A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis, and its product and application

Abstract

The present invention discloses a method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis, as well as its product and application, belonging to the technical field of diglyceride production. The method comprises the following steps: (1) mixing vegetable oil, glycerol, water, and an enzyme preparation to react to obtain a reaction solution; (2) allowing the reaction solution to stand for stratification and collecting the supernatant; (3) distilling and separating the supernatant, decolorizing, and deodorizing the supernatant to obtain diglyceride; the enzyme preparation in step (1) comprises lipase, esterase, and phospholipase C in a weight ratio of 5-10:0.1-0.3:1. Compared with the existing technology, the method of the present invention is simple and low-cost, and the prepared diglyceride has greater advantages in lowering uric acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of diglyceride production, and particularly relates to a method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis, and a product and application thereof. Background Art

[0002] Diacylcerides (DAGs) are natural components of oils and fats and are an intermediate product in oil metabolism. They possess the flavor of regular oils and are highly safe. Diacylcerides have attracted widespread attention for their potential to reduce visceral fat, inhibit weight gain, and lower blood lipids.

[0003] In recent years, the production of DAG oil with nutritional and health functions using ordinary oils as raw materials has become one of the main directions of oil development.

[0004] For example, CN101270375A discloses a process for preparing 1,3-diglycerol by an enzymatic method in a tert-butanol system. The process steps are as follows: adding 20-200% of tert-butanol based on the weight of the acyl acceptor monoglyceride and 2-20% of lipase based on the weight of the acyl acceptor monoglyceride to an acyl acceptor monoglyceride in a molar ratio of 1:0.5-1:3, and placing them together in any biochemical reactor suitable for enzyme reaction and mixing them uniformly; controlling the temperature at 30-65°C and reacting for 1-12 hours; the conversion rate of the 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 a fatty acid, a short-chain fatty acid ester, a monoglyceride, a triglyceride, an animal or plant fat, or an incomplete hydrolyzate of fat.

[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 weight 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 glycerin water content is 3-5wt%; 2) mixing: mixing rapeseed oil, glycerin, biological enzyme and water, heating to 50-80°C, stirring at a speed of 35-45 rpm, and reacting for 1- 6 hours to obtain a mixture; 3) esterification and dehydration of the mixture to obtain an esterified dehydrate (physical method: centrifugation); 4) sedimentation and deglycerolization of the esterified dehydrate to obtain a precipitate; 5) molecular distillation for further screening: ① vacuum dehydration; ② removal of fatty acids; ③ removal of monoglycerides; ④ removal of triglycerides: then vacuum deglycerolization at 100-280°C and a vacuum degree of less than 10Pa for 10-20 minutes to obtain diglyceride. The diglyceride content of this invention is more than 85wt%.

[0006] Gout is a metabolic disease characterized by long-term disturbances in purine metabolism, resulting in persistently elevated blood uric acid (UA) levels and ultimately leading to the deposition of urate crystals in the joints. Clinical manifestations include hyperuricemia (HC), acute gouty arthritis, tophi, and joint deformities. Over time, this condition can lead to chronic nephritis and the formation of renal uric acid stones. Clinically, a blood uric acid concentration >417 μmol / L is considered hyperuricemia, and approximately 5% to 12% of patients with hyperuricemia will develop gout. Uric acid can promote platelet aggregation, inducing the formation of blood clots and atherosclerosis. Elevated uric acid levels can also lead to obesity, lipid metabolism disorders, diabetes, and cerebrovascular disease.

[0007] CN113350328A discloses novel applications for diglycerides and their compositions. Research has found that diglycerides can help lower uric acid levels and can be used to prevent, treat, and improve hyperuricemia. Furthermore, suitable diglyceride compositions have a significant uric acid-lowering effect in patients with hyperuricemia. However, this invention imposes high requirements for diglyceride content, requiring a content of at least 80%, and also places high demands on the diglyceride production process.

[0008] Therefore, there is an urgent need to provide a method for preparing diglyceride with low process requirements and good uric acid-lowering effect. Summary of the Invention

[0009] In order to solve the above technical problems, the present invention provides a method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis, and its product and application.

[0010] In order to implement the above technical solution, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis, comprising the following steps:

[0012] (1) Mixing vegetable oil, glycerol, water and enzyme preparation to obtain a reaction solution;

[0013] (2) Let the reaction solution stand and separate the layers, and collect the upper layer;

[0014] (3) The upper liquid is distilled, separated, decolorized and deodorized to obtain diglyceride.

[0015] Preferably, the vegetable oil in step (1) is at least one of Acer truncatum seed oil, Hippophae rhamnoides seed oil, Rapeseed oil, Peony seed oil, Corn oil and Peanut oil.

[0016] Preferably, the enzyme preparation in step (1) is at least one of lipase, esterase, phospholipase A1, phospholipase A2 and phospholipase C.

[0017] The lipase Lipase in the present invention is derived from Hansenula polymorpha, Aspergillus niger, Mucor circinelloides, Aspergillus oryzae, Candida cylindrica, pig or cattle pancreas, Rhizopus nivalis, Rhizomucor miehei, Rhizopus oryzae, Trichoderma leishmanii, sheep throat, salivary gland or forestomach tissue of calf or lamb; the esterase Esterase is derived from Rhizomucor miehei, Trichoderma leishmanii or Aspergillus niger; the phospholipase A1 (Phospholipase A1) is derived from Aspergillus oryzae or Aspergillus niger; the phospholipase A2 (Phospholipase A2) is derived from pig pancreatic tissue, Aspergillus niger or Trichoderma leishmanii; and the phospholipase C (Phospholipase C) is derived from Pichia pastoris or Bacillus licheniformis.

[0018] More preferably, the enzyme preparation is lipase, esterase and phospholipase C.

[0019] More preferably, the weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 5-10:0.1-0.3:1; most preferably, it is 8:0.12:1.

[0020] Preferably, the weight ratio of the vegetable oil to glycerol in step (1) is 1:1.1-1.5; more preferably 1:1.275.

[0021] Preferably, the amount of the enzyme preparation added in step (1) is 0.1-5% by weight of the vegetable oil; more preferably 0.1-1%.

[0022] Preferably, the amount of water added in step (1) is 6%-30% of the weight of the vegetable oil; more preferably 25%.

[0023] Preferably, the reaction conditions in step (1) are: reaction at 50-55°C for 1-10 hours; more preferably, reaction at 52°C for 8 hours.

[0024] Preferably, the standing time in step (2) is 1-2 hours.

[0025] Preferably, the conditions for the distillation separation in step (3) are: cooling water temperature 35-40°C, vacuum degree 0.8-1.2 Pa, scraping speed 70-80 r / min, and feed flow rate 13-17 kg / min.

[0026] In a second aspect, the present invention provides diglyceride prepared by the above method.

[0027] Experiments have shown that the diglyceride prepared by the present invention has a good technical effect of lowering uric acid.

[0028] In a third aspect, the present invention also provides the use of the above-mentioned diglyceride in the preparation of uric acid-lowering products.

[0029] Preferably, the uric acid-lowering product is a medicine.

[0030] Preferably, the uric acid-lowering product is in the form of liquid, granules, powder, tablets or pills.

[0031] In a fourth aspect, the present invention further provides a uric acid-lowering diglyceride composition, comprising the diglyceride and auxiliary materials.

[0032] Preferably, the diglyceride composition is a pharmaceutical product.

[0033] Preferably, the excipients are conventional excipients compatible with the drug. Specific types and amounts can be selected by those skilled in the art based on the dosage form of the product.

[0034] The present invention also provides a uric acid-lowering diglyceride composition, which comprises, by weight percentage, 60-99% diglyceride oil, 0-20% DHA algae oil, 0-20% grape seed oil and 0-1% vitamins.

[0035] Wherein, the vitamin is at least one of vitamin A, D, E and K.

[0036] Preferably, the diglyceride composition comprises, by weight percentage, 85-99% diglyceride, 0.5-10% DHA algae oil, 0.49-4.9% grape seed oil and 0.01-0.1% vitamins.

[0037] Beneficial effects of the present invention

[0038] (1) The present invention prepares diglyceride by screening enzyme preparations. On the one hand, it can effectively reduce the amount of enzyme preparations used in the preparation process and reduce production costs. On the other hand, the prepared diglyceride has greater advantages in lowering uric acid.

[0039] (2) The preparation method of the diglyceride composition of the present invention is simple, low-cost, and suitable for large-scale promotion.

[0040] (3) The present invention improves the preparation method by using vegetable oil and an enzyme preparation to perform a glycerol hydrolysis reaction to obtain diglycerides. This product is then compounded with DHA algae oil, grape seed oil, and vitamins to prepare a diglyceride composition. Compared with the existing technology, the diglyceride composition prepared by the present invention has a better uric acid-lowering effect. DETAILED DESCRIPTION

[0041] The following examples are only intended to help understand the method and core concepts of the present invention. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0042] Therefore, the present invention will not be limited to the embodiments shown herein, but may be applied in 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 one of ordinary skill in the art to which the present invention belongs.

[0043] As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0044] All ranges relating to 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 herein is intended to include all subranges within that range.

[0045] The term "acceptable" indicates that the composition is compatible with the other ingredients comprising the formulation and / or the subject being treated therewith.

[0046] 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. The enzyme preparations in this embodiment all meet the requirements for the use of food additives in accordance with GB2760-2024. Among them, lipase, CAS No. 9001-62-1, enzyme activity 400,000 U / g, was purchased from Xi'an Darwin Biotechnology Co., Ltd.; the esterase, derived from Rhizomucor miehei, CAS No. 6016-18-6, enzyme activity 10,000 U / g; the phospholipase C (Phospholipase C), enzyme activity 200,000 U / g, was purchased from Xi'an Darwin Biotechnology Co., Ltd.; vitamin K, CAS No.: 12001-79-5, vitamin A, CAS No.: 11103-57-4, grape seed oil, CAS No.: 85594-37-2, DHA algae oil, CAS No.: 68002-87-9, and low-erucic acid rapeseed oil, CAS No.: 120962-03-0.

[0047] Example 1 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis

[0048] Here are the steps:

[0049] (1) Mix 1000 g of vegetable oil, 1275 g of glycerol, 250 g of water, and 20 g of enzyme preparation, and react at 52 °C for 8 h to obtain a reaction solution;

[0050] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0051] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0052] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0053] The enzyme preparation in step (1) is lipase.

[0054] Example 2 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis

[0055] Here are the steps:

[0056] (1) Mix 1000 g of vegetable oil, 1100 g of glycerol, 60 g of water, and 1 g of enzyme preparation, and react at 50°C for 10 h to obtain a reaction solution;

[0057] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0058] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0059] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0060] The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 5:0.1:1.

[0061] Example 3 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis

[0062] Here are the steps:

[0063] (1) Mix 1000 g of vegetable oil, 1500 g of glycerol, 300 g of water, and 10 g of enzyme preparation, and react at 55°C for 1 h to obtain a reaction solution;

[0064] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0065] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0066] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0067] The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 10:0.3:1.

[0068] Example 4 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis

[0069] Here are the steps:

[0070] (1) Mix 1000 g of vegetable oil, 1275 g of glycerol, 250 g of water, and 5 g of enzyme preparation, and react at 52 °C for 6 h to obtain a reaction solution;

[0071] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0072] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0073] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0074] The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 8:0.12:1.

[0075] Comparative Example 1 A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis

[0076] Here are the steps:

[0077] (1) Mix 1000 g of vegetable oil, 1275 g of glycerol, 250 g of water, and 20 g of enzyme preparation, and react at 52 °C for 8 h to obtain a reaction solution;

[0078] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0079] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0080] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0081] The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 12:1:1.

[0082] Comparative Example 2: A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis

[0083] Here are the steps:

[0084] (1) Mix 1000 g of vegetable oil, 1275 g of glycerol, 250 g of water, and 20 g of enzyme preparation, and react at 52 °C for 8 h to obtain a reaction solution;

[0085] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0086] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0087] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0088] The enzyme preparation in step (1) is lipase Lipase and phospholipase C, and the weight ratio of the lipase Lipase to the phospholipase C is 8:1.

[0089] Comparative Example 3 A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis

[0090] Here are the steps:

[0091] (1) Mix 1000 g of vegetable oil, 1200 g of glycerol, 1275 g of water, and 5 g of enzyme preparation, and react at 52°C for 6 h to obtain a reaction solution;

[0092] (2) The reaction solution was allowed to stand for 90 min, separated into layers, and the upper layer was collected;

[0093] (3) The upper liquid was injected into the molecular distillation equipment, and the cooling water temperature was controlled at 37°C, the vacuum degree was 1Pa, the scraping speed was 75r / min, and the feed flow rate was 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride;

[0094] Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil;

[0095] The enzyme preparation in step (1) is esterase Esterase and phospholipase C, and the weight ratio of the esterase Esterase to the phospholipase C is 0.12:1.

[0096] Example 5 Diglyceride Composition

[0097] According to the formula in Table 1, DHA algae oil, grape seed oil and vitamins were added to the diglyceride and mixed to obtain the diglyceride composition.

[0098] The formula of the diglyceride composition is shown in Table 1.

[0099] Table 1

[0100]

[0101] Note: In the table, “* 1 " indicates that the vitamin is vitamin A, "* 2 "Indicates that vitamins are vitamin A and vitamin K in a weight ratio of 6:1," * 3 " indicates that the vitamins are vitamin A and vitamin K in a weight ratio of 2:1; "* 4 " indicates that the vitamins are vitamin A and vitamin K in a weight ratio of 4:1.

[0102] Experimental example: Verification of uric acid-lowering efficacy

[0103] 1. Experimental Animals

[0104] SD rats, SPF grade, male, weighing 200-250 g, were provided by Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0105] 2. Test samples

[0106] The diester oil feed (by weight) is: 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, wherein the diglyceride composition is prepared as in Example 5.

[0107] 3. Experimental methods

[0108] After 7 days of adaptive feeding, the animals in the normal control group were fed with a basal feed (25 parts of flour, 25 parts of oatmeal, 25 parts of cornmeal, 10 parts of soybean flour, 8 parts of fish meal, 4 parts of bone meal and 1 part of refined salt, by weight), and the animals in the model control group and diester oil treatment groups (S1-S4 and D1-D3) were fed with a D12451 yeast feed (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, by weight). The daily food intake of each animal was controlled to be the same (7.5 g / 100 g body weight).

[0109] At the same time, all animals in the control group, except the normal control group, received an intraperitoneal injection of 450 mg / kg / d of potassium oxonate for a total of 8 weeks. After 8 weeks, the diester oil-treated group received a diet changed to a diester oil-based diet, with all other procedures remaining unchanged for another 6 weeks.

[0110] 4. Index detection

[0111] After the experiment, the animals were anesthetized, and blood was collected from the abdominal aorta. Serum was separated and measured for uric acid (UA), triglyceride (TG), and total cholesterol (TC). Statistics were performed using SPSS. Continuous data were expressed as mean ± standard deviation (X ± SD). Comparisons between groups were performed using the independent sample t-test, with p < 0.05 indicating statistical significance.

[0112] 4.1 Uric acid test

[0113] Except for the final blood draw, each subsequent blood draw volume was approximately 0.75 µL. Blood samples were stored at 4°C for 1 hour and then centrifuged at 3000 rpm for 15 minutes. Serum was separated and assayed using a SpectraMax M5 microplate reader according to the instructions for the uric acid (UA) kit provided by the Nanjing Jiancheng Bioengineering Research Institute.

[0114] 4.2. Triglyceride and total cholesterol testing

[0115] Animals were anesthetized and blood was collected from the abdominal aorta before being sacrificed. Blood samples were stored at 4°C for 1 hour and then centrifuged at 3000 rpm for 15 minutes. Serum was separated and triglyceride (TG) and total cholesterol (TC) levels were measured using a BS-240VET biochemical analyzer.

[0116] The results are shown in Table 2 below.

[0117] Table 2

[0118]

[0119] Note: Different letters in the same column indicate significant differences among the groups, P < 0.05.

[0120] The results showed that compared with the normal control group, the serum triglycerides, total cholesterol and uric acid of the rats in the model control group were increased, and the differences were significant (P < 0.05), indicating that the model of dyslipidemia combined with hyperuricemia induced by high-fat combined with yeast feed was successfully established.

[0121] Compared with the model group, the serum triglyceride, total cholesterol and uric acid of the rats in the diester oil treatment group were reduced, and the differences were significant (P < 0.05), indicating that the diglyceride composition provided by the present invention has the effect of lowering serum uric acid levels and improving blood lipids.

[0122] It was also found that there were no significant differences in serum triglycerides and total cholesterol among the rats treated with diester oil (P>0.05). However, the serum uric acid levels of rats in diester oil-treated groups S2-S4 were significantly lower than those in diester oil-treated group S1 and diester oil-treated groups D1-D3 (P<0.05). This indicates that the diglyceride composition prepared from S2-S4 of the present invention has a better serum uric acid-lowering effect than the diglyceride compositions prepared from S1 and D1-D3.

[0123] The above further describes the present invention in conjunction with specific embodiments. However, 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 solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis, characterized in that: The steps include: (1) Mixing vegetable oil, glycerol, water and enzyme preparation to obtain a reaction solution; (2) Let the reaction solution stand and separate the layers, and collect the upper layer; (3) Distilling and separating the upper liquid, decolorizing and deodorizing it to obtain diglyceride; The enzyme preparation in step (1) is in a weight ratio of 5-10: 0.1-0.3:1 lipase, esterase and phospholipase C; The esterase Esterase is Rhizomucor miehei esterase; The vegetable oil in step (1) is low-erucic acid rapeseed oil.

2. The method according to claim 1, characterized in that The weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 8:0.12:

1.

3. The method according to claim 1, characterized in that The weight ratio of the vegetable oil to glycerol in step (1) is 1:1.1-1.5; The amount of water added in step (1) is 6-30% by weight of the vegetable oil; The amount of the enzyme preparation added in step (1) is 0.1-5% by weight of the vegetable oil.

4. The method according to claim 1, wherein The reaction in step (1) is: reacting at 50-55°C for 1-10 hours.

5. The method according to claim 1, wherein The conditions for the distillation separation in step (3) are: cooling water temperature 35-40°C, vacuum degree 0.8-1.2 Pa, scraping speed 70-80 r / min, and feed flow rate 13-17 kg / min.

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