Method for preparing diglyceride with effects of reducing blood fat, total cholesterol, blood sugar and uric acid through enzymolysis as well as product and application of diglyceride

Abstract

The invention discloses a method for preparing diglyceride with the effects of reducing blood fat, total cholesterol, blood sugar and uric acid through enzymolysis and a product and application of the diglyceride, and belongs to the technical field of diglyceride production.The method comprises the following steps that 1, vegetable oil, glycerin, compound lipase and water are mixed to be subjected to a glycerolysis reaction, and a reactant is obtained; (2) separating reactants, and collecting supernatant; (3) performing distillation separation on the supernatant, and performing decoloration and deodorization to obtain diglyceride; the vegetable oil in the step (1) is low-erucic acid rapeseed oil and corn oil; the composite lipase in the step (1) is prepared from mucor javanicus lipase, aspergillus niger lipase and aspergillus oryzae lipase in a mass ratio of (2-4): 1: (5-8). According to the invention, the use amount of enzyme can be reduced, the cost is reduced, and the prepared diglyceride has better activity of reducing blood fat, total cholesterol, blood sugar and uric acid, and has more advantages in the preparation of health care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of diglyceride production, and specifically relates to a method for preparing diglyceride with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis, and a product and application thereof. Background Art

[0002] Diacylcerol (DAG) is a natural component of oils and fats and an intermediate product of oil metabolism. It has the flavor of ordinary oils and is highly safe. Diacylcerol and triglycerides have different absorption and metabolism patterns in the body. It has the effects of reducing visceral fat, inhibiting weight gain, and reducing blood lipids, and therefore has received widespread attention.

[0003] In recent years, the preparation of DAG oil with nutritional and health functions using ordinary oil as raw materials has become one of the main directions of oil development. For example, CN101270375A discloses a process for preparing 1,3-diacerol by enzymatic method in a tert-butyl alcohol system, wherein the process steps are as follows: acyl acceptor monoglyceride and acyl donor with a molar ratio of 1:0.5-1:3, tert-butyl alcohol based on 20-200% of the mass of acyl acceptor monoglyceride, and lipase based on 2-20% of the mass of acyl acceptor monoglyceride are added, and the mixture is uniformly loaded into any biochemical reactor suitable for enzyme reaction, the temperature is controlled at 30-65°C, and the reaction is performed for 1-12 hours, the conversion rate of acyl acceptor monoglyceride to diglyceride reaches 70-90%, and the content of 1,3-diacerol in the generated diglyceride reaches more than 75%; the acyl donor is a fatty acid, a short-chain fatty acid ester, a monoglyceride, a triglyceride, an animal or plant fat or an incomplete hydrolyzate of a fat.

[0004] Hua Di et al. disclosed a method for the continuous preparation of diacylglycerol by enzymatic glycerol hydrolysis in their research on the continuous preparation of diacylglycerol by enzymatic glycerol hydrolysis. By comparing the reaction effects of different lipases and different solvent systems, they chose to use Novozyme 435 immobilized lipase packed column reactor to continuously react in a solvent-free system. The reaction conditions were further optimized and the optimal process conditions were determined to be: the molar ratio of soybean oil to glycerol was 1:2, the reaction temperature was 60°C, and the flow rate was 0.05g / min. Under these conditions, the content of diacylglycerol in the product could reach 58.17%.

[0005] CN103243126A discloses a method for preparing diglyceride, comprising the following steps: 1) selecting raw materials: selecting rapeseed oil, glycerin, biological enzyme and water according to the mass proportion of each raw material: 100 parts of rapeseed oil, 30 parts of glycerin, 1-8 parts of biological enzyme, and 0-2 parts of water, and the water content of glycerin is 3-5wt%; 2) mixing: mixing rapeseed oil, glycerin, biological enzyme and water, heating to 60-80°C, stirring at a speed of 120-130 rpm, reacting for 2-6 hours to obtain a mixture; 3) esterifying and dehydrating the mixture to obtain an esterified dehydrate; 4) sedimenting and deglycerinizing the esterified dehydrate to obtain a precipitate; 5) Further screening by molecular distillation: ① vacuum dehydration; ② removal of fatty acids; ③ removal of monoglycerides; ④ removal of triglycerides: then vacuum deglyceride at 180-240°C and a vacuum degree of 1-5Pa for 10-20 minutes to obtain diglycerides. The content of diglycerides in this invention is above 85wt%.

[0006] Although there are many methods for preparing triglycerides, preparing a more functional diglyceride is still a very challenging task. Summary of the invention

[0007] The present invention is to provide a method for preparing diglyceride with the effects of lowering blood lipids, lowering total cholesterol, lowering blood sugar and lowering uric acid by enzymatic hydrolysis, and its products and applications. The diglyceride prepared by the enzymatic hydrolysis process of the present invention has multiple effects such as lowering blood lipids, lowering total cholesterol, lowering blood sugar and lowering uric acid. Compared with the prior art, the health care product prepared by using the diglyceride has better effects of lowering blood lipids, lowering total cholesterol, lowering blood sugar and lowering uric acid, and has obvious advantages.

[0008] In order to realize the above technical solution, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing diglyceride with the effects of lowering blood lipids, lowering total cholesterol, lowering blood sugar and lowering uric acid by enzymatic hydrolysis, comprising the following steps: (1) mixing vegetable oil, glycerol, compound lipase and water to perform glycerol hydrolysis reaction to obtain a reactant; (2) separating the reactants obtained in step (1) and collecting the upper liquid; (3) The upper liquid obtained in step (2) is distilled, separated, decolorized and deodorized to obtain diglyceride.

[0009] In some embodiments, the mass ratio of the vegetable oil to glycerol in step (1) is 0.6-1.1:1; preferably 0.7-1:1.

[0010] In some embodiments, the vegetable oil in step (1) is at least one of rapeseed oil, corn oil, sunflower oil and peanut oil. Preferably, rapeseed oil and corn oil; more preferably, the mass ratio of rapeseed oil to corn oil is 1.5-5:1; more preferably, 3-3.5:1.

[0011] In some embodiments, the amount of the composite lipase added in step (1) is 0.05-5% of the total mass of the vegetable oil and glycerol; more preferably 0.1-2%.

[0012] In some embodiments, the composite lipase in step (1) is at least one of Hansenula polymorpha lipase, Aspergillus niger lipase, Mucor javanica lipase, Aspergillus oryzae lipase, Candida columnaris lipase, pancreatic lipase, Rhizopus niveus lipase, Rhizomucor miehei lipase, Rhizopus oryzae lipase and Trichoderma reesei lipase.

[0013] In some embodiments, the composite lipase in step (1) is at least one of Mucor javanica lipase, Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase; preferably, the composite lipase is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase; further preferably, the mass ratio of Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase is 2-4:1:5-8; further preferably, the mass ratio of Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase is 3:1:6.

[0014] In some embodiments, the amount of water added in step (1) is 6-26% of the mass of the vegetable oil; preferably 8-20%.

[0015] In some embodiments, the conditions of the glycerolysis reaction in step (1) are: reaction at 30-56° C. for 1-3 h.

[0016] In some embodiments, the separation in step (3) is standing or centrifuging. Preferably, the standing time is 1-4 h; more preferably 1-3 h, and even more preferably 1-2 h.

[0017] In some embodiments, the conditions for the distillation separation in step (3) are: cooling water temperature 35-40°C, vacuum degree 5-10 Pa, scraping speed 50-100 r / min, and feed flow rate 12-18 kg / min.

[0018] In a second aspect, the present invention provides diglyceride prepared by the above method.

[0019] Experiments have shown that the diglyceride prepared by the present invention has more advantages than commercially available diglyceride in preparing health products or medicines with multiple functions such as lowering blood lipids, lowering total cholesterol, lowering blood sugar and lowering uric acid.

[0020] In a third aspect, the present invention provides use of the diglyceride prepared by the above method in the preparation of health products or medicines.

[0021] The diglyceride composition described in the present invention can also be used as a raw material, mixed with health care products or pharmaceutically acceptable auxiliary materials, to prepare corresponding health care products and medicines.

[0022] In some embodiments, the dosage form of the health product is liquid, granules, powder, tablet or pill; preferably liquid.

[0023] In some embodiments, the drug is a liquid, tablet, capsule, granule, dry suspension or pill; preferably a liquid.

[0024] In a fourth aspect, the present invention provides a diglyceride composition, comprising the diglyceride prepared by the above method.

[0025] In some embodiments, the diglyceride composition further includes Acer truncatum seed oil, Hippophae rhamnoides seed oil, vitamin E and high oleic peanut oil.

[0026] Preferably, the diglyceride composition comprises, by mass percentage, 60-99% of diglyceride oil, 0.01-20% of Acer truncatum seed oil, 0.01-20% of Hippophae rhamnoides seed oil, 0.01-0.2% of vitamin E and the balance of high oleic peanut oil.

[0027] Beneficial effects of the present invention (1) The present invention optimizes the glycerol hydrolysis reaction from two aspects: raw material compounding and enzyme preparation screening. Low-erucic acid rapeseed oil and corn oil are used as raw materials, and a composite lipase obtained by compounding Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase is used as an enzyme preparation. Diglycerol is prepared through enzymatic hydrolysis. The diglycerol has better activities of lowering blood lipids, total cholesterol, blood sugar and uric acid, and has more advantages in the preparation of health care products.

[0028] (2) The present invention utilizes a compound of Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase, wherein Aspergillus oryzae lipase can catalyze acyl migration and ester exchange reactions, increase the diversity of diacylglycerol isomers, and optimize lipid metabolic activity; Mucor javanica lipase can specifically hydrolyze Sn-1,3 ester bonds to increase the content of 1,3 diacylglycerol; Aspergillus niger lipase can optimize reaction conditions and further improve the metabolic activities of Mucor javanica lipase and Aspergillus oryzae lipase. The compound of the three enzymes can reduce the amount of enzyme used and reduce costs on the one hand, and can enhance the lipid-lowering, total cholesterol-lowering, blood sugar-lowering and uric acid-lowering activities of diacylglycerol on the other hand.

[0029] (3) Experiments have shown that the product prepared using the diglyceride prepared by the present invention has better effects on lowering blood lipids, total cholesterol, blood sugar and uric acid than the prior art. DETAILED DESCRIPTION

[0030] The following examples are only used to help understand the method of the present invention and its core concept. It should be noted that, for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications may be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0031] Therefore, the present invention will not be limited to these embodiments shown in this article, but can be applied to a wider range consistent with the principles and novel features disclosed herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0032] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033] All ranges involving the same component or property include endpoints, which can be independently combined. Since these ranges are continuous, they include every numerical value between the minimum and maximum values. It should also be understood that any numerical range cited in the present invention is intended to include all subranges within the range.

[0034] The term "nutraceutical or pharmaceutically acceptable" indicates that the composition is compatible with the other ingredients making up the formulation.

[0035] 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 common commercial products in the technical field. In the present invention, the Java Mucor lipase is SIGMA 534803, 100,000 U / g; the Aspergillus niger lipase is SIGMA 62301, 200 U / g; the Aspergillus oryzae lipase is SIGMA 62285, 50 mU / g, low-erucic acid rapeseed oil, purchased from Chengdu Xinxing Grain and Oil Co., Ltd., batch 20240919 (production date), high oleic 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, seabuckthorn 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 sorbifolia oil, purchased from Shandong Lin Yuhong Xanthoceras sorbifolia Co., Ltd.

[0036] Example 1 Method for preparing diglyceride with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis The steps are: (1) Mixing vegetable oil, glycerol, compound lipase and water, and performing glycerol hydrolysis reaction at a temperature of 52±2°C for 1.5 hours to obtain a reactant; The vegetable oil is low-erucic acid rapeseed oil and corn oil in a mass ratio of 3:1; The composite lipase is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 3:1:6; The mass ratio of vegetable oil to glycerol is 0.8:1; The amount of compound lipase added was 0.2% of the total mass of vegetable oil and glycerol; The amount of water added is 10% of the mass of the vegetable oil.

[0037] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5r / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0038] Example 2 Method for preparing diglyceride with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis The steps are: (1) Mixing vegetable oil, glycerol, compound lipase and water, and performing glycerol hydrolysis reaction at a temperature of 32±2°C for 3 hours to obtain a reactant; The vegetable oil is low-erucic acid rapeseed oil and corn oil in a mass ratio of 1.5:1; The composite lipase is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 2:1:8; The mass ratio of vegetable oil to glycerol is 0.6:1; The amount of compound lipase added was 0.1% of the total mass of vegetable oil and glycerol; The amount of water added is 6% of the mass of the vegetable oil.

[0039] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5 / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0040] Example 3 Method for preparing diglyceride with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis The steps are: (1) Mixing vegetable oil, glycerol, compound lipase and water, and performing glycerol hydrolysis reaction at a temperature of 54±2°C for 1 hour to obtain a reactant; The vegetable oil is low-erucic acid rapeseed oil and corn oil in a mass ratio of 5:1; The composite lipase is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 4:1:5; The mass ratio of vegetable oil to glycerol is 1.1:1; The amount of compound lipase added was 2% of the total mass of vegetable oil and glycerol; The amount of water added is 26% of the mass of the vegetable oil.

[0041] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5r / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0042] Example 4 Method for preparing diglyceride with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis The steps are: (1) Mixing vegetable oil, glycerol, lipase and water, and performing glycerol hydrolysis reaction at a temperature of 46±2°C for 2 hours to obtain a reactant; The vegetable oil is low-erucic acid rapeseed oil and corn oil in a mass ratio of 3:1; The lipase is Mucor javanica lipase; The mass ratio of vegetable oil to glycerol is 0.8:1; The amount of lipase added was 5% of the total mass of vegetable oil and glycerol; The amount of water added is 10% of the mass of the vegetable oil.

[0043] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5r / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0044] Comparative Example 1 The difference between this comparative example and Example 4 is that the composition of the composite lipase is different; The steps are: (1) Mixing vegetable oil, glycerol, compound lipase and water, and performing glycerol hydrolysis reaction at a temperature of 52±2°C for 1.5 hours to obtain a reactant; The vegetable oil is low-erucic acid rapeseed oil and corn oil in a mass ratio of 3:1; The composite lipase is Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 31:6; The mass ratio of vegetable oil to glycerol is 0.8:1; The amount of compound lipase added was 5% of the total mass of vegetable oil and glycerol; The amount of water added is 10% of the mass of the vegetable oil.

[0045] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5r / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0046] Comparative Example 2 The difference between this comparative example and Example 1 is that the composition of the composite lipase is different, and Xanthoceras sorbifolia oil is used instead of low-erucic acid rapeseed oil.

[0047] The steps are: (1) Mixing vegetable oil, glycerol, compound lipase and water, and performing glycerol hydrolysis reaction at a temperature of 52±2°C for 1.5 hours to obtain a reactant; The vegetable oil is Xanthoceras sorbifolia oil and corn oil in a mass ratio of 3:1; The composite lipase is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 8:2:1; The mass ratio of vegetable oil to glycerol is 0.8:1; The amount of compound lipase added was 0.2% of the total mass of vegetable oil and glycerol; The amount of water added is 10% of the mass of the vegetable oil.

[0048] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5r / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0049] Comparative Example 3 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.

[0050] The steps are: (1) Mixing vegetable oil, glycerol, compound lipase and water, and performing glycerol hydrolysis reaction at a temperature of 52±2°C for 1.5 hours to obtain a reactant; The vegetable oil is sea buckthorn seed oil and corn oil in a mass ratio of 3:1; The composite lipase is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 1:5:2; The mass ratio of vegetable oil to glycerol is 0.8:1; The amount of compound lipase added was 0.2% of the total mass of vegetable oil and glycerol; The amount of water added is 10% of the mass of the vegetable oil.

[0051] (2) The reaction product obtained in step (1) was allowed to stand for 2 h and the upper liquid was collected; (3) The upper layer liquid obtained in step (2) is distilled and separated, with the cooling water temperature of 37±2°C, the vacuum degree of 8Pa, the scraping rate of 75±5r / min, the feed flow rate of 15kg / min, and decolorization and deodorization to obtain diglyceride.

[0052] Example 5 Diaceride Composition According to the formula in Table 1, Acer truncatum seed oil, Hippophae rhamnoides seed oil, vitamin E and high oleic peanut oil were added to the diglyceride and mixed to obtain a diglyceride composition.

[0053] Table 1 Diaceryl Composition Formula

[0054] 1. Blood lipid lowering efficacy test The effect of the prepared diglyceride composition on zebrafish blood lipids was investigated.

[0055] 1. Experimental Animals Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to each IL of reverse osmosis water, conductivity was 450-550 µS / cm: pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3), bred and provided by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd., the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0056] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 3 days post fertilization (3 dpf) were used to evaluate the effects of the samples on zebrafish blood lipids.

[0057] 2. Test samples and reagents 2.1 Test samples Samples S1-S4 and D1-D3: The diglyceride composition was prepared into a 200 mg / mL stock solution using DMSO and stored at -20°C for later use, wherein the diglyceride composition was prepared according to Example 5; Control sample (D0): A commercially available diglyceride composition was prepared into a 200 mg / mL stock solution using DMSO and stored at -20°C for future use. The commercially available diglyceride composition was provided by Guangdong Shanbainian Special Medical Food Co., Ltd., 80% DAG content diglyceride edible oil, production date 20220902.

[0058] 2.2 Reagents 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).

[0059] 3. Detection method 3.1 MCT determination 3dpf wild-type AB strain zebrafish were randomly selected in beakers, and 30 zebrafish were treated in each beaker (experimental group). Except for the normal control group, the other experimental groups were given high-sugar and high-fat feed in water to establish a zebrafish high-fat model. After 2 days of treatment at 28°C, the high-sugar and high-fat feed was removed, and samples (concentrations are shown in Table 2) were given in water. At the same time, a normal control group was set up, and the capacity of each cup was 25mL. The liquid was changed every morning and evening. During the sample treatment, the number of zebrafish deaths in each experimental group was counted every day and removed in time. After 3 days of treatment at 28°C, the MTC of the sample on the high-fat model zebrafish was determined.

[0060] Table 2 Results of the concentration experiment on the effect of samples on zebrafish blood lipids

[0061] From the above, it can be seen that under the experimental conditions, the MTC of the diglyceride composition on zebrafish blood lipids is 2000 μg / mL.

[0062] 3.2 Effects on zebrafish blood lipids 3 dpf wild-type AB strain zebrafish were randomly selected and placed in beakers, with 10 zebrafish in each beaker. Except for the normal control group, all other experimental groups were given high-sugar and high-fat feed in water to establish a zebrafish high-fat model. After 2 days of treatment at 28℃, except for the model control group, the high-sugar and high-fat feed was removed from the other experimental groups, and the sample concentrations were given in water. At the same time, the normal control group and the model control group were set up. The model control group continued to be given high-sugar and high-fat feed until 8 dpf, and the capacity of each cup was 25mL. The liquid was changed every morning and evening. After 3 days of treatment at 28℃, Oil Red 0 was given for overall fat staining. After decolorization and bleaching, the zebrafish were photographed under a dissecting microscope, and the data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The staining intensity of the zebrafish tail blood vessels was analyzed, and the statistical analysis results of this indicator were used to evaluate the effect of the sample on zebrafish blood lipids. The statistical analysis results are expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0063] The results are shown in Table 3.

[0064] Table 3 Experimental results of the effects of samples on zebrafish blood lipids (n=10)

[0065] Note: Different letters in the same column indicate significant differences among the groups, P < 0.05.

[0066] 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.

[0067] 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.

[0068] Compared with the administration group D0, there was no significant difference in the staining intensity of the zebrafish tail blood vessels in the administration groups D1-D3 (P>0.05), while the staining intensity of the zebrafish tail blood vessels in the administration groups S1-S4 was significantly reduced (P<0.05). This shows that the diglyceride composition prepared by S1-S4 has a better lipid-lowering effect than the commercially available product, and the diglyceride composition prepared by D1-D3 has basically the same lipid-lowering effect as the commercially available product.

[0069] Comparison of the administration groups S1-S4 shows that the lipid-lowering effect of the diglyceride composition prepared by S1-S3 is better than that of S4.

[0070] 2. Blood sugar lowering efficacy test The effect of the prepared diglyceride composition on lowering blood sugar was investigated.

[0071] 1. Experimental Animals Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg instant sea salt was added to each IL of reverse osmosis water, conductivity was 450-550 µS / cm: pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3), bred by the fish farming center of Hangzhou Huante Biotechnology Co., Ltd., the experimental animal use license number is: SYXK (Zhejiang) 2022-0004, and the breeding management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0072] 2. Test samples, instruments and reagents 2.1 Test samples Samples S1-S4 and D1-D3: The diglyceride compositions were prepared into 200 mg / mL stock solutions using DMSO and stored at -20°C for later use, wherein the diglyceride compositions were prepared according to Example 5; Control sample (D0): a commercially available diglyceride composition, prepared into a 200 mg / mL stock solution using DMSO and stored at -20°C for future use. The commercially available diglyceride composition was provided by Guangdong Shanbainian Special Medical Food Co., Ltd., 80% DAG content diglyceride edible oil, production date 20220902; Positive control: metformin hydrochloride tablets (hereinafter referred to as metformin), white tablets, batch number ACL1077, Shanghai Bristol-Myers Squibb Co., Ltd., the solvent is ultrapure water.

[0073] 2.2 Instruments 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 Diagnostics (Shanghai) Co., Ltd., China); blood glucose meter test strips (Batch No. 670452, Roche Diagnostics (Shanghai) Co., Ltd., China).

[0074] 2.3 Reagents Egg yolk powder (Batch No. 20230203, Zhejiang Aiger Biotechnology Co., Ltd., China); anhydrous glucose (Batch No. C15778026, Shanghai McLean Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, Batch No. BCCD8942, Sigma, Switzerland).

[0075] 3. Blood sugar lowering efficacy test method 5 dpf wild-type AB strain zebrafish were randomly selected in beakers, and 10 zebrafish were treated in each beaker (experimental group). The sample concentration was 1000μg / mL, and the positive control metformin concentration was 400μg / mL. A normal control group and a model control group were set up at the same time, and the capacity of each cup was 25mL. Except for the normal control group, the other experimental groups were given a high-sugar and high-fat diet to establish a zebrafish hyperglycemia model. After 2 days of treatment at 28℃, data were collected using a blood glucose meter, and the glucose level of zebrafish was analyzed and statistically analyzed. The statistical analysis results of this indicator were used to evaluate the hypoglycemic efficacy of the sample. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0076] The results are shown in Table 4.

[0077] Table 4 Results of the blood sugar lowering efficacy test of the samples (n=10)

[0078] Note: Different letters in the same column indicate significant differences among the groups, P < 0.05.

[0079] From the above, we can see that compared with the normal control group, the glucose level of zebrafish in the model control group was significantly increased (P < 0.05), indicating that the zebrafish hyperglycemia model was successfully established.

[0080] Compared with the model control group, the glucose levels of zebrafish in each group were significantly increased or decreased (P < 0.05), indicating that in the technical scheme claimed for protection of the present invention, the diglyceride compositions prepared by S1-S4 and D1-D3 of the present invention and the commercially available diglycerides all have the effect of lowering blood sugar.

[0081] At the same time, compared with the positive control group, there was no significant change in the glucose level of zebrafish in the drug-treated groups S1-S4 (P>0.05), indicating that the diglyceride composition prepared by S1-S4 of the present invention has an efficacy equivalent to that of metformin.

[0082] 3. Uric acid and total cholesterol lowering efficacy test The effect of the prepared diglyceride composition on serum uric acid (UA), serum total cholesterol (TC) and triglyceride (TG) in hyperuricemia model rats fed a high-fat diet was investigated.

[0083] 1. Experimental Animals SD rats, SPF grade, male, 7-9 weeks old, were provided by Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0084] 2. Test samples and reagent consumables 2.1 Test samples The diglyceride compositions were prepared according to Example 5.

[0085] Commercially available diglyceride composition (D0), provided by Guangdong Shanbainian Special Medical Food Co., Ltd., 80% DAG content diglyceride edible oil, production date 20220902.

[0086] 2.2 Reagents Uric Acid (UA) kit (enzyme colorimetric method), batch number: 20221107, Nanjing Jiancheng Bioengineering Institute.

[0087] Triglyceride (TG) assay kit (oxidase method): batch number: 141722001, Shenzhen Mindray Animal Medical Technology Co., Ltd.

[0088] Total cholesterol (TC) assay kit (oxidase method): batch number: 141622003, Shenzhen Mindray Animal Medical Technology Co., Ltd.; Potassium oxythioate: Batch number: #B2226363, Aladdin.

[0089] 2.3 Feed Basic feed: 25 parts of flour, 25 parts of oatmeal, 25 parts of cornmeal, 10 parts of soybean meal, 8 parts of fish meal, 4 parts of bone meal and 1 part of refined salt, by mass.

[0090] 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 basic feed and 11.28 parts of yeast powder.

[0091] Diester oil feed: 30 parts of diglyceride composition, 16.7 parts of sucrose, 19.6 parts of casein, 5.6 parts of minerals, 52.2 parts of basic feed and 11.28 parts of yeast powder, calculated by mass.

[0092] 3. Experimental methods The animals were adaptively fed for 7 days and randomly divided into a normal control group, a model control group, and a drug-treated group (S1-S4 and D0-D3) according to their body weight, with 8 animals in each group. The animals in the normal control group were fed with a basic feed, and the animals in the other groups were fed with D12451 yeast feed, and the daily food intake of each animal was controlled to be 15 g.

[0093] At the same time, except for the normal control group, the other groups were intraperitoneally injected with 450 mg / kg / d potassium oxonate for 8 weeks. After 8 weeks, the treated group was changed to diester oil feed, and the other operations remained unchanged for a total of 6 weeks.

[0094] 4. Index detection After the experiment, the animals were anesthetized, blood was collected from the abdominal aorta, and serum was separated to measure uric acid (UA), triglyceride (TG), and total cholesterol (TC). SPSS was used for relevant mathematical statistics. The quantitative data were expressed as mean ± standard deviation (mean ± SD), and the independent sample t test was used for comparison between groups. p < 0.05 was considered statistically significant.

[0095] 4.1 Uric acid test Except for the last blood collection, the volume of each blood collection was 0.75 μL. After blood collection, the blood samples were stored at 4°C for 1 hour and centrifuged at 3000 r / min for 15 minutes. Serum was separated and measured using the SpectraMax M5 microplate reader according to the instructions of the uric acid (UA) kit provided by Nanjing Jiancheng Bioengineering Research Institute.

[0096] 4.2. Triglyceride and total cholesterol testing After the animals were anesthetized and blood was collected from the abdominal aorta until they died, the blood samples were stored at 4°C for 1 hour and then centrifuged at 3000 r / min for 15 minutes. The serum was separated and the triglyceride (TG) and total cholesterol (TC) contents in the serum were detected using a BS-240VET biochemical analyzer.

[0097] 5. Test results The results are shown in Table 5.

[0098] Table 5 Effects of samples on serum uric acid UA, serum total cholesterol TC and triglyceride TG in rats

[0099] Note: Different letters in the same column indicate significant differences among the groups, P < 0.05.

[0100] From the above, we can see that compared with the normal control group, the serum uric acid UA, serum total cholesterol TC and triglyceride TG of the rats in the model control group were significantly increased (P < 0.05), indicating that the model was successfully established.

[0101] Compared with the model control group, there were significant differences in serum uric acid UA, serum total cholesterol TC and triglyceride TG in rats in each group (P < 0.05), indicating that the diglyceride composition prepared by S1-S4, the diglyceride composition prepared by D1-D3 and the commercially available diglyceride all have the effects of lowering uric acid, total cholesterol and triglycerides.

[0102] It is worth noting that the serum uric acid UA, serum total cholesterol TC and triglyceride TG of rats in the drug-treated groups S1-S4 were significantly lower than those in the drug-treated groups D0-D3 (P < 0.05), indicating that the diglyceride composition prepared by S1-S4 is significantly better than D1-D3 and commercially available diglycerides in lowering uric acid, total cholesterol and triglycerides.

[0103] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing diglyceride with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis, characterized in that: The steps include: (1) mixing vegetable oil, glycerol, compound lipase and water to perform glycerol hydrolysis reaction to obtain a reactant; (2) separating the reactants obtained in step (1) and collecting the upper liquid; (3) distilling and separating the upper layer liquid obtained in step (2), decolorizing and deodorizing to obtain diglyceride; The vegetable oil in step (1) is low-erucic acid rapeseed oil and corn oil; The composite lipase in step (1) is a lipase of Mucor javanica, Aspergillus niger and Aspergillus oryzae in a mass ratio of 2-4:1:5-8.

2. The method according to claim 1, characterized in that: The mass ratio of the Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase is 3:1:

6.

3. The method according to claim 1, characterized in that The vegetable oil in step (1) is low-erucic acid rapeseed oil and corn oil in a mass ratio of 1.5-5:

1.

4. The method according to claim 1, characterized in that: The mass ratio of the vegetable oil to glycerol in step (1) is 0.6-1.1:

1.

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

6. The method according to claim 1, characterized in that The amount of water added in step (1) is 6-26% of the mass of the vegetable oil; And / or the conditions of the glycerol hydrolysis reaction in step (1) are: reaction at 30-56° C. for 1-3 hours.

7. diglyceride prepared by the method described in any one of claims 1 to 6.

8. Use of the diglyceride according to claim 7 in the preparation of health products or medicines.

9. A diglyceride composition comprising the diglyceride according to claim 7 and a health product or a pharmaceutically acceptable excipient.

10. The diglyceride composition according to claim 9, characterized in that Calculated by mass percentage, it comprises 60-99% of diglyceride oil, 0.01-20% of Acer truncatum seed oil, 0.01-20% of seabuckthorn seed oil, 0.01-0.2% of vitamin E and the balance of high oleic peanut oil.

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