Method for preparing diglyceride for reducing uric acid through enzymolysis as well as product and application of diglyceride

Abstract

The invention discloses a method for preparing uric acid-reducing diglyceride through enzymolysis and a product and application thereof, and belongs to the technical field of diglyceride production, the method comprises the following steps: (1) mixing vegetable oil, glycerol, water and an enzyme preparation for reaction to obtain a reaction solution; (2) standing and layering the reaction liquid, and collecting supernatant; (3) performing distillation separation on the supernatant, and performing decoloration and deodorization to obtain diglyceride; the enzyme preparation in the step (1) is prepared from lipase Lipase, esterase Esterase and phospholipase C in a weight ratio of (5-10): (0.1-0.3): 1. Compared with the prior art, the method disclosed by the invention is simple and low in cost, and the prepared diglyceride has more advantages in the aspect of reducing uric acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of diglyceride production, and specifically relates to a method for preparing uric acid-lowering diglyceride 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 fats and is highly safe. Diacylcerol has the effects of reducing visceral fat, inhibiting weight gain, and reducing blood lipids, and therefore has received widespread attention.

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

[0004] 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 in a molar ratio of 1:0.5-1:3, tert-butyl alcohol in an amount of 20-200% based on the weight of the acyl acceptor monoglyceride, and lipase in an amount of 2-20% based on the weight of the 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, 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-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.

[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 water content of glycerin 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) the mixture is esterified and dehydrated to obtain an esterified dehydrate (physical method: centrifugation); 4) the esterified dehydrate is precipitated and deglycerolized to obtain a precipitate; 5) molecular distillation is further screened: ① vacuum dehydration; ② removal of fatty acids; ③ removal of monoglycerides; ④ removal of triglycerides: then at 100-280°C, with a vacuum degree of less than 10pa, vacuum deglycerol triglycerides are removed for 10-20 minutes to obtain diglycerides. The diglyceride content of the invention is more than 85wt%.

[0006] Gout is a metabolic disease caused by long-term purine metabolism disorder, which causes a continuous increase in the level of uric acid (UA) in the blood, and eventually leads to the deposition of urate crystals in the joints. Clinical manifestations include: hyperuricemia (HC), acute gouty arthritis, tophi deposition, joint deformities, and chronic nephritis and the formation of renal uric acid stones over time. Clinically, it is believed that when the uric acid concentration in the blood is >417μmol / L, it can be judged as hyperuricemia, and about 5% to 12% of patients with hyperuricemia will develop gout. Uric acid can promote platelet aggregation and induce the formation of thrombosis and atherosclerosis. Elevated uric acid levels can also lead to obesity, lipid metabolism disorders, diabetes and cerebrovascular diseases.

[0007] CN113350328A discloses a new application of diglyceride and its composition. The study found that diglyceride can help reduce uric acid levels and can be used to prevent and treat hyperuricemia; moreover, suitable diglyceride compositions have a good uric acid-lowering effect for patients with hyperuricemia. However, the invention has high requirements for diglyceride, the content must reach more than 80%, and the requirements for the production process of diglyceride are high. 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

[0008] 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.

[0009] 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 uric acid-lowering diglyceride by enzymatic hydrolysis, comprising the following steps: (1) mixing vegetable oil, glycerol, water and enzyme preparation to obtain a reaction solution; (2) Allow the reaction solution to stand and separate into layers, and collect the upper layer; (3) The upper liquid is distilled, separated, decolorized and deodorized to obtain diglyceride.

[0010] 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.

[0011] Preferably, the enzyme preparation in step (1) is at least one of lipase Lipase, esterase Esterase, phospholipase A1, phospholipase A2 and phospholipase C.

[0012] The lipase Lipase in the present invention is derived from Hansenula polymorpha, Aspergillus niger, Mucor circinelloides, Aspergillus oryzae, Candida cylindrica, pancreas of pig or cattle, Rhizopus niveus, Rhizomucor miehei, Rhizopus oryzae, Trichoderma reesei, sheep throat, salivary gland or forestomach tissue of calf or lamb; the esterase Esterase is derived from Rhizomucor miehei, Trichoderma reesei 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 reesei; the phospholipase C (Phospholipase C) is derived from Pichia pastoris or Bacillus licheniformis.

[0013] More preferably, the enzyme preparation is lipase Lipase, esterase Esterase and phospholipase C.

[0014] More preferably, the weight ratio of the lipase Lipase, esterase Esterase and phospholipase C is 5-10:0.1-0.3:1; most preferably 8:0.12:1.

[0015] Preferably, the weight ratio of the vegetable oil to glycerol in step (1) is 1:1.1-1.5, and more preferably 1:1.275.

[0016] 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%.

[0017] Preferably, the amount of water added in step (1) is 6%-30% by weight of the vegetable oil; more preferably 25%.

[0018] 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.

[0019] Preferably, the standing time in step (2) is 1-2 hours.

[0020] 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.

[0021] In a second aspect, the present invention provides diglyceride prepared by the above method.

[0022] Experiments have shown that the diglyceride prepared by the present invention has a good technical effect of lowering uric acid.

[0023] In a third aspect, the present invention also provides the use of the above-mentioned diglyceride in the preparation of uric acid-lowering products.

[0024] Preferably, the uric acid-lowering product is a food, a health product or a medicine.

[0025] Preferably, the uric acid-lowering product is in the form of liquid, granules, powder, tablets or pills.

[0026] In a fourth aspect, the present invention further provides a uric acid-lowering diglyceride composition, comprising the diglyceride and auxiliary materials.

[0027] Preferably, the diglyceride composition is a food, a health product or a medicine.

[0028] Preferably, the auxiliary materials are conventional auxiliary materials compatible with food, health products or medicines. The specific types and dosages can be conventionally selected by those skilled in the art according to the dosage form of the product.

[0029] The present invention also provides a uric acid-lowering diglyceride composition, which comprises, by weight percentage, 60-99% of diglyceride oil, 0-20% of DHA algae oil, 0-20% of grape seed oil and 0-1% of vitamins.

[0030] Wherein, the vitamin is at least one of vitamin A, D, E and K.

[0031] 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.

[0032] Beneficial effects of the present invention (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 more advantages in lowering uric acid.

[0033] (2) The preparation method of the diglyceride composition of the present invention is simple, low-cost, and suitable for large-scale promotion.

[0034] (3) The present invention improves the preparation method, uses vegetable oil and enzyme preparation to carry out glycerol hydrolysis reaction to obtain diglyceride, and uses the product to prepare a diglyceride composition by compounding with DHA algae oil, grape seed oil and vitamins. Compared with the prior art, the diglyceride composition prepared by the present invention has a better uric acid-lowering effect. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0038] 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.

[0039] The term "acceptable" indicates that the composition is compatible with the other ingredients making up the formulation and / or the subject being treated therewith.

[0040] 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 GB2760-2024, wherein the lipase Lipase, CAS No. 9001-62-1, enzyme activity 400,000 / g, is purchased from Xi'an Darwen Biotechnology Co., Ltd.; the esterase Esterase is 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, is purchased from Xi'an Darwen 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, low erucic acid rapeseed oil, CAS No.: 120962-03-0.

[0041] Example 1 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis Here are the steps: (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; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; The enzyme preparation in step (1) is lipase.

[0042] Example 2 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis Here are the steps: (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; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of lipase Lipase, esterase Esterase and phospholipase C is 5:0.1:1.

[0043] Example 3 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis Here are the steps: (1) Mix 1000 g of vegetable oil, 1500 g of glycerol, 300 g of water and 10 g of enzyme preparation, react at 55°C for 1 h to obtain a reaction solution; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of lipase Lipase, esterase Esterase and phospholipase C is 10:0.3:1.

[0044] Example 4 A method for preparing uric acid-lowering diglycerides by enzymatic hydrolysis Here are the steps: (1) 1000 g of vegetable oil, 1275 g of glycerol, 250 g of water and 5 g of enzyme preparation were mixed and reacted at 52 °C for 6 h to obtain a reaction solution; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of lipase Lipase, esterase Esterase and phospholipase C is 8:0.12:1.

[0045] Comparative Example 1 A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis Here are the steps: (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; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; The enzyme preparation in step (1) is lipase Lipase, esterase Esterase and phospholipase C, and the weight ratio of lipase Lipase, esterase Esterase and phospholipase C is 12:1:1.

[0046] Comparative Example 2 A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis Here are the steps: (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; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; The enzyme preparation in step (1) is lipase Lipase and phospholipase C, and the weight ratio of lipase Lipase to phospholipase C is 8:1.

[0047] Comparative Example 3 A method for preparing uric acid-lowering diglyceride by enzymatic hydrolysis Here are the steps: (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; (2) Let the reaction solution stand for 90 minutes, separate the layers, and collect the upper layer; (3) Inject the upper liquid into the molecular distillation equipment, control the cooling water temperature at 37°C, the vacuum degree at 1Pa, the scraping rate at 75r / min, and the feed flow rate at 15kg / min for distillation separation, decolorization and deodorization to obtain diglyceride; Wherein, the vegetable oil in step (1) is low-erucic acid rapeseed oil; 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.

[0048] Example 5 Diaceride Composition According to the formula in Table 1, DHA algae oil, grape seed oil and vitamins are added to the diglyceride and mixed to obtain the diglyceride composition.

[0049] The formula of the diglyceride composition is shown in Table 1.

[0050] Table 1

[0051] Note: In the table, “* 1 " indicates that the vitamin is vitamin A, "* 2 "Indicates that the 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.

[0052] Experimental example: Verification of uric acid lowering efficacy 1. Experimental Animals SD rats, SPF grade, male, weighing 200-250 g, were provided by Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.

[0053] 2. Test samples 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 according to Example 5.

[0054] 3. Experimental methods After 7 days of adaptive feeding, the animals in the normal control group were fed with a basal feed (by weight: 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), 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 (by weight: 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), and the daily food intake of each animal was controlled to be the same (7.5 g / 100 g body weight).

[0055] At the same time, except for the animals in the normal control group, the other groups were intraperitoneally injected with 450 mg / kg / d potassium oxonate for a total of 8 weeks. After 8 weeks, the feed of the diester oil treatment group was changed to diester oil feed, and the other operations remained unchanged for 6 weeks.

[0056] 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 (X ± SD). The independent sample t test was used for comparison between the groups. p < 0.05 was considered statistically significant.

[0057] 4.1 Uric acid test Except for the last blood collection, the volume of each blood collection was about 0.75 μL. After blood collection, the blood sample was stored at 4°C for 1 hour and then 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.

[0058] 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 3000r / 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.

[0059] The results are shown in Table 2 below.

[0060] Table 2

[0061] Note: Different letters in the same column indicate significant differences among the groups, P < 0.05.

[0062] 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 difference was significant (P < 0.05), indicating that the model of dyslipidemia combined with hyperuricemia induced by high-fat combined with yeast feed was successfully established.

[0063] Compared with the model group, the serum triglycerides, total cholesterol and uric acid of the rats in the diester oil treatment group were all reduced, and there was a significant difference (P < 0.05), indicating that the diglyceride composition provided by the present invention has the effect of reducing serum uric acid levels and improving blood lipids.

[0064] At the same time, it was found that there was no significant difference in serum triglyceride and total cholesterol between rats in the diester oil treatment groups (P>0.05), but the serum uric acid level of rats in diester oil treatment groups S2-S4 was lower than that in diester oil treatment group S1 and diester oil treatment groups D1-D3, and there was a significant difference (P<0.05). This shows that the diglyceride composition prepared by S2-S4 of the present invention has a better effect of lowering serum uric acid than the diglyceride composition prepared by S1 and D1-D3.

[0065] 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 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) Allow the reaction solution to stand and separate into 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) has a weight ratio of 5-10: 0.1-0.3:1 lipase, esterase and phospholipase C.

2. The method according to claim 1, characterized in that The vegetable oil in step (1) is low-erucic acid rapeseed oil.

3. 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.

4. 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% of the 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.

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

6. The method according to claim 1, characterized in that The conditions for 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.

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

8. Use of diglyceride prepared by the method according to any one of claims 1 to 6 in the preparation of a uric acid-lowering product, wherein the product is a food, a health product or a medicine.

9. A diglyceride composition for lowering uric acid, characterized in that: The invention comprises the diglyceride according to claim 7 and a health product or a pharmaceutically acceptable auxiliary material.

10. The diglyceride composition according to claim 9, characterized in that Calculated by weight percentage, it comprises 85-99% diglyceride oil, 0.5-10% DHA algae oil, 0.49-4.9% grape seed oil and 0.01-0.1% vitamins, wherein the vitamins are at least one of vitamins A, D, E and K.

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