Diglyceride oil for reducing blood fat, uric acid and blood sugar as well as preparation method and application of diglyceride oil

Abstract

The invention relates to the technical field of functional grease processing, in particular to diglyceride oil capable of reducing blood fat, uric acid and blood sugar as well as a preparation method and application of the diglyceride oil. The preparation method comprises the following steps: mixing the raw material oil and the nutrient solution, inoculating a compound bacteria solution, and carrying out fermentation treatment to obtain a fermentation product; uniformly mixing the fermentation product with glycerol to obtain a mixed material 1, and performing ultrasonic treatment on the mixed material 1 to obtain a mixed material 2; adding a compound enzyme, and reacting to obtain a reactant; heating and centrifuging a reactant, taking an upper-layer oil sample to obtain a crude product, and carrying out molecular distillation, decoloration and deodorization on the crude product to obtain the diglyceride oil for reducing blood fat, uric acid and blood sugar. The diglyceride oil has good effects of reducing blood fat, uric acid and blood sugar, and is high in diglyceride content and lower in preparation cost.

Description

Technical Field

[0001] This invention relates to the field of functional oil processing technology, specifically to a diglyceride oil that lowers blood lipids, uric acid, and blood sugar, as well as its preparation method and applications. Background Technology

[0002] Diacylglycerol (DAG) is a class of products synthesized by the esterification of one molecule of glycerol and two molecules of free fatty acids, or a structural lipid in which one fatty acid in triglyceride (TAG) is replaced by a hydroxyl group. Studies have shown that edible oils rich in DAG, as a novel type of functional oil, possess various physiological activities and functions, such as preventing and treating fatty liver, cardiovascular and cerebrovascular diseases, and preventing obesity.

[0003] Currently, the main methods for producing DAG include chemical transesterification and enzymatic catalysis. Chemical transesterification uses a strong alkaline substance as a catalyst to exchange the fatty acid structure of one triglyceride with that of another triglyceride, resulting in two new triglyceride molecules. However, the yield of diglyceride is low, and side reactions are prone to occur. Enzymatic catalysis utilizes enzymes to catalyze the reaction. Depending on the reactants, it includes esterification, glycerolysis, hydrolysis, and transesterification. Glycerolysis involves the reaction of fats and glycerol under the catalysis of lipases. The acyl group of the fat transfers to the glycerol, first forming monoglycerides and diglycerides, then undergoing acyl transfer to form triglycerides. The triglycerides then undergo transesterification with the fats to form diglycerides. This method has a short reaction time and the products are easy to separate, making it one of the more common methods currently used.

[0004] For example, Chinese patent CN120384106A discloses a method for preparing diglycerides that lower triglycerides, cholesterol, and uric acid, as well as their bio-enzymatic 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 a 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, and the carrier includes loofah sponge, soybean meal, and bentonite in a mass ratio of 1:2-4:3-6. The mass concentration of the lipase is 6%-10%.

[0005] For example, Chinese patent CN120138072A provides a diglyceride oil with hypoglycemic effects and its preparation method. The preparation method includes the following steps: mixing sea buckthorn seed oil, maple seed oil, rapeseed oil, corn oil, and high-oleic peanut oil, then adding glycerol and water to obtain material A; adding *Pseudomonas cepacia* lipase and *Candida antarcticis* lipase B to material A for a first reaction to obtain material B; adding *Candida lipolyticis* lipase and *Candida antarcticis* lipase B to material B for a second reaction to obtain material C; adding *Thermophilus spp.* lipase to material C for a third reaction to obtain material D; and processing material D after the reaction to obtain diglyceride oil. The diglyceride oil obtained by this invention has good hypoglycemic effects. In step (2), the total weight of *Pseudomonas cepacia* lipase and *Candida antarcticis* lipase B is 7% of the total weight of the sea buckthorn seed oil, *Acer truncatum* seed oil, rapeseed oil, corn oil, and high-oleic peanut oil. In step (3), the total weight of *Candida lipolyticis* lipase and *Candida antarcticis* lipase B is 5% of the total weight of the sea buckthorn seed oil, *Acer truncatum* seed oil, rapeseed oil, corn oil, and high-oleic peanut oil. In step (4), the *Thermophilic Mycospora* lipase is 3% of the total weight of the sea buckthorn seed oil, *Acer truncatum* seed oil, rapeseed oil, corn oil, and high-oleic peanut oil.

[0006] Current bio-enzymatic preparation methods generally suffer from the common problem of high enzyme preparation costs. To improve the quality of diglyceride oil, lipase is immobilized, and multiple steps and types of reactions are involved, which further increases the preparation cost and the difficulty of controlling the enzymatic hydrolysis conditions.

[0007] Therefore, a preparation process that yields high-quality diglyceride oil with high content and lower preparation cost is still needed. Summary of the Invention

[0008] This invention addresses the problems existing in the prior art by providing a diglyceride oil that lowers blood lipids, uric acid, and blood sugar, along with its preparation method and applications. This diglyceride oil has good effects in lowering blood lipids, uric acid, and blood sugar, and also has a high diglyceride content and lower preparation cost.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] On the one hand, the present invention provides a method for preparing diglyceride oil that lowers blood lipids, uric acid, and blood sugar, comprising the following steps: (1) Mix the raw oil and nutrient solution, inoculate with compound bacterial solution, and carry out fermentation treatment to obtain fermentation product; the compound bacterial solution is a mixture of Mucor truncatula bacterial solution and Lactobacillus plantarum bacterial solution in a volume ratio of 2-6:1-3. (2) Mix the fermentation product obtained in step (1) with glycerol to obtain mixture 1. Sonicate mixture 1 to obtain mixture 2. (3) Add a compound enzyme to the mixture 2 obtained in step (2), react, and obtain the reactant; the compound enzyme is a mixture of lipase from *Thermophilus spp.* and papain in a mass ratio of 5-10:0.1-2. (4) The reactants obtained in step (3) are heated and centrifuged, and the upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation, decolorization and deodorization to obtain the lipid-lowering, uric acid-lowering and blood sugar-lowering diglyceride oil.

[0011] Preferably, in step (1), the raw material oil is selected from one or more of the following: 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, Chinese tallow tree seed oil, sea buckthorn seed oil, DHA algae oil, hemp seed oil, Sichuan pepper seed oil, Sichuan pepper oil, pumpkin seed oil, chili oil, almond oil, sacha inchi oil, and animal oil.

[0012] Preferably, in step (1), the volume ratio of the raw oil and the nutrient solution is 1-3:1-3, and more preferably 1:1.

[0013] Preferably, in step (1), the nutrient solution includes: yeast powder, sodium dihydrogen phosphate, potassium dihydrogen phosphate and water.

[0014] More preferably, the nutrient solution comprises: 0.1g / L-1g / L yeast powder, 0.5g / L-2g / L sodium dihydrogen phosphate, 0.5g / L-2g / L potassium dihydrogen phosphate, and the remainder water.

[0015] Most preferably, the nutrient solution comprises: 0.5 g / L yeast powder, 1.0 g / L sodium dihydrogen phosphate, 5.0 g / L potassium dihydrogen phosphate, and the remainder water.

[0016] Preferably, in step (1), the inoculation amount of the compound bacterial solution is 1%v / v-10%v / v, and more preferably 5%v / v.

[0017] Preferably, in step (1), the composite bacterial solution is a mixture of Mucor mold and Lactobacillus plantarum in a volume ratio of 4:1.

[0018] Preferably, in step (1), the fermentation conditions are: fermentation at 25℃-40℃ for 40h-100h, and more preferably: fermentation at 30℃ for 72h.

[0019] Preferably, in step (2), the volume ratio of the fermentation product to glycerol is 1:1-5, and more preferably 1:2.

[0020] Preferably, in step (2), the conditions for ultrasonic treatment are: ultrasonic power 800W-1200W, time 10min-40min, and more preferably: ultrasonic power 1000W, time 20min.

[0021] Preferably, in step (3), the mass of the added compound enzyme is 0.1%-1% of the mass of the mixture 2, and more preferably 0.2%.

[0022] Preferably, in step (3), the complex enzyme is a mixture of Myxocytophytic lipase and papain in a mass ratio of 6:1.

[0023] Preferably, in step (3), the reaction conditions are: 35℃-50℃ for 1-3 hours, and more preferably: 40℃ for 2 hours.

[0024] As a best example of the present invention, the following steps are included: (1) Mix the raw oil and nutrient solution at a volume ratio of 1:1, inoculate with 5% v / v compound bacterial solution (mixed with Mucor mold and Lactobacillus plantarum at a volume ratio of 4:1), ferment at 30℃ for 72h, sterilize at 121℃ for 20min after fermentation, and filter through a 0.45μm filter plate to obtain the fermentation product; the nutrient solution is prepared as follows: mix 0.5g / L yeast powder, 1.0g / L sodium dihydrogen phosphate, 5.0g / L potassium dihydrogen phosphate and the remaining water, and sterilize at 121℃ for 20min.

[0025] (2) Mix the fermentation product obtained in step (1) and glycerol in a volume ratio of 1:2 to obtain mixture 1. Perform ultrasonic treatment on mixture 1 with an ultrasonic power of 1000W and a time of 20min to obtain mixture 2. (3) Add 0.2% of the mass of the compound enzyme to the mixture 2 obtained in step (2), and react at a constant temperature of 40℃ for 2 hours to obtain the reactant; the compound enzyme is a mixture of lipase from Thermophilic filamentosa and papain in a mass ratio of 6:1. (4) The reactants obtained in step (3) are heated at 85°C and centrifuged for 3 hours. The upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation at 160°C-230°C and 10 Pa. The heavy phase is collected, decolorized and deodorized to obtain diglyceride oil.

[0026] In another aspect, the present invention provides a diglyceride oil prepared according to the above preparation method.

[0027] In another aspect, the present invention provides the application of the above-mentioned diglyceride oil in the preparation of lipid-lowering products.

[0028] In another aspect, the present invention provides the application of the above-mentioned diglyceride oil in the preparation of uric acid-lowering products.

[0029] Finally, this invention provides the application of the above-mentioned diglyceride oil in the preparation of hypoglycemic products.

[0030] The beneficial effects of this invention are: (1) The present invention uses a fermentation-ultrasound-enzymatic hydrolysis method to prepare diglyceride oil. Through fermentation with specific strains, some triglycerides are degraded. Then, ultrasonic treatment is used to generate acoustic cavitation effect, increase the reaction interface, generate microbubbles, provide attachment points for enzymes, and make subsequent enzymatic hydrolysis more complete. Through the synergistic effect of fermentation-ultrasound-enzymatic hydrolysis, the reaction process is significantly accelerated, the amount of enzyme used is reduced, and the content of diglycerides in crude product is significantly increased.

[0031] (2) The present invention uses lipase and papain of the cottony thermophilic filamentous fungus for synergistic enzymatic hydrolysis, which has higher enzymatic hydrolysis efficiency than single lipase hydrolysis.

[0032] (3) Animal experiments have verified that the diglyceride oil prepared by this invention has good effects in lowering blood lipids, lowering uric acid and lowering blood sugar. Detailed Implementation

[0033] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0035] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. 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 this invention pertains. Unless otherwise defined, percentages and whole contents in this invention refer to mass content, and the raw materials used in this invention can be obtained by conventional preparation methods or through conventional commercial channels.

[0036] In the following embodiments, both *Mucor* sp. and *Lactobacillus plantarum* cultures can be prepared using conventional methods in the art. As an example, the preparation method of this embodiment is as follows: The *Mucor* filamentosa culture was prepared as follows: Activated *Mucor* filamentosa (CGMCC 11578, purchased from the CGMCC website) was inoculated into a culture medium (prepared by sterilizing a mixture of 5 g / L corn steep liquor, 35 g / L dextrin, 1.0 g / L NaNO3, 0.5 g / L KCl, and the remainder water), and cultured in a constant-temperature shaker until the bacterial concentration in the culture was 1.0 × 10⁻⁶. 8 Bacterial count / mL, collect bacterial solution for later use.

[0037] The *Lactobacillus plantarum* bacterial culture was prepared as follows: Activated *Lactobacillus plantarum* (CICC2076, purchased from the CICC website) was inoculated into a culture medium (prepared by sterilizing a mixture of 10% peptone (g / L), yeast extract (5g / L), potassium dihydrogen phosphate (2g / L), KCl (0.5g / L), and the remainder water), and cultured in a constant-temperature shaker until the bacterial concentration in the culture was 1.3 × 10⁻⁶. 8 Bacterial count / mL, collect bacterial solution for later use.

[0038] In the following examples, the lipases from *Thermophilus spp.* and *Rhizopus oryzae* were Novozymes immobilized lipases using Lipozyme TLIM, both with a transesterification activity of 250 IUN / g; papain was purchased from Xiasheng, with an activity of 100,000 u / g; alkaline protease was purchased from Xiasheng, with an activity of 200,000 u / g. Corn oil was purchased from Shandong Yuhuang Grain and Oil Food Co., Ltd.; peanut oil was purchased from Shandong Jinsheng Grain and Oil Food Co., Ltd.; sea buckthorn seed oil was purchased from Heze Zhonghe Jianyuan Biotechnology Co., Ltd.; and Acer truncatum seed oil was purchased from Heze Zhonghe Jianyuan Biotechnology Co., Ltd.

[0039] In the following embodiments, the decolorization and deodorization steps are conventional methods in the art, specifically as follows (not limiting the present invention): (1) Decolorization with activated carbon: The collected heavy phase and activated carbon (addition amount 3%wt) are stirred at 70℃-100℃ for 1 hour, filtered to remove activated carbon and adsorbed impurities; (2) Deodorization with steam distillation: The air in the oil is exhausted, and at 70℃-100℃ and 0.2kPa, the odor substances are removed by steam jet stirring and the difference in volatility is utilized, and the mixture is cooled under vacuum conditions.

[0040] Example 1 A method for preparing a diglyceride oil that lowers blood lipids, uric acid, and blood sugar, comprising the following steps: (1) Mix corn oil and nutrient solution at a volume ratio of 1:1, inoculate with 5% v / v compound bacterial solution (mixed with Mucor mold and Lactobacillus plantarum at a volume ratio of 4:1), ferment at 30℃ for 72h, sterilize at 121℃ for 20min after fermentation, and filter through a 0.45μm filter plate to obtain fermentation product; the nutrient solution is prepared as follows: mix 0.5g / L yeast powder, 1.0g / L sodium dihydrogen phosphate, 5.0g / L potassium dihydrogen phosphate and the remaining water, and sterilize at 121℃ for 20min.

[0041] (2) Mix the fermentation product obtained in step (1) and glycerol in a volume ratio of 1:2 to obtain mixture 1. Perform ultrasonic treatment on mixture 1 with an ultrasonic power of 1200W and a time of 10min to obtain mixture 2. (3) Add 0.2% of the mass of the compound enzyme to the mixture 2 obtained in step (2), and react at a constant temperature of 40℃ for 2 hours to obtain the reactant; the compound enzyme is a mixture of lipase from Thermophilic filamentosa and papain in a mass ratio of 6:1. (4) The reactants obtained in step (3) are heated at 85°C and centrifuged for 3 hours. The upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation at 160°C-230°C and 10Pa vacuum. The heavy phase is collected and decolorized and deodorized to obtain diglyceride oil.

[0042] Example 2 (1) Mix peanut oil and sea buckthorn seed oil (peanut oil and walnut oil volume ratio 2:1) and nutrient solution at a volume ratio of 1:3, inoculate with 10% v / v compound bacterial solution (mixed with Mucor mold and Lactobacillus plantarum bacterial solution at a volume ratio of 2:3), ferment at 30℃ for 72h, sterilize at 121℃ for 20min after fermentation, and filter through a 0.45μm filter plate to obtain fermentation product; the nutrient solution is prepared as follows: mix 0.5g / L yeast powder, 1.0g / L sodium dihydrogen phosphate, 5.0g / L potassium dihydrogen phosphate and the remaining water, and sterilize at 121℃ for 20min.

[0043] (2) Mix the fermentation product obtained in step (1) and glycerol in a volume ratio of 1:1 to obtain mixture 1. Perform ultrasonic treatment on mixture 1 with an ultrasonic power of 1000W and a time of 20min to obtain mixture 2. (3) Add 0.2% of the mass of the compound enzyme to the mixture 2 obtained in step (2), and react at a constant temperature of 40℃ for 2 hours to obtain the reactant; the compound enzyme is a mixture of lipase from Thermophilic filamentosa and papain in a mass ratio of 5:2. (4) The reactants obtained in step (3) are heated at 85°C and centrifuged for 3 hours. The upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation at 160°C-230°C and 10Pa vacuum. The heavy phase is collected and decolorized and deodorized to obtain diglyceride oil.

[0044] Example 3 (1) Mix Acer truncatum seed oil and nutrient solution at a volume ratio of 3:1, inoculate with 1% v / v compound bacterial solution (mixed with Mucor mold and Lactobacillus plantarum at a volume ratio of 6:1), ferment at 40℃ for 48h, sterilize at 121℃ for 20min after fermentation, and filter through a 0.45μm filter plate to obtain fermentation product; the nutrient solution is prepared as follows: mix 0.5g / L yeast powder, 1.0g / L sodium dihydrogen phosphate, 5.0g / L potassium dihydrogen phosphate and the remaining water, and sterilize at 121℃ for 20min.

[0045] (2) The fermentation product obtained in step (1) and glycerol are mixed at a volume ratio of 1:5 to obtain mixture 1. Mixture 1 is subjected to ultrasonic treatment with an ultrasonic power of 800W and a time of 40min to obtain mixture 2. (3) Add 0.2% of the mass of the compound enzyme to the mixture 2 obtained in step (2), and react at a constant temperature of 40℃ for 2 hours to obtain the reactant; the compound enzyme is a mixture of lipase from Thermophilic filamentosa and papain in a mass ratio of 10:0.1. (4) The reactants obtained in step (3) are heated at 85°C and centrifuged for 3 hours. The upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation at 160°C-230°C and 10Pa vacuum. The heavy phase is collected and decolorized and deodorized to obtain diglyceride oil.

[0046] Examples 4-10 Unlike Example 1, the amount of compound enzyme added in step (3) is different, while the other steps are the same. A detailed comparison with Example 1 is as follows: Table 1.

[0047] Comparative Example 1 A method for preparing a diglyceride oil that lowers blood lipids, uric acid, and blood sugar, comprising the following steps: (1) Mix corn oil and glycerin in a volume ratio of 1:2 to obtain mixture 1. Perform ultrasonic treatment on mixture 1 with an ultrasonic power of 1200W and a time of 10min to obtain mixture 2. (2) Add 3% of the mass of the compound enzyme to the mixture 2 obtained in step (2), and react at a constant temperature of 40°C for 2 hours to obtain the reactant; the compound enzyme is a mixture of lipase from Thermophilic filamentosa and papain in a mass ratio of 6:1. (3) The reactants obtained in step (2) are heated at 85°C and centrifuged for 3 hours. The upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation at 160°C-230°C and 10 Pa vacuum. The heavy phase is collected and decolorized and deodorized to obtain diglyceride oil.

[0048] Comparative Example 2 A method for preparing a diglyceride oil that lowers blood lipids, uric acid, and blood sugar, comprising the following steps: (1) Mix corn oil and nutrient solution at a volume ratio of 1:1, inoculate with 5% v / v compound bacterial solution (mixed with Mucor mold and Lactobacillus plantarum at a volume ratio of 4:1), ferment at 30℃ for 72h, sterilize at 121℃ for 20min after fermentation, and filter through a 0.45μm filter plate to obtain fermentation product; the nutrient solution is prepared as follows: mix 0.5g / L yeast powder, 1.0g / L sodium dihydrogen phosphate, 5.0g / L potassium dihydrogen phosphate and the remaining water, and sterilize at 121℃ for 20min.

[0049] (2) Mix the fermentation product obtained in step (1) and glycerol at a volume ratio of 1:2 to obtain mixture 1; (3) Add 0.2% of the mass of the compound enzyme to the mixture 1 obtained in step (2), and react at a constant temperature of 40℃ for 2 hours to obtain the reactant; the compound enzyme is a mixture of lipase from Thermophilic filamentosa and papain in a mass ratio of 6:1. (4) The reactants obtained in step (3) are heated at 85°C and centrifuged for 3 hours. The upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation at 160°C-230°C and 10Pa vacuum. The heavy phase is collected and decolorized and deodorized to obtain diglyceride oil.

[0050] Comparative Example 3 Unlike Example 1, in step (1), the bacterial solution used is a single Mucor mold culture. All other aspects are the same.

[0051] Comparative Example 4 Unlike Example 1, in step (1), the composite bacterial solution is a mixture of *Mucor* sp. and *Lactobacillus plantarum* bacterial solutions at a volume ratio of 1:5. All other steps are the same.

[0052] Comparative Example 5 Unlike Example 1, in step (1), the bacterial solution used is a single Lactobacillus plantarum bacterial solution. All other aspects are the same.

[0053] Comparative Example 6 Unlike Example 1, in step (3), the enzyme used is a single *Thermophilic lipase*. All other steps are the same.

[0054] Comparative Example 7 Unlike Example 1, in step (3), the enzyme used is a mixture of Rhizopus miltiorrhiza lipase and alkaline protease in a mass ratio of 6:1. All other steps are the same.

[0055] Comparative Example 8 Unlike Example 1, in step (3), the complex enzyme is a mixture of Myxocytophytic lipase and papain in a mass ratio of 2:1. All other aspects are the same.

[0056] Result detection 1. Diacylglycerol content detection The diglyceride content in the crude products obtained in the examples and comparative examples was determined according to GB / T 26636.

[0057] The test results are shown in Table 2: Table 2

[0058] The results show that the combined fermentation-ultrasound-enzymatic hydrolysis process used in this invention to prepare diglyceride oil can selectively obtain diglycerides and increase the diglyceride content in the crude product.

[0059] The results of Examples 4-10 show that in the preparation method of the present invention, as the amount of enzyme increases, the content of diglycerides shows a trend of first increasing and then decreasing. This indicates that keeping the amount of enzyme within a reasonable range is more conducive to selectively obtaining diglycerides, and the low amount of compound enzyme also significantly reduces the preparation cost.

[0060] The results of Comparative Examples 1 and 2 indicate that fermentation and ultrasonic treatment are key steps in obtaining high-content diglyceride products, which can greatly promote enzymatic hydrolysis efficiency. The results of Comparative Examples 3-5 show that specific microbial complexes are a key means to increase diglyceride content, and the results of Comparative Examples 6-8 show that the selection of specific enzyme complexes is also a key means to increase diglyceride content with less enzyme dosage.

[0061] 2. Blood sugar lowering experiment Diglyceride oils prepared using the three processes described in Example 1, Comparative Example 1, and Comparative Example 2 were used to conduct a hypoglycemic experiment.

[0062] The zebrafish were provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use license number is: SYXK (Zhe) 2022-0004, and the feeding management meets the requirements of international AAALAC accreditation (accreditation number: 001458). The zebrafish were raised in fish culture water at 28°C, with 200 mg of instant sea salt added to every 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm, the pH was 6.5-8.5, and the hardness was 50-100 mg / L CaCO3.

[0063] The diglyceride oils prepared by the three processes of Example 1, Comparative Example 1, and Comparative Example 2 were respectively made into stock solutions with a concentration of 200 mg / mL using DMSO and stored at -20°C for later use.

[0064] 2.1 Determination of the maximum detection concentration (MTC) Wild-type AB strain zebrafish at 5 days post-fertilization (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish in each beaker (experimental group). The samples were administered by water dissolution, and a normal control group and a model control group were set up simultaneously. The volume of each beaker was 25 mL. Except for the normal control group, the other experimental groups were given a high-sugar and high-fat diet by water dissolution to establish a hyperglycemic model in zebrafish. After treatment at 28°C for 2 days, the MTC of different diglyceride oil samples on the model zebrafish was measured.

[0065] Under the conditions of this experiment, the MTC of the hypoglycemic efficacy of each group was 2000 μg / mL. See Table 3 for details.

[0066] Table 3.

[0067] 2.2 Verification of hypoglycemic efficacy 30 wild-type AB strain zebrafish at 5 dpf were randomly selected in each group and placed in beakers. The samples were administered by water dissolution. The experimental groups were given the samples prepared in Example 1, Comparative Example 1, and Comparative Example 2 (concentration 1000 μg / mL) by water dissolution respectively, and the positive control group was given metformin (concentration 400 μg / mL) by water dissolution. At the same time, a normal control group and a model control group were set up, and the volume of each beaker was 25 mL. Except for the normal control group, the other experimental groups were given a high-sugar and high-fat diet by water dissolution to establish a hyperglycemic model in zebrafish. After treatment at 28°C for 2 days, data were collected using a blood glucose meter, and the glucose levels of zebrafish were analyzed and statistically analyzed. The hypoglycemic efficacy of the samples was evaluated based on the statistical analysis results of this index. The statistical processing results were expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0068] The results are shown in Table 4: Table 4

[0069] Note: Compared with the normal control group, ##P<0.01; compared with the model control group, positive control group, Example 1 group, comparative example 1 group, and comparative example 2 group, &P<0.05, &&P<0.01; compared with Example 1 group, comparative example 1 group and comparative example 2 group, P < 0.05.

[0070] The results showed that the diglyceride oils prepared in Example 1, Comparative Example 1, and Comparative Example 2 all had hypoglycemic effects, and the hypoglycemic effect of the diglyceride oil prepared in Example 1 was significantly improved compared with that of Comparative Example 1 and Comparative Example 2.

[0071] 3. Uric acid-lowering test Male SD rats, weighing 180-200g, were acclimatized for one week and randomly divided into four groups: normal control group, model control group, Example 1 group, Comparative Example 1 group, and Comparative Example 2 group, with 10 rats in each group. The rearing environment was kept well-ventilated, with a temperature of 22±1℃ and humidity maintained at 55%-65%. The light and dark cycles were repeated every 12 hours, and rats had free access to water and food during the experiment.

[0072] After the experiment began, the normal control group was fed a basal diet, while the other groups were fed D12451 yeast feed, with each animal consuming 15 g of feed per day. Simultaneously, except for the normal control group, the other groups received intraperitoneal injections of 450 mg / kg / day of potassium oxonate for 8 weeks. After 8 weeks, the animals in Example 1, Comparative Example 1, and Comparative Example 2 were switched to a diglyceride oil diet, with all other feeding conditions remaining unchanged, for a total of 6 weeks.

[0073] Basic feed: by weight, 25 parts wheat flour, 25 parts oatmeal, 25 parts cornmeal, 10 parts soybean meal, 8 parts fish meal, 4 parts bone meal, and 1 part refined salt.

[0074] D12451 Yeast Feed: By weight, it contains 29 parts lard, 2.8 parts soybean oil, 16.7 parts sucrose, 19.6 parts casein, 5.6 parts minerals, 52.2 parts basic feed, and 11.28 parts yeast powder.

[0075] Diglyceride oil feed: by weight, 30 parts diglyceride oil, 16.7 parts sucrose, 19.6 parts casein, 5.6 parts minerals, 52.2 parts basic feed and 11.28 parts yeast powder.

[0076] After the experiment, the animals were anesthetized, blood was collected from the abdominal aorta, and serum uric acid (UA) was measured. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0077] Table 5

[0078] Note: When comparing the model control group with the normal control group, ##P < 0.01; when comparing the positive control group, the group of Example 1, the group of Comparative Example 1, and the group of Comparative Example 2 with the model control group, &&P < 0.01; when comparing the group of Comparative Example 1 and the group of Comparative Example 2 with the group of Example 1, P < 0.01.

[0079] The results show that the diglyceride oils prepared in Example 1, Comparative Example 1, and Comparative Example 2 all have the effect of reducing uric acid, and the effect of reducing uric acid of the diglyceride oil prepared in Example 1 is significantly improved compared with Comparative Example 1 and Comparative Example 2.

[0080] 4. Hypolipidemic experiment The diglyceride oils prepared by the three processes of Example 1, Comparative Example 1, and Comparative Example 2 were selected for hypolipidemic experiments.

[0081] The zebrafish were provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The license number for the use of experimental animals was: SYXK (Zhe) 2022 - 0004, and the feeding management met the requirements of international AAALAC certification (certification number: 001458). The zebrafish were raised in fish-raising water at 28°C. 200 mg of instant sea salt was added to each liter of reverse osmosis water, with a conductivity of 450 - 550 μS / cm, a pH of 6.5 - 8.5, and a hardness of 50 - 100 mg / L CaCO3.

[0082] The diglyceride oils prepared by the three processes of Example 1, Comparative Example 1, and Comparative Example 2 were respectively formulated into stock solutions with a concentration of 200 mg / mL using DMSO and stored at -20°C for later use.

[0083] Thirty wild-type AB strain zebrafish with a 3-day pf (dpf) count were randomly selected from each group and placed in beakers. Except for the normal control group, all experimental groups were fed a high-sugar, high-fat diet dissolved in water to establish a zebrafish hyperlipidemia model. After treatment at 28℃ for 2 days, except for the model control group, the high-sugar, high-fat diet was removed from all other groups, and samples were dissolved in water and administered to each group. The experimental groups were given samples prepared in Example 1, Comparative Example 1, and Comparative Example 2 (concentration 1000 μg / mL), respectively. The positive control group was given fenofibrate dissolved in water (concentration 1.5 mg / L). A normal control group and a model control group were also set up. The model control group continued to be fed a high-sugar, high-fat diet until 8 dpf. Each beaker contained 25 mL. The liquid was changed twice daily, morning and evening. After treatment at 28℃ for 3 days, the zebrafish were stained with Oil Red 0 for overall fat staining. Following destaining and bleaching, the zebrafish were photographed under a dissecting microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The staining intensity of the zebrafish tail vessels was analyzed, and the statistical analysis results of this index were used to evaluate the effect of the samples on zebrafish blood lipids. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0084] Table 6

[0085] Note: Compared with the normal control group, ##P<0.01; compared with the model control group, the positive control group, Example 1 group, Comparative Example 1 group, and Comparative Example 2 group, &&P<0.01; compared with Example 1 group, Comparative Example 1 group and Comparative Example 2 group, P < 0.05 P < 0.01.

[0086] The results showed that the diglyceride oils prepared in Example 1, Comparative Example 1, and Comparative Example 2 all had lipid-lowering effects, and the lipid-lowering effect of the diglyceride oil prepared in Example 1 was significantly improved compared with that of Comparative Example 1 and Comparative Example 2.

[0087] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a diglyceride oil that lowers blood lipids, uric acid, and blood sugar, characterized in that, Includes the following steps: (1) Mix the raw oil and nutrient solution, inoculate with compound bacterial solution, and carry out fermentation treatment to obtain fermentation product; the compound bacterial solution is a mixture of Mucor truncatula bacterial solution and Lactobacillus plantarum bacterial solution in a volume ratio of 2-6:1-3. (2) Mix the fermentation product obtained in step (1) with glycerol to obtain mixture 1. Sonicate mixture 1 to obtain mixture 2. (3) Add a compound enzyme to the mixture 2 obtained in step (2), react, and obtain the reactant; the compound enzyme is a mixture of lipase from *Thermophilus spp.* and papain in a mass ratio of 5-10:0.1-2. (4) The reactants obtained in step (3) are heated and centrifuged, and the upper oil sample is taken to obtain the crude product. The crude product is subjected to molecular distillation, decolorization and deodorization to obtain the lipid-lowering, uric acid-lowering and blood sugar-lowering diglyceride oil.

2. The preparation method according to claim 1, characterized in that, In step (1), the raw material oil is selected from one or more of the following: 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, Chinese tallow tree seed oil, sea buckthorn seed oil, DHA algae oil, hemp seed oil, Sichuan pepper seed oil, Sichuan pepper oil, pumpkin seed oil, chili oil, almond oil, sacha inchi oil, and animal oil.

3. The preparation method according to claim 1, characterized in that, In step (1), the volume ratio of the raw oil and the nutrient solution is 1-3:1-3, the inoculation amount of the compound bacterial solution is 1%v / v-10%v / v, and the fermentation conditions are: 25℃-40℃ fermentation for 40h-100h.

4. The preparation method according to claim 1, characterized in that, In step (2), the volume ratio of the fermentation product to glycerol is 1:1-5.

5. The preparation method according to claim 1, characterized in that, In step (2), the conditions for ultrasonic treatment are: ultrasonic power 800W-1200W, time 10min-40min.

6. The preparation method according to claim 1, characterized in that, In step (3), the mass of the added compound enzyme is 0.1%-1% of the mass of the mixture 2, and the reaction conditions are: 35℃-50℃ for 1-3 hours.

7. The diglyceride oil prepared by the method according to any one of claims 1-6.

8. The use of the diglyceride oil of claim 7 in the preparation of lipid-lowering products.

9. The use of the diglyceride oil of claim 8 in the preparation of uric acid-lowering products.

10. The use of the diglyceride oil of claim 9 in the preparation of hypoglycemic products.

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