhizopus niveus lipase, Rhizomucor miehei lipase, Rhizopus oryzae lipase, and Trichoderma reesei lipase.

[0017] In some embodiments, the composite lipase in step (1) is at least one of Mucor javanicus lipase, Aspergillus niger lipase, and Aspergillus oryzae lipase; preferably, the composite lipase is Mucor javanicus lipase, Aspergillus niger lipase, and Aspergillus oryzae lipase; further preferably, the mass ratio of Mucor javanicus lipase, Aspergillus niger lipase, and Aspergillus oryzae lipase is 2 - 4:1:5 - 8; even more preferably, the mass ratio of Mucor javanicus lipase, Aspergillus niger lipase, and Aspergillus oryzae lipase is 3:1:6.

[0018] In some embodiments, the addition amount of the water in step (1) is 6 - 26% of the mass of the vegetable oil; preferably, it is 8 - 20%.

[0019] In some embodiments, the conditions for the glycerolysis reaction in step (1) are: reacting at 30 - 56 °C for 1 - 3 h.

[0020] In some embodiments, the separation in step (3) is by standing or centrifugation. Preferably, the standing time is 1 - 4 h; further preferably, it is 1 - 3 h, and even more preferably, it is 1 - 2 h.

[0021] In some embodiments, the conditions for the distillation separation in step (3) are: the cooling water temperature is 35 - 40 °C, the vacuum degree is 5 - 10 Pa, the scraping film rate is 50 - 100 r / min, and the feed flow rate is 12 - 18 kg / min.

[0022] In a second aspect, the present invention provides the diglyceride prepared by the above method.

[0023] Experiments have proved that the diglyceride prepared by the present invention has more advantages than the commercially available diglyceride in the preparation of drugs with multiple effects such as reducing blood lipid, total cholesterol, blood sugar, and uric acid.

[0024] In a third aspect, the present invention provides the use of the diglyceride prepared by the above method in the preparation of pharmaceuticals.

[0025] The diglyceride composition described in the present invention can also be used as a raw material and mixed with pharmaceutically acceptable excipients to prepare corresponding pharmaceuticals.

[0026] In some embodiments, the pharmaceutical is a liquid preparation, tablet, capsule, granule, dry suspension or pill; preferably a liquid preparation.

[0027] In a fourth aspect, the present invention provides a diglyceride composition comprising the diglyceride prepared by the above method.

[0028] In some embodiments, the diglyceride composition further comprises Acer truncatum seed oil, Hippohgae rhamnoides seed oil, vitamin E and high oleic acid peanut oil.

[0029] Preferably, the diglyceride composition comprises 60-99% of diglyceride oil, 0.01-20% of Acer truncatum seed oil, 0.01-20% of Hippohgae rhamnoides seed oil, 0.01-0.2% of vitamin E and the balance of high oleic acid peanut oil by mass percentage.

[0030] Advantages of the present invention

[0031] (1) The present invention optimizes the glycerolysis reaction from two aspects of raw material compounding and screening of enzyme preparations. Using low erucic acid rapeseed oil and corn oil as raw materials, and a composite lipase obtained by compounding Rhizomucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase as the enzyme preparation, diglyceride is prepared through an enzymatic reaction. This diglyceride has better activities of reducing blood lipid, total cholesterol, blood sugar and uric acid, and has more advantages in the preparation of pharmaceuticals.

[0032] (2) The present invention uses a compound of Rhizomucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase. Among them, Aspergillus oryzae lipase can catalyze acyl migration and transesterification reactions, increase the diversity of diglyceride isomers, and optimize lipid metabolism activity. Rhizomucor javanicus lipase can specifically hydrolyze the ester bond at the Sn-1,3 position to increase the content of 1,3-diglyceride. Aspergillus niger lipase can optimize the reaction conditions and further improve the metabolic activities of Rhizomucor javanicus lipase and Aspergillus oryzae lipase. The compound of the three enzymes can, on the one hand, reduce the amount of enzyme used and lower the cost, and on the other hand, enhance the activities of diglyceride in reducing blood lipid, total cholesterol, blood sugar and uric acid.

[0033] (3) Experiments have proved that the product prepared by using the diglyceride prepared by the present invention has better effects of reducing blood lipid, total cholesterol, blood sugar and uric acid than the prior art. Detailed implementation manners

[0034] The descriptions of the following embodiments are only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can still be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0035] Therefore, the present invention will not be limited to these embodiments shown in this article, but can be applied to a wider range that conforms to the principles and novel features disclosed in this article. Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0036] As used in the specification and claims, the singular forms "a", "an", and "the" include the plural forms unless the context clearly dictates otherwise.

[0037] All ranges involving the same components or properties include the endpoints, and these endpoints can be independently combined. Since these ranges are continuous, they include every value between the minimum and maximum values. It should also be understood that any numerical range cited in the present invention is expected to include all sub-ranges within that range.

[0038] The term "pharmaceutically acceptable" indicates that the composition is compatible with other components constituting the preparation.

[0039] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products in this technical field. In the present invention, Mucor javanicus lipase is SIGMA 534803, 100,000 U / g; Aspergillus niger lipase is SIGMA 62301, 200 U / g; Aspergillus oryzae lipase is SIGMA 62285, 50 mU / g, low erucic acid rapeseed oil, purchased from Chengdu Xinxing Grain and Oil Co., Ltd., batch number 20240919 (production date), high oleic acid peanut oil, purchased from Shandong Jinsheng Grain and Oil Food Co., Ltd., batch number GY2405, Acer truncatum seed oil, purchased from Heze Zhonghe Jianyuan Biotechnology Co., Ltd., batch number 22T20B48801, sea buckthorn seed oil, purchased from Heze Zhonghe Jianyuan Biotechnology Co., Ltd., batch number 08T20B48801, corn oil, purchased from Shandong Yuhuang Grain and Oil Food Co., Ltd., batch number PY240916107, Xanthoceras sorbifolium Bunge oil, purchased from Shandong Linyuhong Xanthoceras sorbifolium Bunge Co., Ltd.

[0040] Example 1 Method for enzymatically preparing diglyceride with the effects of reducing blood lipid, total cholesterol, blood sugar and uric acid

[0041] The steps are as follows:

[0042] (1) Mix vegetable oil, glycerol, complex lipase and water, and carry out glycerolysis reaction at a temperature of 52 ± 2 °C for 1.5 h to obtain a reactant;

[0043] Among them, the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 3:1;

[0044] The complex lipase is Rhizomucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 3:1:6;

[0045] The mass ratio of vegetable oil to glycerol is 0.8:1;

[0046] The addition amount of the complex lipase is 0.2% of the total mass of vegetable oil and glycerol;

[0047] The addition amount of water is 10% of the mass of vegetable oil.

[0048] (2) Let the reactant obtained in step (1) stand for 2 h, and collect the upper layer liquid;

[0049] (3) Carry out distillation separation on the upper layer liquid obtained in step (2), cool the water temperature to 37 ± 2 °C, the vacuum degree is 8 Pa, the scraping film rate is 75 ± 5 r / min, the feeding flow rate is 15 kg / min, and carry out decolorization and deodorization to obtain diglyceride.

[0050] Example 2 Method for enzymatically preparing diglyceride with effects of reducing blood lipid, total cholesterol, blood glucose and uric acid

[0051] The steps are as follows:

[0052] (1) Mix vegetable oil, glycerol, complex lipase and water, and carry out glycerolysis reaction at a temperature of 32 ± 2 °C for 3 h to obtain a reactant;

[0053] Among them, the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 1.5:1;

[0054] The complex lipase is Rhizomucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 2:1:8;

[0055] The mass ratio of vegetable oil to glycerol is 0.6:1;

[0056] The addition amount of the complex lipase is 0.1% of the total mass of vegetable oil and glycerol;

[0057] The addition amount of water is 6% of the mass of vegetable oil.

[0058] (2) Let the reactant obtained in step (1) stand for 2 h, and collect the upper layer liquid;

[0059] (3) Distill and separate the supernatant obtained in step (2), with the cooling water temperature at 37 ± 2 °C, the vacuum degree at 8 Pa, the scraping film rate at 75 ± 5 / min, the feeding flow rate at 15 kg / min, and decolorize and deodorize to obtain diglyceride.

[0060] Example 3 Method for Preparing Diglyceride with Functions of Reducing Blood Lipid, Total Cholesterol, Blood Glucose and Uric Acid by Enzymatic Hydrolysis

[0061] The steps are as follows:

[0062] (1) Mix vegetable oil, glycerol, composite lipase and water, and carry out glycerolysis reaction at a temperature of 54 ± 2 °C for 1 h to obtain a reaction product;

[0063] Among them, the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 5:1;

[0064] The composite lipase is Mucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 4:1:5;

[0065] The mass ratio of vegetable oil to glycerol is 1.1:1;

[0066] The addition amount of the composite lipase is 2% of the total mass of vegetable oil and glycerol;

[0067] The addition amount of water is 26% of the mass of vegetable oil.

[0068] (2) Let the reaction product obtained in step (1) stand for 2 h, and collect the supernatant;

[0069] (3) Distill and separate the supernatant obtained in step (2), with the cooling water temperature at 37 ± 2 °C, the vacuum degree at 8 Pa, the scraping film rate at 75 ± 5 r / min, the feeding flow rate at 15 kg / min, and decolorize and deodorize to obtain diglyceride.

[0070] Example 4 Method for Preparing Diglyceride with Functions of Reducing Blood Lipid, Total Cholesterol, Blood Glucose and Uric Acid by Enzymatic Hydrolysis

[0071] The steps are as follows:

[0072] (1) Mix vegetable oil, glycerol, lipase and water, and carry out glycerolysis reaction at a temperature of 46 ± 2 °C for 2 h to obtain a reaction product;

[0073] Among them, the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 3:1;

[0074] The lipase is Mucor javanicus lipase;

[0075] The mass ratio of vegetable oil to glycerol is 0.8:1;

[0076] The addition amount of the lipase is 5% of the total mass of vegetable oil and glycerol;

[0077] The addition amount of water is 10% of the mass of the vegetable oil.

[0078] (2) Let the reactant obtained in step (1) stand for 2 h, and collect the upper layer liquid;

[0079] (3) Carry out distillation separation on the upper layer liquid obtained in step (2), cool the water temperature to 37±2 °C, the vacuum degree is 8 Pa, the scraping film rate is 75±5 r / min, the feed flow rate is 15 kg / min, decolorize and deodorize to obtain diglyceride.

[0080] Comparative Example 1

[0081] The difference between this comparative example and Example 4 lies in: the composition of the composite lipase is different;

[0082] The steps are as follows:

[0083] (1) Mix vegetable oil, glycerol, composite lipase and water, and carry out glycerolysis reaction at a temperature of 52±2 °C for 1.5 h to obtain a reactant;

[0084] Among them, the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 3:1;

[0085] The composite lipase is Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 31:6;

[0086] The mass ratio of vegetable oil to glycerol is 0.8:1;

[0087] The addition amount of the composite lipase is 5% of the total mass of vegetable oil and glycerol;

[0088] The addition amount of water is 10% of the mass of the vegetable oil.

[0089] (2) Let the reactant obtained in step (1) stand for 2 h, and collect the upper layer liquid;

[0090] (3) Carry out distillation separation on the upper layer liquid obtained in step (2), cool the water temperature to 37±2 °C, the vacuum degree is 8 Pa, the scraping film rate is 75±5 r / min, the feed flow rate is 15 kg / min, decolorize and deodorize to obtain diglyceride.

[0091] Comparative Example 2

[0092] The difference between this comparative example and Example 1 lies in: the composition of the composite lipase is different, and Xanthoceras sorbifolia oil is used instead of rapeseed oil with low erucic acid.

[0093] The steps are as follows:

[0094] (1) Mix vegetable oil, glycerol, composite lipase and water, and carry out glycerolysis reaction at a temperature of 52±2 °C for 1.5 h to obtain a reactant;

[0095] Among them, the vegetable oil is Xanthoceras sorbifolia oil and corn oil with a mass ratio of 3:1;

[0096] The composite lipase is Rhizopus javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 8:2:1;

[0097] The mass ratio of the vegetable oil to glycerol is 0.8:1;

[0098] The addition amount of the composite lipase is 0.2% of the total mass of the vegetable oil and glycerol;

[0099] The addition amount of water is 10% of the mass of the vegetable oil.

[0100] (2) Let the reactant obtained in step (1) stand for 2 h, and collect the upper layer liquid;

[0101] (3) Carry out distillation separation on the upper layer liquid obtained in step (2), cool the water temperature to 37 ± 2 °C, the vacuum degree is 8 Pa, the scraping film rate is 75 ± 5 r / min, the feeding flow rate is 15 kg / min, and carry out decolorization and deodorization to obtain diglyceride.

[0102] Comparative Example 3

[0103] The difference between this comparative example and Example 1 is that: the composition of the composite lipase is different, and seabuckthorn seed oil is used to replace low erucic acid rapeseed oil.

[0104] The steps are as follows:

[0105] (1) Mix the vegetable oil, glycerol, composite lipase and water, and carry out glycerolysis reaction at a temperature of 52 ± 2 °C for 1.5 h to obtain a reactant;

[0106] Among them, the vegetable oil is seabuckthorn seed oil and corn oil with a mass ratio of 3:1;

[0107] The composite lipase is Rhizopus javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 1:5:2;

[0108] The mass ratio of the vegetable oil to glycerol is 0.8:1;

[0109] The addition amount of the composite lipase is 0.2% of the total mass of the vegetable oil and glycerol;

[0110] The addition amount of water is 10% of the mass of the vegetable oil.

[0111] (2) Let the reactant obtained in step (1) stand for 2 h, and collect the upper layer liquid;

[0112] (3) Carry out distillation separation on the upper layer liquid obtained in step (2), cool the water temperature to 37 ± 2 °C, the vacuum degree is 8 Pa, the scraping film rate is 75 ± 5 r / min, the feeding flow rate is 15 kg / min, and carry out decolorization and deodorization to obtain diglyceride.

[0113] Example 5 Diglyceride Composition

[0114] According to the formulation amounts in Table 1, Acer truncatum seed oil, sea buckthorn seed oil, vitamin E, and high-oleic acid peanut oil were added to diglyceride and mixed to obtain a diglyceride composition.

[0115] Table 1 Formulation of Diglyceride Composition

[0116]

[0117] I. Hypolipidemic Efficacy Test

[0118] Explore the effect of the prepared diglyceride composition on the blood lipid of zebrafish.

[0119] 1. Test Animals

[0120] 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, the pH was 6.5 - 8.5, and the hardness was 50 - 100 mg / L CaCO3). They were provided by the fish culture center of Hangzhou Huante Biotechnology Co., Ltd. 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).

[0121] Wild-type AB strain zebrafish were bred by natural paired mating. Zebrafish at 3 days post-fertilization (3 dpf) were used to evaluate the effect of the sample on the blood lipid of zebrafish.

[0122] 2. Test Samples and Reagents

[0123] 2.1 Test Samples

[0124] Samples S1 - S4 and D1 - D3: The diglyceride composition was formulated into a 200 mg / mL stock solution using DMSO and stored at -20°C for later use. Among them, the diglyceride composition was prepared from Example 5;

[0125] Control sample (D0): A commercially available diglyceride composition was formulated into a 200 mg / mL stock solution using DMSO and stored at -20°C for later use. Among them, the commercially available diglyceride composition was provided by Guangdong Shanbainian Special Medical Food Co., Ltd., an 80% DAG content diglyceride edible oil with a production date of 20220902.

[0126] 2.2 Reagents

[0127] Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Oil Red 0 (batch number F2116210, Shanghai Aladdin Biotechnology Co., Ltd., China); 1,2-propylene glycol (batch number 20211117, Sinopharm Chemical Reagent Co., Ltd., China); pure egg yolk powder (batch number 20200809, Zhejiang Aige Biotechnology Co., Ltd., China); D-(+)-glucose (batch number 12209335, Shanghai Aladdin Biochemical Technology Co., Ltd., China); 4% tissue cell fixative (batch number 20221014, Beijing Solebold Co., Ltd., China).

[0128] 3. Detection method

[0129] 3.1 MCT determination

[0130] Wild-type AB zebrafish (3 dpf) were randomly selected and placed in beakers. Thirty zebrafish were treated in each beaker (experimental group). All experimental groups, except the normal control group, were fed a high-sugar, high-fat diet in water to establish a high-fat diet model in zebrafish. After two days of treatment at 28°C, the high-sugar, high-fat diet was removed and the samples (concentrations shown in Table 2) were administered in water. A normal control group was also established. Each beaker held 25 mL of water. The medium was changed every morning and evening. During the sample treatment period, the number of zebrafish that died in each experimental group was counted daily and removed promptly. After a further three days of treatment at 28°C, the MTC of the samples in the high-fat diet model zebrafish was measured.

[0131] Table 2 Results of the experimental study on the effect of sample concentration on zebrafish blood lipids

[0132]

[0133] From the above, it can be seen that under the experimental conditions, the MTC of the diglyceride composition on the blood lipids of zebrafish is 2000 μg / mL.

[0134] 3.2 Effects on zebrafish blood lipids

[0135] 3-dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 10 zebrafish per beaker. All experimental groups, except the normal control group, were fed a high-sugar, high-fat diet in water to establish a high-fat diet model in zebrafish. After two days of treatment at 28°C, the high-sugar, high-fat diet was removed from all experimental groups, except the model control group, and the sample concentrations were administered in water. A normal control group and a model control group were also established. The model control group continued to receive the high-sugar, high-fat diet until 8 dpf. Each beaker held 25 mL of water. The medium was changed daily in the morning and evening. After three days of treatment at 28°C, the fish were stained with Oil Red 0 for whole-body lipid staining. After decolorization and bleaching, the zebrafish were photographed under a dissecting microscope and analyzed and acquired using NIS-Elements D 3.20 advanced image processing software. The intensity of vascular staining in the tail was analyzed, and the effects of the samples on zebrafish lipids were statistically analyzed using this indicator. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0136] The results are shown in Table 3.

[0137] Table 3 Experimental results of the effects of samples on zebrafish blood lipids (n=10)

[0138]

[0139] Note: Different letters in the same column indicate significant differences between the groups, P < 0.05.

[0140] From the above, we can see that compared with the normal control group, the staining intensity of the blood vessels in the zebrafish tail of the model control group was significantly increased (P < 0.05), indicating that the zebrafish high-fat model was successfully established.

[0141] Compared with the model control group, the staining intensity of the zebrafish tail blood vessels in each drug-treated group was significantly reduced (P < 0.05), indicating that the diglyceride composition in each group has the effect of lowering blood lipids.

[0142] Compared with the treatment group D0, the staining intensity of the zebrafish tail vessels in treatment groups D1-D3 showed no significant difference (P>0.05), while the staining intensity of the zebrafish tail vessels in treatment groups S1-S4 was significantly reduced (P<0.05). This indicates that the diglyceride composition prepared with S1-S4 has a better lipid-lowering effect than the commercially available product, and the diglyceride composition prepared with D1-D3 has essentially the same lipid-lowering effect as the commercially available product.

[0143] Comparison of the drug administration groups S1-S4 showed that the lipid-lowering effect of the diglyceride composition prepared by S1-S3 was better than that of S4.

[0144] 2. Blood sugar lowering efficacy test

[0145] Explore the hypoglycemic effect of the prepared diglyceride composition.

[0146] 1. Test animals

[0147] Zebrafish were all raised in fish-raising water at 28°C (water quality: 200 mg of instant sea salt was added to each liter of reverse osmosis water, the conductivity was 450 - 550 μS / cm, the pH was 6.5 - 8.5, and the hardness was 50 - 100 mg / L CaCO3). They were provided by the fish-raising center of Hangzhou Huante Biotechnology Co., Ltd. The license number for the use of experimental animals was: SYXK (Zhe) 2022 - 0004, and the feeding management met the requirements of international AAALAC accreditation (accreditation number: 001458).

[0148] 2. Test samples, instruments, and reagents

[0149] 2.1 Test samples

[0150] Samples S1 - S4 and D1 - D3: The diglyceride composition was formulated into a 200 mg / mL stock solution using DMSO and stored at -20°C for later use. Among them, the diglyceride compositions were respectively prepared from Example 5.

[0151] Control sample (D0): A commercially available diglyceride composition was formulated into a 200 mg / mL stock solution using DMSO and stored at -20°C for later use. Among them, the commercially available diglyceride composition was provided by Guangdong Shanbainian Special Medical Food Co., Ltd., an 80% DAG content diglyceride edible oil with a production date of 20220902.

[0152] Positive control: Metformin hydrochloride tablets (hereinafter referred to as metformin), white tablets, batch number ACL1077, produced by Sino-American Shanghai Squibb Co., Ltd., and the solvent was ultrapure water.

[0153] 2.2 Instruments

[0154] Dissecting microscope (SZX7, OLYMPUS, Japan); Precision electronic balance (CP214, OHAUS, USA); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Blood glucose meter (ACCU-CHEK Performa, Roche Diagnostic Products (Shanghai) Co., Ltd., China); Blood glucose meter test strips (batch number 670452, Roche Diagnostic Products (Shanghai) Co., Ltd., China).

[0155] 2.3 Reagents

[0156] Egg yolk powder (batch number 20230203, Zhejiang Aige Biotechnology Co., Ltd., China); anhydrous glucose (batch number C15778026, Shanghai Macklin Biochemical Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland).

[0157] 3. Test method for blood glucose lowering effect

[0158] Randomly select 5 dpf wild-type AB strain zebrafish into a beaker, and 10 zebrafish are treated in each beaker (experimental group). The sample concentration of 1000 μg / mL and the positive control metformin concentration of 400 μg / mL are given by water solution respectively. At the same time, a normal control group and a model control group are set up, and the volume of each cup is 25 mL. Except for the normal control group, a hyperglycemic model of zebrafish is established by giving a high-sugar and high-fat diet by water solution in the remaining experimental groups. After treatment at 28 °C for 2 days, data is collected with a blood glucose meter, and the glucose level of zebrafish is analyzed and statistically analyzed. The blood glucose lowering effect of the sample 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, and p<0.05 indicates that the difference is statistically significant.

[0159] The results are shown in Table 4.

[0160] Table 4 Experimental results of the blood glucose lowering effect of the sample (n = 10)

[0161]

[0162] Note: Different letters in the same column represent significant differences in data between groups, P < 0.05.

[0163] As can be seen from the above, compared with the normal control group, the glucose level of zebrafish in the model control group increased significantly (P < 0.05), indicating that the hyperglycemic model of zebrafish was successfully established.

[0164] Compared with the model control group, the glucose level of zebrafish in each group decreased significantly (P < 0.05), indicating that in the technical solution claimed in the present invention, the diglyceride composition prepared by S1-S4 and D1-D3 of the present invention and the commercially available diglyceride all have the effect of lowering blood glucose.

[0165] At the same time, compared with the positive control group, there was no significant change in the glucose level of zebrafish in the S1-S4 groups of the administration group (P > 0.05), indicating that the diglyceride composition prepared by S1-S4 of the present invention has the same efficacy as metformin.

[0166] III. Tests on uric acid and total cholesterol lowering effects

[0167] To investigate the effects of the prepared diglyceride composition on serum uric acid (UA), total cholesterol (TC), and triglyceride (TG) in hyperuricemia model rats fed a high-fat diet.

[0168] 1. Experimental animals

[0169] SD rats, SPF grade, male, 7 - 9 weeks old, provided by Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0170] 2. Test samples, reagents, and consumables

[0171] 2.1 Test samples

[0172] The diglyceride compositions were prepared respectively by Example 5.

[0173] Commercially available diglyceride composition (D0), provided by Guangdong Shanbainian Special Medical Food Co., Ltd., 80% DAG - content diglyceride edible oil, production date: 20220902.

[0174] 2.2 Reagents

[0175] Uric Acid (UA) kit (enzymatic colorimetric method), batch number: 20221107, Nanjing Jiancheng Bioengineering Institute.

[0176] Triglyceride (TG) assay kit (oxidase method): batch number: 141722001, Shenzhen Mindray Animal Medical Technology Co., Ltd.

[0177] Total Cholesterol (TC) assay kit (oxidase method): batch number: 141622003, Shenzhen Mindray Animal Medical Technology Co., Ltd.;

[0178] Potassium oxonate: batch number: #B2226363, Aladdin.

[0179] 2.3 Feed

[0180] Basal feed: by mass, 25 parts of flour, 25 parts of oatmeal, 25 parts of cornmeal, 10 parts of bean flour, 8 parts of fish meal, 4 parts of bone meal, and 1 part of refined salt.

[0181] D12451 yeast feed: by mass, 29 parts of lard, 2.8 parts of soybean oil, 16.7 parts of sucrose, 19.6 parts of casein, 5.6 parts of minerals, 52.2 parts of basal feed, and 11.28 parts of yeast powder.

[0182] Diacylglycerol oil feed: 30 parts of diacylglycerol composition, 16.7 parts of sucrose, 19.6 parts of casein, 5.6 parts of minerals, 52.2 parts of basal feed, and 11.28 parts of yeast powder, by mass.

[0183] 3. Experimental methods

[0184] Animals were adaptively fed for 7 days and randomly divided into a normal control group, a model control group, and dosing groups (S1 - S4 and D0 - D3) according to body weight, with 8 animals in each group. Animals in the normal control group were fed basal feed, while animals in the other groups were fed D12451 yeast feed, and the food intake per animal per day was controlled at 15 g.

[0185] Meanwhile, except for the animals in the normal control group, the other groups were intraperitoneally injected with potassium oxonate at 450 mg / kg / d for a total of 8 weeks. After 8 weeks, the dosing groups were switched to diacylglycerol oil feed, and the other operations remained unchanged, for a total of 6 weeks.

[0186] 4. Index detection

[0187] After the experiment, the animals were anesthetized, and blood was collected from the abdominal aorta. Serum was separated to measure the indexes of uric acid (UA), triglyceride (TG), and total cholesterol (TC), and relevant mathematical statistics were performed using SPSS. Measurement data were expressed as mean ± standard deviation (mean ± SD). Independent - sample t - test was used for comparison between groups, and p < 0.05 indicated statistical significance.

[0188] 4.1. Uric acid detection

[0189] Except for the last blood collection, the blood collection volume each time was 0.75 μL. After blood collection, the blood sample was stored at 4°C for 1 h and then centrifuged at 3000 r / min for 15 min. Serum was separated, and the operation was carried out according to the instruction manual of the uric acid (UA) kit provided by Nanjing Jiancheng Bioengineering Institute, and measured using a SpectraMax M5 microplate reader.

[0190] 4.2. Triglyceride and total cholesterol detection

[0191] After the animals were anesthetized and blood was collected from the abdominal aorta until death, the blood sample was stored at 4°C for 1 h and then centrifuged at 3000 r / min for 15 min. Serum was separated, and the contents of triglyceride (TG) and total cholesterol (TC) in the serum were detected using a BS - 240VET biochemical analyzer.

[0192] 5. Test results

[0193] The results are shown in Table 5.

[0194] Table 5 Effects of samples on uric acid (UA), total cholesterol (TC) and triglyceride (TG) in rat serum

[0195]

[0196] Note: Different letters in the same column represent significant differences in data between groups, P < 0.05.

[0197] As can be seen from the above, compared with the normal control group, the levels of uric acid (UA), total cholesterol (TC) and triglyceride (TG) in the serum of rats in the model control group were significantly increased (P < 0.05), indicating successful modeling.

[0198] Compared with the model control group, there were significant differences in the levels of uric acid (UA), total cholesterol (TC) and triglyceride (TG) in the serum of rats in each group (P < 0.05), indicating that the diglyceride compositions prepared from S1 - S4, the diglyceride compositions prepared from D1 - D3, and the commercially available diglycerides all have the effects of reducing uric acid, total cholesterol and triglyceride.

[0199] It is worth noting that the levels of uric acid (UA), total cholesterol (TC) and triglyceride (TG) in the serum of rats in the S1 - S4 group of the dosing group were significantly lower than those in the D0 - D3 group of the dosing group (P < 0.05), indicating that the diglyceride composition prepared from S1 - S4 has significantly better effects of reducing uric acid, total cholesterol and triglyceride than the diglycerides prepared from D1 - D3 and the commercially available diglycerides.

[0200] The above is a further description of the present invention in combination with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

Claims

1. A method for preparing diglycerides with the efficacy of reducing blood lipid, blood sugar and uric acid by enzymatic hydrolysis, characterized in that, It includes the following steps: (1) Mix vegetable oil, glycerol, compound lipase and water to carry out glycerolysis reaction to obtain a reactant; (2) Separate the reactant obtained in step (1) and collect the upper layer liquid; (3) Distill, separate, decolorize and deodorize the upper layer liquid obtained in step (2) to obtain diglyceride; In step (1), the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 1.5 - 5:1; In step (1), the mass ratio of the vegetable oil to glycerol is 0.6 - 1.1:1; In step (1), the addition amount of the compound lipase is 0.05 - 5% of the total mass of the vegetable oil and glycerol; In step (1), the addition amount of the water is 6 - 26% of the mass of the vegetable oil; In step (1), the conditions of the glycerolysis reaction are: reacting at 30 - 56°C for 1 - 3 h; In step (1), the compound lipase is Rhizomucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase with a mass ratio of 2 - 4:1:5 - 8.

2. The method according to claim 1, wherein The mass ratio of the Rhizomucor javanicus lipase, Aspergillus niger lipase and Aspergillus oryzae lipase is 3:1:

6.

3. The method according to claim 1, wherein In step (1), the vegetable oil is rapeseed oil with low erucic acid and corn oil with a mass ratio of 3 - 3.5:

1.

4. The method according to claim 1, wherein In step (1), the mass ratio of the vegetable oil to glycerol is 0.7 - 1:

1.

5. The method according to claim 1, characterized in that, In step (1), the addition amount of the compound lipase is 0.1 - 2% of the total mass of the vegetable oil and glycerol.

6. The method according to claim 1, characterized in that, In step (1), the addition amount of the water is 8 - 20% of the mass of the vegetable oil.

7. Diglyceride prepared by the method according to any one of claims 1 - 6.

8. Use of the diglyceride according to claim 7 in the preparation of a medicament having the effects of reducing blood lipid, reducing blood sugar and reducing uric acid.

9. Use of the diglyceride according to claim 7 in the preparation of a medicament having the effect of reducing total cholesterol.

10. A diglyceride composition, comprising the diglyceride according to claim 7 and a pharmaceutically acceptable excipient.

11. The diglyceride composition according to claim 10, wherein By mass percentage, it includes 60 - 99% of diglyceride, 0.01 - 20% of Acer truncatum seed oil, 0.01 - 20% of sea buckthorn seed oil, 0.01 - 0.2% of vitamin E and the balance of high oleic acid peanut oil.

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