Preparation method and application of diglyceride oil with improved cardiovascular and cerebrovascular health effect

Abstract

The present application relates to the technical field of oil and fat chemistry, and particularly relates to a preparation method and application of a diglyceride oil with the function of improving cardiovascular and cerebrovascular health. The preparation method of the diglyceride oil comprises the following steps: (1) mixing oil and water, adding a first hydrolytic enzyme to perform first hydrolysis, and then adding a second hydrolytic enzyme to perform second hydrolysis, to obtain a hydrolysis product; (2) adding the hydrolysis product to glycerol and a lipase to perform reaction, and then distilling, to obtain the diglyceride oil; in step (1), the first hydrolytic enzyme is Lipozyme RM IM, and the second hydrolytic enzyme is Candida antarctica lipase B; in step (2), the lipase is a mixture of lipase Lipase G50 and Rhizomucor niveus lipase, and the mass ratio of the two is 1:1-3. The diglyceride oil prepared by the method has the function of improving cardiovascular and cerebrovascular health.

Description

Technical Field

[0001] This invention relates to the field of oleochemical technology, specifically to a method for preparing and applying diglyceride oil that improves cardiovascular health. Background Technology

[0002] Diacylglycerol (DAG), also known as diglyceride, is a class of structural lipids synthesized by the esterification of one molecule of glycerol and two molecules of free fatty acids, or by the substitution of a hydroxyl group in one fatty acid of triacylglycerol (TAG). Edible oils rich in DAG possess various physiological activities and functions, including the prevention and treatment of fatty liver and cardiovascular diseases. They do not accumulate in the body after consumption and play an important role in preventing obesity, making them widely used.

[0003] The main components of edible oils are triglycerides and diglycerides. Diglycerides are trace components of natural plant oils and endogenous intermediate products of fat metabolism in the body; they are natural components found in edible oils. They differ from triglycerides in molecular structure and therefore in their metabolic pathways.

[0004] Currently, the main methods for preparing diglycerides are divided into chemical methods and bio-enzymatic methods. Chemical methods require high temperatures, and the finished product has a dark color and poor flavor. Bio-enzymatic methods do not require high temperatures, have mild reaction conditions, and consume less energy. Therefore, bio-enzymatic methods for preparing diglycerides are a green and environmentally friendly synthetic process.

[0005] The preparation of diglycerides by bio-enzyme mainly involves further decomposing commonly consumed oils with bio-enzymes, breaking down the original triglycerides into diglycerides, thereby increasing the diglyceride content in edible oils to about 80%. However, this process has the drawbacks of difficulty in controlling the degree of hydrolysis, which is prone to over-hydrolysis, and the presence of excessive byproducts such as free fatty acids and monoglycerides, which affect the quality of the oils.

[0006] Chinese Patent Publication No. CN120384106A discloses a method for preparing diglycerides that lower triglycerides, cholesterol, and uric acid, and their applications. The preparation method includes the following steps: (1) adding water and glycerol to vegetable oil, and then adding immobilized lipase to react and obtain the reactant; (2) centrifuging the reactant, collecting the light phase product, performing molecular distillation, and collecting the heavy phase product to obtain diglycerides; the immobilized lipase includes lipase and a carrier, the carrier including loofah sponge, soybean meal, and bentonite, with a mass ratio of 1:2-4:3-6, and the lipase including lipase from Aspergillus oryzae and lipase from Rhizopus spp., with a mass ratio of 3-5:1. However, it requires the preparation of a special immobilized carrier, the preparation process is relatively complex, and the effect of improving cardiovascular health needs to be further improved.

[0007] Chinese Patent Publication No. CN119955870A discloses a method for preparing diglycerides with lipid-lowering, total cholesterol-lowering, blood glucose-lowering, and uric acid-lowering effects through enzymatic hydrolysis. The method includes the following steps: (1) mixing vegetable oil, glycerol, a compound lipase, and water to carry out a glycerol hydrolysis reaction to obtain a reactant; (2) separating the reactant and collecting the supernatant; (3) distilling, decolorizing, and deodorizing the supernatant to obtain diglycerides; the vegetable oil mentioned in step (1) is low-erucic acid rapeseed oil and corn oil; the compound lipase mentioned in step (1) is a mixture of *Rhizopus guarana* 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 lipid-lowering, total cholesterol-lowering, blood glucose-lowering, and uric acid-lowering activities. However, the cardiovascular health-improving effect of the prepared diglycerides needs further improvement.

[0008] Therefore, it is essential to develop a method for preparing and applying diglycerides that can solve the above-mentioned technical problems and have a better effect on improving cardiovascular and cerebrovascular health. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a simpler preparation process for diglycerides that have a better effect on improving cardiovascular and cerebrovascular health, as well as their application. Previous research has shown that the diglyceride content in diglyceride oil is not positively correlated with the improvement of cardiovascular and cerebrovascular health, but is closely related to the composition of the diglyceride oil. Therefore, this invention optimizes the preparation process of diglyceride oil to enhance its effect on improving cardiovascular and cerebrovascular health.

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

[0011] The first aspect of this invention provides a method for preparing diglyceride oil with the effect of improving cardiovascular and cerebrovascular health, comprising the following steps:

[0012] (1) Mix oil and water, add the first hydrolytic enzyme for the first hydrolysis, and then add the second hydrolytic enzyme for the second hydrolysis to obtain the hydrolysis product;

[0013] (2) Add the hydrolysis product to glycerol and lipase for reaction, and then distill to obtain diglyceride oil;

[0014] In step (1), the first hydrolase is Lipozyme RM IM and the second hydrolase is Candida antarcticis lipase B; in step (2), the lipase is a mixture of lipase Lipase G50 and Rhizopus spp. lipase, with a mass ratio of 1:1-3.

[0015] In one embodiment of the present invention, the amount of the first hydrolytic enzyme used in step (1) accounts for 5-10% of the oil mass.

[0016] As one embodiment of the present invention, the conditions for the first hydrolysis in step (1) are: temperature 55-65℃, time 2-4h.

[0017] In one embodiment of the present invention, the amount of the second hydrolytic enzyme used in step (1) accounts for 1-5% of the oil mass.

[0018] As one embodiment of the present invention, the conditions for the second hydrolysis in step (1) are: temperature 40-45℃, time 2-4h.

[0019] In one embodiment of the present invention, the mass ratio of oil to water in step (1) is 1:0.6-1.2.

[0020] In one embodiment of the present invention, the oil mentioned in step (1) is selected from at least one of vegetable oil and animal oil.

[0021] In a preferred embodiment of the present invention, the vegetable oil is selected from at least one of flaxseed oil, soybean oil, rapeseed oil, peanut oil, corn oil, sunflower seed oil, camellia seed oil, coconut oil, palm oil, palm kernel oil, olive oil, olive pomace oil, walnut oil, rice bran oil, rice bran oil, cottonseed oil, perilla seed oil, safflower seed oil, grape seed oil, tea seed oil, peony seed oil, sesame oil, wheat germ oil, maple seed oil, *Xanthoceras sorbifolium* oil, sea buckthorn seed oil, DHA algal oil, medium-chain triglycerides, medium- and long-chain fatty acid edible oils, hemp seed oil, Sichuan pepper seed oil, Sichuan pepper oil, pumpkin seed oil, chili oil, almond oil, and sacha inchi oil; and / or the animal oil is selected from at least one of duck fat, lard, mutton fat, goose fat, and chicken fat.

[0022] In one embodiment of the present invention, the mass ratio of the hydrolysis product to glycerol in step (2) is 1:2-4.

[0023] In one embodiment of the present invention, the amount of lipase used in step (2) is 5-15% of the mass of glycerol.

[0024] In one embodiment of the present invention, the reaction temperature in step (2) is 40-60°C and the time is 2-6h.

[0025] As one embodiment of the present invention, the distillation method described in step (2) is molecular distillation.

[0026] In a preferred embodiment of the present invention, the conditions for molecular distillation are as follows: two-stage distillation is performed, with the temperature of the first stage distillation being 150-170℃ and the vacuum degree being less than 10Pa; and the temperature of the second stage distillation being 200-280℃ and the vacuum degree being less than 2Pa.

[0027] As one embodiment of the present invention, step (2) further includes a decolorization and / or deodorization step after distillation.

[0028] In a preferred embodiment of the present invention, the decolorization and / or deodorization steps are carried out using activated clay and / or activated carbon.

[0029] In a more preferred embodiment of the present invention, the amount of activated clay and activated carbon used is 0.5-4% of the mass of the product after distillation.

[0030] A second aspect of the present invention provides the application of the diglyceride oil prepared by the above-described preparation method in the preparation of food or pharmaceuticals.

[0031] As one embodiment of the present invention, the food or medicine has the effect of improving cardiovascular and cerebrovascular health or improving blood lipids.

[0032] In one embodiment of the present invention, the food is a general food, a health food, or a food formulated for special medical purposes.

[0033] The beneficial effects of this invention are:

[0034] This invention first uses Lipozyme RM IM and Candida antarcticis lipase B to hydrolyze the lipase under specific conditions, and then uses a specific mass ratio of lipase Lipase G50 to react with Rhizopus spp. The resulting diglyceride oil significantly reduced the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in experimental rats, while significantly increasing the level of high-density lipoprotein cholesterol (HDL-C), thus exhibiting a significant effect on improving cardiovascular health. Detailed Implementation

[0035] 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 a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0036] This invention provides a method for preparing diglyceride oil with the effect of improving cardiovascular and cerebrovascular health, comprising the following steps:

[0037] (1) Mix oil and water, add the first hydrolytic enzyme for the first hydrolysis, and then add the second hydrolytic enzyme for the second hydrolysis to obtain the hydrolysis product;

[0038] (2) Add the hydrolysis product to glycerol and lipase for reaction, and then distill to obtain diglyceride oil;

[0039] In step (1), the first hydrolase is Lipozyme RM IM and the second hydrolase is Candida antarcticis lipase B; in step (2), the lipase is a mixture of lipase Lipase G50 and Rhizopus spp. lipase, with a mass ratio of 1:1-3.

[0040] The lipase Lipase G50 of this invention is a glycerol lipase, also known as "Lipase G Amano 50".

[0041] In one embodiment of the present invention, the amount of the first hydrolytic enzyme used in step (1) accounts for 5-10% of the oil mass.

[0042] As one embodiment of the present invention, the conditions for the first hydrolysis in step (1) are: temperature 55-65℃, time 2-4h.

[0043] In one embodiment of the present invention, the amount of the second hydrolytic enzyme used in step (1) accounts for 1-5% of the oil mass.

[0044] As one embodiment of the present invention, the conditions for the second hydrolysis in step (1) are: temperature 40-45℃, time 2-4h.

[0045] In one embodiment of the present invention, the mass ratio of oil to water in step (1) is 1:0.6-1.2.

[0046] In one embodiment of the present invention, the oil mentioned in step (1) is selected from at least one of vegetable oil and animal oil.

[0047] In a preferred embodiment of the present invention, the vegetable oil is selected from at least one of flaxseed oil, soybean oil, rapeseed oil, peanut oil, corn oil, sunflower seed oil, camellia seed oil, coconut oil, palm oil, palm kernel oil, olive oil, olive pomace oil, walnut oil, rice bran oil, rice bran oil, cottonseed oil, perilla seed oil, safflower seed oil, grape seed oil, tea seed oil, peony seed oil, sesame oil, wheat germ oil, maple seed oil, *Xanthoceras sorbifolium* oil, sea buckthorn seed oil, DHA algal oil, medium-chain triglycerides, medium- and long-chain fatty acid edible oils, hemp seed oil, Sichuan pepper seed oil, Sichuan pepper oil, pumpkin seed oil, chili oil, almond oil, and sacha inchi oil; and / or the animal oil is selected from at least one of duck fat, lard, mutton fat, goose fat, and chicken fat.

[0048] In one embodiment of the present invention, the mass ratio of the hydrolysis product to glycerol in step (2) is 1:2-4.

[0049] In one embodiment of the present invention, the amount of lipase used in step (2) is 5-15% of the mass of glycerol.

[0050] In one embodiment of the present invention, the reaction temperature in step (2) is 40-60°C and the time is 2-6h.

[0051] As one embodiment of the present invention, the distillation method described in step (2) is molecular distillation.

[0052] In a preferred embodiment of the present invention, the conditions for molecular distillation are as follows: two-stage distillation is performed, with the temperature of the first stage distillation being 150-170℃ and the vacuum degree being less than 10Pa; and the temperature of the second stage distillation being 200-280℃ and the vacuum degree being less than 2Pa.

[0053] As one embodiment of the present invention, step (2) further includes a decolorization and / or deodorization step after distillation.

[0054] In a preferred embodiment of the present invention, the decolorization and / or deodorization steps are performed using activated clay and / or activated carbon. Any commercially available activated clay or activated carbon can achieve the effects of the present invention.

[0055] In a more preferred embodiment of the present invention, the amounts of activated clay and activated carbon are 0.5-4% of the mass of the distilled product, respectively; the treatment temperature is 80-160℃, the treatment time is 30-60 min, and the vacuum degree is 60-150 Pa.

[0056] In the following examples, Lipozyme RM IM (enzyme activity 275 IUN / g) was purchased from Novozymes. Candida antarcticis lipase B (enzyme activity 150,000 U / g) was purchased from Guangdong Maike Baisheng Biotechnology Co., Ltd. Lipase G50 (enzyme activity 50,000 U / g) was purchased from Amano Enzyme Group, Japan. Rhizopus spp. lipase was purchased from Hong Kong Gissner International Trading Co., Ltd.

[0057] Example 1

[0058] A method for preparing diglyceride oil with the effect of improving cardiovascular and cerebrovascular health, the preparation steps are as follows:

[0059] (1) Mix oil (flaxseed oil, soybean oil, rapeseed oil and peanut oil in equal mass ratio) and water in a mass ratio of 1:0.6. Add 10% of the oil mass of Lipozyme RM IM for the first hydrolysis at 55℃ for 4 hours. Then add 1% of the oil mass of Candida antarctica lipase B for the second hydrolysis at 40℃ for 4 hours to obtain the hydrolysis product.

[0060] (2) The hydrolysis product was added to glycerol and lipase (lipase G50: Rhizopus spp. lipase mass ratio 1:1) for reaction. The mass ratio of hydrolysis product to glycerol was 1:2. The amount of lipase was 5% of the mass of glycerol. The reaction temperature was 40℃ and the time was 6h. After the reaction was completed, the product was centrifuged and the upper layer was collected. Two-stage distillation was performed. The temperature of the first distillation was 150℃ and the vacuum degree was 9Pa. The temperature of the second distillation was 200℃ and the vacuum degree was 1.5Pa. The heavy phase product was collected. 2% of the mass of the heavy phase product was added to activated clay and 2% of the mass of activated carbon for decolorization and deodorization treatment. The treatment temperature was 100℃ and the treatment time was 40min. The vacuum degree was 100Pa. Diglyceride oil was obtained.

[0061] Example 2

[0062] A method for preparing diglyceride oil with the effect of improving cardiovascular and cerebrovascular health, the preparation steps are as follows:

[0063] (1) Mix oils (corn oil, sunflower seed oil, camellia seed oil, coconut oil and palm oil in equal mass ratio) and water in a mass ratio of 1:1.2. Add 5% of Lipozyme RM IM by oil mass for the first hydrolysis at 65℃ for 2 hours. Then add 5% of Candida antarcticis lipase B by oil mass for the second hydrolysis at 45℃ for 2 hours to obtain the hydrolysis product.

[0064] (2) The hydrolysis product was added to glycerol and lipase (lipase G50: Rhizopus spp. lipase mass ratio 1:3) for reaction. The mass ratio of hydrolysis product to glycerol was 1:4. The amount of lipase was 15% of the mass of glycerol. The reaction temperature was 60℃ and the time was 2h. After the reaction was completed, the product was centrifuged and the upper layer was collected. Two-stage distillation was performed. The temperature of the first distillation was 170℃ and the vacuum degree was 6Pa. The temperature of the second distillation was 280℃ and the vacuum degree was 0.5Pa. The heavy phase product was collected. 2% of the mass of the heavy phase product was added to activated clay and 2% of the mass of activated carbon for decolorization and deodorization treatment. The treatment temperature was 100℃ and the treatment time was 40min. The vacuum degree was 100Pa. Diglyceride oil was obtained.

[0065] Example 3

[0066] A method for preparing diglyceride oil with the effect of improving cardiovascular and cerebrovascular health, the preparation steps are as follows:

[0067] (1) Mix oil (soybean oil and Acer truncatum seed oil in equal mass ratio) and water in a mass ratio of 1:0.9. Add 8% of Lipozyme RM IM by oil mass for the first hydrolysis at 60℃ for 3 hours. Then add 3% of Candida antarctica lipase B by oil mass for the second hydrolysis at 40℃ for 3 hours to obtain the hydrolysis product.

[0068] (2) The hydrolysis product was added to glycerol and lipase (lipase G50: Rhizopus spp. lipase mass ratio 1:2) for reaction. The mass ratio of hydrolysis product to glycerol was 1:3. The amount of lipase was 10% of the mass of glycerol. The reaction temperature was 50℃ and the time was 4h. After the reaction was completed, the product was centrifuged and the upper layer was collected. Two-stage distillation was performed. The temperature of the first distillation was 160℃ and the vacuum degree was 8Pa. The temperature of the second distillation was 240℃ and the vacuum degree was 1Pa. The heavy phase product was collected. 2% of the mass of the heavy phase product was added to activated clay and 2% of the mass of activated carbon for decolorization and deodorization treatment. The treatment temperature was 100℃ and the treatment time was 40min. The vacuum degree was 100Pa. Diglyceride oil was obtained.

[0069] Comparative Example 1

[0070] The only difference from Example 3 is that in step (1), only Lipozyme RM IM is added, and Candida antarcticis lipase B is not added. The total amount of hydrolytic enzyme and the total hydrolysis time are kept unchanged. All other conditions are the same. Step (1) is as follows:

[0071] Oils (soybean oil and Acer truncatum seed oil mixed in equal mass ratio) and water were mixed at a mass ratio of 1:0.9. Lipozyme RM IM (11% by mass of oil) was added for hydrolysis at 60℃ for 6 hours to obtain the hydrolysis product.

[0072] Comparative Example 2

[0073] The only difference from Example 3 is that in step (1), only Candida antarcticis lipase B is added, and Lipozyme RM IM is not added. The total amount of hydrolytic enzyme and the total hydrolysis time are kept the same. All other conditions are the same. Step (1) is as follows:

[0074] Oils (soybean oil and Acer truncatum seed oil mixed in equal mass ratio) and water were mixed at a mass ratio of 1:0.9. Antarctic Candida lipase B at 11% of the oil mass was added for hydrolysis at 40℃ for 6 hours to obtain the hydrolysis product.

[0075] Comparative Example 3

[0076] The only difference from Example 3 is the order of enzymatic hydrolysis of the two hydrolases in step (1). All other conditions are the same. Step (1) is as follows:

[0077] Oils (soybean oil and Acer truncatum seed oil mixed in equal mass ratio) and water were mixed at a mass ratio of 1:0.9. Antarctic Candida lipase B was added at 3% of the oil mass for the first hydrolysis at 40℃ for 3 hours. Then, Lipozyme RM IM at 8% of the oil mass was added for the second hydrolysis at 60℃ for 3 hours to obtain the hydrolysis product.

[0078] Comparative Example 4

[0079] The only difference from Example 3 is the mass ratio of lipase G50 to Rhizopus spp. lipase in step (2). All other conditions are the same, as shown in Table 1.

[0080] Table 1. Mass ratio of lipase G50 to Rhizopus spp. lipase in Comparative Example 4.

[0081]

[0082] Comparative Example 5

[0083] The only difference from Example 3 is that the lipase used in step (2) is different; only Rhizopus spp. lipase is used, while the total amount of lipase remains unchanged.

[0084] Comparative Example 6

[0085] The only difference from Example 3 is that the lipase used in step (2) is different; only lipase Lipase G50 is used, while the total amount of lipase remains the same.

[0086] Test Example 1

[0087] Healthy male SD rats, weighing 200±20g, were selected. Maintenance diets were purchased from Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd. The high-fat diet was prepared by adding 15% lard to the maintenance diet. The diglyceride diet was prepared by adding 15% diglyceride oil (prepared in each example or comparative example) to the maintenance diet. The temperature in the rearing room was maintained at 22±2℃, and the humidity at 55±5%, with light and darkness adjusted at 12-hour intervals.

[0088] After being fed a maintenance diet for one week, six mice were randomly selected as the normal control group and continued to be fed the maintenance diet. The remaining mice were fed a high-fat diet as the high-fat diet group. After 15 days of feeding, compared with the normal control group, the high-fat diet group showed significantly higher levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) (p<0.05), and significantly lower levels of high-density lipoprotein cholesterol (HDL-C) (p<0.05), indicating successful model establishment.

[0089] The high-fat diet group was randomly divided into 13 groups, with 6 rats in each group. There were no significant differences in TC, TG, LDL-C, and HDL-C values ​​among the rats in the high-fat diet group (p > 0.05). One group served as the model control group and continued to be fed a high-fat diet, while the other 12 groups were fed diets containing diglycerides prepared in Examples 1-3 and Comparative Examples 1-6, respectively. Subsequent experiments were conducted after 30 days of feeding.

[0090] During the experiment, there were no significant differences in food intake, water consumption, and body weight among the groups of mice (p > 0.05). After the experiment, the rats were fasted for 8.5 hours, and each group was anesthetized with 3% sodium pentobarbital (30 mg / kg). After weighing and blood collection, they were euthanized by cervical dislocation. Blood samples were centrifuged at 4000 rpm, and serum was collected for the detection of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). The results are shown in Tables 2-5.

[0091] Table 2. Results of Total Cholesterol (TC) Detection

[0092]

[0093] Note: Compared with the model control group, # p < 0.05 ## p < 0.01; compared with Example 3 group, & p < 0.05.

[0094] Total cholesterol (TC) refers to the sum of cholesterol contained in all lipoproteins in the blood. Elevated total cholesterol levels generally indicate an increased risk of hyperlipidemia and atherosclerosis. Compared with the model group, Examples 1-3 showed a significant decrease in total cholesterol content (p < 0.01), indicating a reduction in the risk of atherosclerosis to some extent. Furthermore, the total cholesterol content of Example 1 was significantly lower than that of Comparative Examples 1-3 (p < 0.05), indicating that Lipozyme RM IM and Candida antarcticis lipase B must be used simultaneously and hydrolyzed under specific conditions to obtain diglyceride oil that significantly reduces total cholesterol content. Comparative Examples 4-1 to 4-4 investigated the effect of the mass ratio of lipase Lipase G50 to Rhizopus spp. lipase, showing that the two lipases must be within the specific mass ratio range of this invention to exert a synergistic effect and significantly reduce total cholesterol content.

[0095] Table 3. Triglyceride (TG) level test results

[0096]

[0097] Note: Compared with the model control group, # p < 0.05 ## p < 0.01; compared with Example 3 group, & p < 0.05.

[0098] Triglycerides (TG) are an independent risk factor for cardiovascular and cerebrovascular diseases. Elevated triglyceride levels increase the risk of cardiovascular and cerebrovascular diseases. Compared with the model group, the triglyceride content in Examples 1-3 was significantly reduced (p < 0.01), indicating a certain degree of reduction in the risk of cardiovascular and cerebrovascular diseases. Furthermore, the triglyceride content in Example 1 was significantly lower than that in Comparative Examples 1-3 (p < 0.05), indicating that Lipozyme RM IM and Candida antarcticis lipase B must be used simultaneously and hydrolyzed under specific conditions to obtain diglyceride oil that significantly reduces triglyceride content. Comparative Examples 4-1 to 4-4 investigated the effect of the mass ratio of lipase Lipase G50 to Rhizopus spp. lipase. The results showed that the two lipases must be within the specific mass ratio range of this invention to exert a synergistic effect and significantly reduce triglyceride content.

[0099] Table 4. Results of Low-Density Lipoprotein Cholesterol (LDL-C) Detection

[0100]

[0101] Note: Compared with the model control group, # p < 0.05 ## p < 0.01; compared with Example 3 group,& p < 0.05.

[0102] Low-density lipoprotein cholesterol (LDL-C) is the carrier that transports cholesterol from the liver to tissues throughout the body. When LDL-C levels in the blood are too high, excess cholesterol deposits on the endothelium of blood vessel walls, leading to endothelial damage, inflammation, and macrophage infiltration. Over time, this cholesterol accumulates in the blood vessel walls, forming lipid streaks that eventually develop into fibrous plaques (atherosclerotic plaques). Elevated LDL-C levels are one of the most important independent risk factors for cardiovascular and cerebrovascular diseases, especially coronary heart disease and cerebral infarction.

[0103] Compared with the model group, Examples 1-3 showed a significant decrease in LDL-C content (p < 0.01), indicating a reduction in the risk of cardiovascular and cerebrovascular diseases to some extent. Furthermore, the LDL-C content in Example 1 was significantly lower than that in Comparative Examples 1-3 (p < 0.05), indicating that Lipozyme RM IM and Candida antarcticis lipase B must be used simultaneously and hydrolyzed under specific conditions to obtain diglyceride oil that significantly reduces LDL-C content. Comparative Examples 4-1 to 4-4 investigated the effect of the mass ratio of lipase Lipase G50 to Rhizopus spp. lipase. The results showed that the two lipases must be within the specific mass ratio range of this invention to exert a synergistic effect and significantly reduce LDL-C content.

[0104] Table 5 Results of High-Density Lipoprotein Cholesterol (HDL-C) Detection

[0105]

[0106] Note: Compared with the model control group, # p < 0.05 ## p < 0.01; compared with Example 3 group, & p < 0.05.

[0107] High-density lipoprotein cholesterol (HDL-C) has a "cholesterol reversal transport" function. Like a "scavenger" team, it collects cholesterol deposited on the walls of blood vessels and transports it back to the liver for metabolism and excretion. High levels of HDL-C help slow the progression of atherosclerotic plaques and may even promote plaque reversal and regression, thereby protecting blood vessels and reducing the risk of cardiovascular and cerebrovascular diseases.

[0108] Compared with the model group, Examples 1-3 showed significantly higher HDL-C levels (p < 0.01), indicating a reduction in the risk of cardiovascular and cerebrovascular diseases to some extent. Furthermore, the HDL-C level in Example 1 was significantly higher than that in Comparative Examples 1-3 (p < 0.05), indicating that Lipozyme RM IM and Candida antarcticis lipase B must be used simultaneously and hydrolyzed under specific conditions to obtain diglyceride oil that significantly increases HDL-C levels. Comparative Examples 4-1-4 investigated the effect of the mass ratio of lipase Lipase G50 to Rhizopus spp. lipase. The results showed that the two lipases must be within the specific mass ratio range of this invention to exert a synergistic effect and significantly increase HDL-C levels.

[0109] In addition, the experimental results of Comparative Examples 5-6 and Example 3 above show that when lipase Lipase G50 and Rhizopus spp. lipase are used simultaneously, the synergistic effect is significant. The prepared diglyceride oil significantly reduces the content of total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in experimental rats, and significantly increases the content of high-density lipoprotein cholesterol (HDL-C).

[0110] The diglyceride oil prepared in Examples 1-3 of this invention was used to reduce the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in experimental rats. The levels of the three indicators were comparable to those of the normal control group (p>0.05), while the level of high-density lipoprotein cholesterol (HDL-C) was significantly better than that of the normal control group (p<0.05), indicating that this invention achieved unexpected technical effects.

[0111] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing diglyceride oil with the effect of improving cardiovascular and cerebrovascular health, characterized in that, Includes the following steps: (1) Mix oil and water, add the first hydrolytic enzyme for the first hydrolysis, and then add the second hydrolytic enzyme for the second hydrolysis to obtain the hydrolysis product; (2) Add the hydrolysis product to glycerol and lipase for reaction, and then distill to obtain diglyceride oil; In step (1), the first hydrolase is Lipozyme RM IM and the second hydrolase is Candida antarcticis lipase B; in step (2), the lipase is a mixture of lipase Lipase G50 and Rhizopus spp. lipase, with a mass ratio of 1:1-3.

2. The preparation method according to claim 1, characterized in that, In step (1), the amount of the first hydrolytic enzyme is 5-10% of the oil mass; and / or the conditions for the first hydrolysis are: temperature 55-65℃, time 2-4h.

3. The preparation method according to claim 1, characterized in that, In step (1), the amount of the second hydrolytic enzyme used accounts for 1-5% of the oil mass; and / or the conditions for the second hydrolysis are: temperature 40-45℃, time 2-4h.

4. The preparation method according to claim 1, characterized in that, The mass ratio of oil to water in step (1) is 1:0.6-1.

2.

5. The preparation method according to claim 1, characterized in that, The oils mentioned in step (1) are selected from at least one of vegetable oils and animal oils.

6. The preparation method according to claim 5, characterized in that, The vegetable oil is selected from at least one of flaxseed oil, soybean oil, rapeseed oil, peanut oil, corn oil, sunflower seed oil, camellia seed oil, coconut oil, palm oil, palm kernel oil, olive oil, olive pomace oil, walnut oil, rice bran oil, rice bran oil, cottonseed oil, perilla seed oil, safflower seed oil, grape seed oil, tea seed oil, peony seed oil, sesame oil, wheat germ oil, maple seed oil, *Xanthoceras sorbifolium* oil, sea buckthorn seed oil, DHA algal oil, medium-chain triglycerides, medium- and long-chain fatty acid edible oils, hemp seed oil, Sichuan pepper seed oil, Sichuan pepper oil, pumpkin seed oil, chili oil, almond oil, and sacha inchi oil; and / or the animal oil is selected from at least one of duck fat, lard, mutton fat, goose fat, and chicken fat.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the hydrolysate to glycerol in step (2) is 1:2-4; and / or the amount of lipase used is 5-15% of the mass of glycerol; and / or the distillation process also includes decolorization and / or deodorization steps.

8. The preparation method according to claim 1, characterized in that, The reaction temperature in step (2) is 40-60℃ and the time is 2-6h; and / or the distillation method is molecular distillation, specifically two-stage distillation, with the temperature of the first stage distillation being 150-170℃ and the vacuum degree being less than 10Pa; and the temperature of the second stage distillation being 200-280℃ and the vacuum degree being less than 2Pa.

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