A triglyceride, cholesterol and uric acid lowering diglyceride and its bioenzymatic preparation method and application

Abstract

The present invention relates to the field of food technology, and specifically to a diglyceride for lowering triglycerides, cholesterol, and uric acid, and a bioenzymatic preparation method and application thereof. The preparation method comprises the following steps: (1) adding water and glycerol to vegetable oil, and then adding immobilized lipase to react to obtain a reactant; (2) centrifuging the reactant, collecting the light phase product, performing molecular distillation, and collecting the heavy phase product to obtain the diglyceride; the immobilized lipase comprises lipase and a carrier, and the carrier comprises loofah sponge, soybean meal, and bentonite, with a mass ratio of 1:2-4:3-6. The present invention has the advantages of good triglyceride-lowering, total cholesterol-lowering, and uric acid-lowering effects, and high yield and purity.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and in particular to a diglyceride capable of lowering triglycerides, cholesterol and uric acid, and a bioenzymatic preparation method and application thereof. Background Art

[0002] Diacylglycerol (DAG), also known as diglyceride, diglyceride, or diglyceride, is a structural lipid formed by the esterification of one glycerol molecule with two free fatty acid molecules, or by the replacement of one fatty acid in a triacylglycerol (TAG) with a hydroxyl group. Recent studies have shown that edible oils rich in DAG, as a new type of functional oil, possess a variety of physiological activities and functions, including the prevention and treatment of fatty liver disease, cardiovascular and cerebrovascular diseases, and the fact that they do not accumulate in the body after consumption, playing an important role in preventing obesity and enjoying widespread application.

[0003] Diglycerides account for over 40% of edible oil. The main components of edible oil are triglycerides and diglycerides. Traditional vegetable oils generally contain around 98% triglycerides and 2% diglycerides. Diaglycerides are trace components of natural plant oils and endogenous intermediates in fat metabolism. They are naturally present in edible oils. They differ from triglycerides in molecular structure and, therefore, in their metabolic pathways.

[0004] At present, the preparation methods of diglyceride are mainly divided into chemical method and bio-enzymatic method. The chemical method requires high temperature conditions, and the finished product has a dark color and poor flavor. The bio-enzymatic method does not require high temperature conditions, the reaction conditions are mild, and the energy consumption is low. Therefore, the bio-enzymatic method for preparing diglyceride is a green and environmentally friendly synthesis process.

[0005] The bio-enzymatic method for preparing diglycerides mainly involves further decomposing daily edible oils with bio-enzymes, breaking down the original triglycerides into diglycerides, thereby increasing the diglyceride content in the edible oil to about 80%. However, this process has the disadvantages of being difficult to control the degree of hydrolysis and prone to over-hydrolysis. In addition, the product contains excessive by-products, such as free fatty acids and monoglycerides, which affect the quality of the oil.

[0006] Chinese Patent Publication No. CN1544412A discloses a method for producing diglyceride oil, comprising the following steps: (1) adsorbing glycerol onto an adsorption material; (2) mixing the adsorption material obtained in (1) with glycerol and triglycerides, and using immobilized lipase as a catalyst to catalyze the reaction of triglycerides and glycerol at 20-90°C; (3) separating, removing the immobilized lipase and glycerol adsorption material from the product obtained in (2), and then separating and removing monoglycerides and fatty acids to obtain diglyceride oil. This method uses triglycerides and adsorbed glycerol as raw materials, does not add water, and produces diglyceride oil by catalyzing the glycerol decomposition reaction at low temperature by immobilized lipase. The generated product contains few by-products and is light in color. However, the yield and purity of diglycerides need to be further improved, and the effects of lowering triglycerides, total cholesterol, and uric acid also need to be further improved.

[0007] Chinese Patent Publication No. CN119955870A discloses a method for preparing diglycerides with the effects of lowering blood lipids, total cholesterol, blood sugar and uric acid by enzymatic hydrolysis, the method comprising the following steps: (1) mixing vegetable oil, glycerol, composite lipase and water to carry out glycerolysis reaction to obtain a reactant; (2) separating the reactant and collecting the supernatant; (3) distilling and separating the supernatant, decolorizing and deodorizing to obtain diglycerides; the vegetable oils described in step (1) are low-erucic acid rapeseed oil and corn oil; the composite lipase described in step (1) is Mucor javanica lipase, Aspergillus niger lipase and Aspergillus oryzae lipase in a mass ratio of 2-4:1:5-8. This method reduces the amount of enzyme used, and the prepared diglycerides have certain activities of lowering blood lipids, total cholesterol, blood sugar and uric acid. However, the yield and purity of the prepared diglycerides are low, and the effects of lowering triglycerides, total cholesterol and uric acid need to be further improved.

[0008] Therefore, it is very necessary to develop a diglyceride and its bioenzymatic preparation method and application that can solve the above technical problems and lower triglycerides, total cholesterol and uric acid. Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a diglyceride having good triglyceride-lowering, total cholesterol-lowering and uric acid-lowering effects, high yield and purity, and a bioenzymatic preparation method and application thereof.

[0010] The present invention is achieved through the following technical solutions:

[0011] The first aspect of the present invention provides a method for preparing diglyceride by enzymatic means for lowering triglycerides, cholesterol and uric acid, comprising the following steps:

[0012] (1) Water and glycerol are added to vegetable oil, and then immobilized lipase is added to react to obtain a reactant;

[0013] (2) Centrifuging the reactants, collecting the light phase product, performing molecular distillation, and collecting the heavy phase product to obtain diglyceride;

[0014] The immobilized lipase comprises lipase and a carrier, wherein the carrier comprises loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:2-4:3-6.

[0015] As an embodiment of the present invention, the mass ratio of the lipase to the carrier is 1:15-20.

[0016] As an embodiment of the present invention, the lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizoma nivalis, with a mass ratio of 3-5:1.

[0017] As an embodiment of the present invention, the vegetable oil includes at least one of peanut oil, soybean oil, rapeseed oil, olive oil and corn oil.

[0018] As an embodiment of the present invention, in step (1), 1-2 times the amount of water and 0.4-0.8 times the amount of glycerol are added to the vegetable oil, and then 4-7% of the immobilized lipase is added to react at 50-70° C. for 2-6 hours based on the mass percentage of the vegetable oil.

[0019] As an embodiment of the present invention, the specific parameters of the molecular distillation in step (2) are: temperature of 110-150° C., and vacuum degree of less than 50 Pa.

[0020] As an embodiment of the present invention, the preparation method of the immobilized lipase comprises the following steps:

[0021] S1 adds lipase to the buffer solution to obtain a mixed solution;

[0022] S2: add loofah, soybean meal and bentonite to the mixed solution in sequence, mix well, filter and vacuum dry to obtain.

[0023] Preferably, the pH of the buffer solution in step S1 is 6.8-7.5.

[0024] Preferably, the mass concentration of lipase in the mixed solution in step S1 is 6-10%.

[0025] Preferably, the mixing process parameters in step S2 are: mixing at 25-35° C. for 1-3 hours.

[0026] The second aspect of the present invention provides a diglyceride prepared by the above-mentioned diglyceride bioenzymatic preparation method.

[0027] The third aspect of the present invention provides the use of the above-mentioned diglyceride in the preparation of a product for lowering triglycerides and / or cholesterol and / or uric acid.

[0028] The beneficial effects of the present invention are:

[0029] The present invention screens the composition and dosage ratio of the lipase in the immobilized lipase, thereby improving the purity and yield of diglyceride and simultaneously improving the effects of lowering triglyceride, total cholesterol and uric acid.

[0030] The present invention further improves the purity and yield of diglyceride by optimizing the carrier of immobilized lipase, and at the same time further improves the effects of lowering triglycerides, total cholesterol and uric acid. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to 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 fall within the scope of protection of the present invention.

[0032] In the following examples, the lipase derived from Aspergillus oryzae was purchased from Hebei Qiansheng Biotechnology Co., Ltd., and the lipase derived from Rhizoma Nasturtium was purchased from Hong Kong Gisenbergia International Trading Co., Ltd.

[0033] Example 1

[0034] A method for preparing diglyceride by bioenzymatic reaction for lowering triglycerides, cholesterol and uric acid, comprising the following steps:

[0035] (1) Add 1.5 times the amount of water and 0.6 times the amount of glycerol to peanut oil, then add 5% immobilized lipase (based on the amount of peanut oil) and react at 60°C for 4 hours to obtain the reactant;

[0036] (2) The reactants were centrifuged, the light phase products were collected, and molecular distillation was performed at a temperature of 130°C and a vacuum degree of 40 Pa. The heavy phase products were collected to obtain diglyceride.

[0037] The lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizoma nivalis, with a mass ratio of 4:1.

[0038] The immobilized lipase includes lipase and a carrier, and the mass ratio of the two is 1:18; the carrier includes loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:3:5.

[0039] The preparation method of the immobilized lipase comprises the following steps:

[0040] S1: adding lipase to sodium phosphate buffer solution (pH=7.5) with a mass concentration of lipase of 8% to obtain a mixed solution;

[0041] S2 is prepared by sequentially adding loofah, soybean meal and bentonite to the mixed solution, mixing at 30°C for 2 h, filtering and vacuum drying.

[0042] The yield of the prepared diglyceride was 83.7% and the purity was 97.4%.

[0043] Example 2

[0044] A method for preparing diglyceride by bioenzymatic reaction for lowering triglycerides, cholesterol and uric acid, comprising the following steps:

[0045] (1) Add 1 times the amount of water and 0.4 times the amount of glycerol to soybean oil, then add 4% immobilized lipase (based on the amount of soybean oil) and react at 50°C for 6 hours to obtain the reactant;

[0046] (2) The reactants were centrifuged, the light phase products were collected, and molecular distillation was performed at a temperature of 110°C and a vacuum degree of 20 Pa. The heavy phase products were collected to obtain diglyceride.

[0047] The lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizoma nivalis, with a mass ratio of 3:1.

[0048] The immobilized lipase includes lipase and a carrier, and the mass ratio of the two is 1:15; the carrier includes loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:2:3.

[0049] The preparation method of the immobilized lipase comprises the following steps:

[0050] S1: adding lipase to sodium phosphate buffer solution (pH=7.5) with a mass concentration of lipase of 6% to obtain a mixed solution;

[0051] S2 is prepared by sequentially adding loofah, soybean meal and bentonite to the mixed solution, mixing at 25°C for 3 hours, filtering and vacuum drying.

[0052] The yield of the prepared diglyceride was 81.4% and the purity was 97.0%.

[0053] Example 3

[0054] A method for preparing diglyceride by bioenzymatic reaction for lowering triglycerides, cholesterol and uric acid, comprising the following steps:

[0055] (1) Add 2 times the amount of water and 0.8 times the amount of glycerol to corn oil, then add 7% immobilized lipase (based on the amount of corn oil) and react at 70°C for 2 h to obtain the reactant;

[0056] (2) The reactants were centrifuged, the light phase products were collected, and molecular distillation was performed at a temperature of 150°C and a vacuum degree of 45 Pa. The heavy phase products were collected to obtain diglyceride.

[0057] The lipase includes lipase derived from Aspergillus oryzae and lipase derived from Rhizoma nivalis, with a mass ratio of 5:1.

[0058] The immobilized lipase includes lipase and a carrier, and the mass ratio of the two is 1:20; the carrier includes loofah sponge, soybean meal and bentonite, and the mass ratio of the three is 1:4:6.

[0059] The preparation method of the immobilized lipase comprises the following steps:

[0060] S1: adding lipase to sodium phosphate buffer solution (pH=7.5) with a mass concentration of lipase of 10% to obtain a mixed solution;

[0061] S2 is prepared by sequentially adding loofah, soybean meal and bentonite to the mixed solution, mixing at 35°C for 1 hour, filtering and vacuum drying.

[0062] The yield of the prepared diglyceride was 82.6% and the purity was 96.7%.

[0063] Comparative Example 1

[0064] The only difference from Example 1 is that the amount of lipase used remains unchanged, but the composition is different, that is, the lipase is from Aspergillus oryzae, and the other conditions are the same.

[0065] The yield of the prepared diglyceride was 74.3% and the purity was 92.5%.

[0066] Comparative Example 2

[0067] The only difference from Example 1 is that the amount of lipase used remains unchanged, but the composition is different, that is, the lipase is derived from Rhizoma Cibotii, and the other conditions are the same.

[0068] The yield of the prepared diglyceride was 72.9% and the purity was 91.8%.

[0069] Comparative Example 3

[0070] The only difference from Example 1 is that the amount of the carrier in the immobilized lipase remains unchanged, but the composition is different, namely, loofah sponge and soybean meal, with a mass ratio of 1:3.

[0071] The yield of the prepared diglyceride was 75.7% and the purity was 93.4%.

[0072] Comparative Example 4

[0073] The only difference from Example 1 is that the amount of the carrier in the immobilized lipase remains unchanged, but the composition is different, namely soybean meal and bentonite, with a mass ratio of 3:5.

[0074] The yield of the prepared diglyceride was 75.1% and the purity was 92.7%.

[0075] Comparative Example 5

[0076] The only difference from Example 1 is that the amount of the carrier in the immobilized lipase remains unchanged, but the composition is different, namely, loofah and bentonite, with a mass ratio of 1:5.

[0077] The yield of the prepared diglyceride was 74.4% and the purity was 92.5%.

[0078] Test Example 1: Triglyceride and total cholesterol lowering effect test

[0079] Healthy male Sprague-Dawley rats weighing 200 ± 20 g were selected. A maintenance diet consisted of corn, soybean meal, fish meal, and other ingredients. A high-fat diet was prepared by adding 15% lard to the maintenance diet. A diglyceride diet was prepared by adding 15% diglyceride (prepared in the Examples or Comparative Examples) to the maintenance diet. The temperature in the housing was maintained at 22 ± 2°C, the humidity at 55 ± 5%, and the light and dark cycles were adjusted at 12-hour intervals.

[0080] After one week of normal feeding with the maintenance diet, 8 mice were randomly selected as the normal control group and continued to be fed with the maintenance diet. The remaining mice were fed with a high-fat diet as the high-fat diet group. After 15 days of feeding, the total cholesterol (TC) and triglyceride (TG) values ​​of the high-fat diet group were significantly increased compared with the normal control group, indicating that the model was successfully established. The high-fat diet group was randomly divided into 9 groups, each with 8 rats. There was no significant difference in TC and TG values ​​between the rats in the high-fat diet group (p>0.05). One group continued to be fed with the high-fat diet as the model control group, and the remaining 8 groups were fed with diglyceride diets containing diglycerides prepared in Examples 1-3 and Comparative Examples 1-5. Subsequent experiments were performed after 30 days of feeding.

[0081] After the experiment, rats were fasted for 8.5 hours. Each group was anesthetized with 3% sodium pentobarbital (30 mg / kg), and blood was collected before being sacrificed by cervical dislocation. Blood samples were centrifuged at 4000 rpm and serum was collected for analysis of total cholesterol (TC) and triglycerides (TG). The results are shown in Table 1.

[0082] Table 1 Test results of various indicators

[0083]

[0084] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001; compared with the Example 1 group, &p<0.05.

[0085] As shown in Table 1, the TC and TG of the model control group were significantly higher than those of the normal control group, indicating an abnormality. However, under the intervention of diglyceride, the TC and TG indicators of each embodiment decreased significantly, indicating that the diglyceride prepared in each embodiment can reduce triglycerides and total cholesterol. The effects of each indicator of each embodiment are significantly better than those of the comparative examples, indicating that the intervention effect of the diglyceride prepared in Examples 1 to 3 on triglycerides and total cholesterol is significantly better than that of the comparative examples, especially the lipase composition of the present application and the composition of the carrier in the immobilized lipase have a significant synergistic effect in reducing triglycerides and total cholesterol.

[0086] Test Example 2: Uric Acid Lowering Effect Test

[0087] Male Kunming mice, weighing 18-22g, were fed a maintenance diet for one week and then randomly divided into 10 groups of 8 mice each. Nine groups were gavaged daily with 100mg / kg of potassium oxonate for modeling. The remaining group, serving as a blank control group, was given an equal volume of water for 7 days, during which all mice were fed a maintenance diet. The serum uric acid levels of the modeled mice were significantly higher than those of the blank control group, indicating successful modeling. There were no significant differences in serum uric acid levels among the modeled mice (p>0.05). Subsequently, eight groups of modeled mice were gavaged with 0.3g / kg of the diglycerides prepared in Examples 1-3 and Comparative Examples 1-5. One group of modeled mice, serving as the model control group, was gavaged with an equal volume of water containing the diglycerides and the blank control group. Ten days after the final dose, blood was collected from the eyeballs and centrifuged to obtain serum for uric acid measurement. The results are shown in Table 2.

[0088] Table 2 Uric acid lowering test results

[0089]

[0090] Note: Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001; compared with the Example 1 group, &p<0.05.

[0091] As shown in Table 2, the uric acid value of the model control group is significantly higher than that of the blank control group, and anomaly occurs. And in each embodiment, under the intervention of diglyceride, the uric acid value significantly decreases, indicating that the diglyceride prepared by each embodiment can reduce uric acid. The uric acid value of each embodiment is significantly lower than that of each comparative example, indicating that the intervention effect of the diglyceride prepared by Example 1-Example 3 on uric acid is significantly better than that of each comparative example, especially the composition of the lipase of the present application and the composition of the carrier in the immobilized lipase are remarkable in reducing uric acid.

[0092] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride by bioenzymatic reaction for lowering triglyceride, cholesterol and uric acid, characterized in that: The steps include: (1) Water and glycerol are added to vegetable oil, and then immobilized lipase is added to react to obtain a reactant; (2) Centrifuging the reactants, collecting the light phase product, performing molecular distillation, and collecting the heavy phase product to obtain diglyceride; The immobilized lipase comprises lipase and a carrier, wherein the carrier is loofah, soybean meal and bentonite, and the mass ratio of the three is 1:2-4:3-6; The lipase is a lipase derived from Aspergillus oryzae and a lipase derived from Rhizoma nivalis, with a mass ratio of 3-5:1; The vegetable oil is at least one of peanut oil, soybean oil and corn oil.

2. The method for preparing diglyceride by bioenzymatic conversion according to claim 1, wherein The mass ratio of the lipase to the carrier is 1:15-20.

3. The method for preparing diglyceride by bioenzymatic conversion according to claim 1, wherein In step (1), 1-2 times the amount of water and 0.4-0.8 times the amount of glycerol are added to the vegetable oil, and then 4-7% of the immobilized lipase is added based on the mass percentage of the vegetable oil, and the reaction is carried out at 50-70° C. for 2-6 hours.

4. The method for preparing diglyceride by bioenzymatic conversion according to claim 1, wherein: The specific parameters of the molecular distillation in step (2) are: temperature of 110-150°C and vacuum degree of less than 50Pa.

5. The method for preparing diglyceride by bioenzymatic conversion according to claim 1, wherein: The preparation method of the immobilized lipase comprises the following steps: S1 adds lipase to the buffer solution to obtain a mixed solution; S2: add loofah, soybean meal and bentonite to the mixed solution in sequence, mix well, filter and vacuum dry to obtain.

6. The method for preparing diglyceride by bioenzymatic conversion according to claim 5, wherein: The pH of the buffer solution in step S1 is 6.8-7.5; the mass concentration of lipase in the mixed solution is 6-10%; and the mixing process parameters in step S2 are: mixing at 25-35° C. for 1-3 hours.

7. A diglyceride, characterized in that The diglyceride is prepared by the enzymatic preparation method of diglyceride according to any one of claims 1 to 6.

8. Use of the diglyceride according to claim 7 in the preparation of products for lowering triglycerides and / or cholesterol and / or uric acid.

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