Method for the preparation of a penta-upgraded blood vessel health improving diglyceride oil by enzymatic hydrolysis, uses and products

Abstract

The application provides a method, application and product for preparing a triglyceride oil with five effects of improving blood vessel health by enzymolysis, and belongs to the technical field of enzyme engineering. The preparation method comprises the following steps: mixing low erucic acid rapeseed oil, glycerol, water, fatty acid and lipase, performing enzymolysis reaction, performing molecular distillation, and performing decolorization and deodorization to obtain the triglyceride oil; the lipase is a mixture of Candida antarctica black Aspergillus niger lipase, Candida cylindracea lipase and bovine pancreas lipase; and the weight ratio of the low erucic acid rapeseed oil, glycerol, water, fatty acid and lipase is 500:80-100:1-2:30-60:10-20. The triglyceride oil prepared by the method can be used to prepare a triglyceride oil composition, and has the five effects of lowering blood uric acid, lowering blood total cholesterol, lowering blood triglyceride, lowering blood sugar and improving blood flow speed, thereby improving blood vessel health.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a method, application and product for preparing diglyceride oil with five functions of improving vascular health through enzymatic hydrolysis. Background Technology

[0002] Diglycerides are a class of fatty acids in which one acyl group of a triglyceride is replaced by a hydrogen atom. The structural formula of diglycerides is:

[0003] or .

[0004] R1 and R2 are the same or different saturated or unsaturated aliphatic hydrocarbon groups.

[0005] Triglycerides are important energy storage molecules in the human body and are essential components of adipose tissue. The main pathways for triglyceride synthesis in the human body include the monoglyceride pathway and the phosphatidic acid pathway. In the monoglyceride pathway, 2-monoacylglycerol is converted to diacylglycerol (i.e., diglyceride) by acyltransferase, and then diglyceride is converted to triglycerides by acyltransferase. Therefore, diglycerides are involved in human fat metabolism, and they play a role in regulating lipid metabolism. Furthermore, diglycerides are also a novel food ingredient.

[0006] Existing technologies for producing diglycerides mainly fall into two categories: chemical methods and enzymatic hydrolysis. The principle of chemical diglyceride synthesis involves using a catalyst to accelerate the esterification reaction of fatty acids with glycerol or monoglycerides, or utilizing the transesterification reaction of oils. However, chemical diglyceride synthesis is limited by low reaction specificity, solvent consumption, and potential side reactions that could harm food safety. Enzymatic hydrolysis produces diglycerides under mild conditions and with high selectivity, making it suitable for producing food-grade diglycerides. However, existing enzymatic hydrolysis methods have limited enzyme production efficiency, and the types of substances that can be used to increase the hydrolysis rate in food production are also limited. Furthermore, the post-hydrolysis process is relatively cumbersome and may lead to the oxidation of carbon-carbon double bonds in unsaturated fatty acids, resulting in a decrease in iodine value. Therefore, there is an urgent need for a production process that rationally combines existing enzymes and auxiliaries in appropriate proportions to improve the yield, purity, and iodine value of diglycerides. Summary of the Invention

[0007] In view of the above-mentioned problems in the prior art, the purpose of this invention is to provide a method, application, and product for preparing diglyceride oil with five benefits for improving vascular health through enzymatic hydrolysis. This invention utilizes a mixture of three enzymes—Candida antarcticis lipase, Candida columnaris lipase, and bovine pancreatic lipase—as the medium for the reaction. Simultaneously, it optimizes the proportions of low-erucic acid rapeseed oil, glycerol, water, fatty acids, and lipase, successfully improving the yield, purity, and iodine value of the enzymatic hydrolysis reaction for preparing low-erucic acid rapeseed diglycerides from low-erucic acid rapeseed oil. This reduces overall production costs while improving product quality.

[0008] To achieve the above-mentioned objectives, the present invention provides a method for preparing diglyceride oil with five benefits for improving vascular health through enzymatic hydrolysis, comprising the following steps:

[0009] Low-erucic acid rapeseed oil, glycerol, water, fatty acids and lipase are mixed, enzymatically hydrolyzed, molecularly distilled, decolorized and deodorized to obtain diglyceride oil;

[0010] The erucic acid content in the low-erucic acid rapeseed oil is less than or equal to 3%.

[0011] The lipase is a mixture of Candida antarcticis lipase, Candida columnaris lipase, and bovine pancreatic lipase, and the weight ratio of the low-erucic acid rapeseed oil, glycerol, water, fatty acids, and lipase is 500:80-100:1-2:30-60:10-20.

[0012] Preferably, the fatty acid is selected from at least one of linoleic acid, α-linolenic acid, oleic acid and eicosapentaenoic acid, more preferably a mixture of linoleic acid and α-linolenic acid.

[0013] As an example of the present invention, the fatty acid is selected from a mixture of linoleic acid and α-linolenic acid in a weight ratio of 1:1.

[0014] Preferably, the lipase is a mixture of Candida antarcticis lipase, Candida columnaris lipase, and bovine pancreatic lipase in a weight ratio of 7:1-2:1-2.

[0015] More preferably, and as an example of the present invention, the lipase is a mixture of Candida antarcticis lipase, Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 7:1.5:1.5.

[0016] Preferably, the weight ratio of the low-erucic acid rapeseed oil, glycerol, water, fatty acids and lipase is 500:90:1.5:45:15.

[0017] Preferably, the temperature of the enzymatic hydrolysis reaction is 50-65℃, and the hydrolysis time is 1-9h.

[0018] As a specific example of the present invention, the temperature of the enzymatic hydrolysis reaction is 60°C and the time of the enzymatic hydrolysis reaction is 8 hours.

[0019] Preferably, the molecular distillation includes a first-stage molecular distillation, a second-stage molecular distillation, and a third-stage molecular distillation;

[0020] The temperature of the first-stage molecular distillation is 100-160℃, and the vacuum degree is less than 500Pa;

[0021] The temperature of the second-stage molecular distillation is 170-200℃, and the vacuum degree is less than 10Pa;

[0022] The temperature of the third-stage molecular distillation is 220-280℃, and the vacuum degree is less than 10Pa.

[0023] The condensation surface temperature of the molecular distillation is 35-55℃, more preferably 40℃; the molecular distillation is repeated 3-5 times, more preferably 4 times.

[0024] On the other hand, the present invention provides a diglyceride oil obtained by the above method.

[0025] Furthermore, the present invention provides the application of the diglyceride oil obtained by the above method in the production of oil and fat products.

[0026] The oil products include, but are not limited to, liquid edible oils, oil microcapsule powders, and gels.

[0027] Preferably, the oil product is a liquid edible oil.

[0028] Finally, the present invention provides a diglyceride oil composition comprising low-erucic acid rapeseed diglyceride oil, corn diglyceride oil, edible vegetable oil, and food additives.

[0029] The low-erucic acid rapeseed diglyceride oil is the diglyceride oil obtained by the above method.

[0030] The term "edible vegetable oil" refers to edible oils made from edible vegetable oilseeds or crude vegetable oils. This includes, but is not limited to, soybean oil, sunflower seed oil, flaxseed oil, sesame oil, grapeseed oil, pumpkin seed oil, peanut oil, walnut oil, almond oil, hazelnut oil, macadamia nut oil, olive oil, coconut oil, palm oil, avocado oil, rice bran oil, corn oil, wheat germ oil, tea oil, peony seed oil, perilla seed oil, hemp seed oil, sea buckthorn fruit oil, tiger nut oil, and *Vernicia fordii* fruit oil, etc.

[0031] Preferably, the edible vegetable oil is selected from one or more of soybean oil, sunflower seed oil, flaxseed oil, grape seed oil, peanut oil, olive oil, corn oil, wheat germ oil, tea oil, peony seed oil, tiger nut oil, and *Sapindus mukorossi* oil.

[0032] More preferably, the edible vegetable oil is selected from peanut oil and / or corn oil.

[0033] More preferably, and as a specific example of the present invention, the edible vegetable oil is high-oleic peanut oil.

[0034] More preferably, the oleic acid content in the high-oleic peanut oil is >75%.

[0035] The term "food additive" refers to substances applied to food that can improve or help improve the sensory requirements, physicochemical properties, or nutritional characteristics of the food. These food additives include, but are not limited to, additives that can be added to edible oil products as specified in GB2760-2024, the National Standard of the People's Republic of China for the Use of Food Additives.

[0036] The food additives include antioxidants, emulsifiers, defoamers, anti-caking agents, acidity regulators, color protectants, metal ion chelating agents, natural extracts, etc.

[0037] Preferably, the food additive is selected from one or more of the following:

[0038] Tert-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate, vitamin E, rosemary extract, tea polyphenols, licorice antioxidants, soybean lecithin, sunflower lecithin, polyglycerol fatty acid esters, citric acid, sodium citrate, curcumin, beta-carotene.

[0039] More preferably, the food additive is vitamin E.

[0040] Further, the diglyceride oil composition comprises:

[0041] Low-erucic acid rapeseed diglyceride oil, corn diglyceride oil, high-oleic peanut oil, and vitamin E.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention provides a technique for the enzymatic hydrolysis production of diglycerides using a specific enzyme combination and reactant ratio, effectively improving the mass percentage content, yield, and iodine value of the produced diglycerides. Specifically, this is reflected in the following aspects:

[0044] (1) Three enzymes, namely Candida antarcticis lipase, Candida columnaris lipase and bovine pancreatic lipase, were combined to increase the mass percentage content and yield of diglycerides through the synergistic effect of the three enzymes.

[0045] (2) This invention establishes a suitable microenvironment for the enzymatic hydrolysis of diglycerides by adjusting the ratio of low erucic acid rapeseed oil, glycerol, water, fatty acids and lipase, which is conducive to maximizing the enzymatic hydrolysis effect after the three enzymes are combined.

[0046] (3) The inventors unexpectedly discovered that during the enzymatic production of diglycerides using the above-mentioned enzyme preparations and enzymatic reaction raw materials, the entire enzyme reaction system (the specific types and weight ratios of the vegetable oil, glycerol, fatty acids, water and lipase used) has a certain selective effect on different types of fatty acids in the fatty acid exchange reaction, which is reflected in the increase of the iodine value of the final diglycerides. Attached Figure Description

[0047] Figure 1 This is a bar chart showing the results of serum uric acid testing in mice on day 42 of the experiment in Example 2.

[0048] Figure 2 This is a bar chart showing the difference in serum uric acid levels between mice on day 42 and day 0 of the experiment in Example 2.

[0049] Figure 3 This is a bar chart showing the results of total cholesterol testing in mice on day 42 of the experiment in Example 2.

[0050] Figure 4 This is a bar chart showing the difference in total cholesterol levels in mice on day 42 of the experiment compared to day 0 of the experiment in Example 2.

[0051] Figure 5 This is a bar chart showing the results of blood triglyceride testing in mice on day 42 of the experiment in Example 2.

[0052] Figure 6 This is a bar chart showing the difference in blood triglyceride levels between mice on day 42 and day 0 of the experiment in Example 2.

[0053] Figure 7 This is a bar chart showing the blood glucose test results of zebrafish in Example 3. Detailed Implementation

[0054] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0055] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels, and different brands of the same chemical reagent have no significant impact on the technical solution and technical effect of the present invention. Unless otherwise specified, the contents mentioned below are mass contents. Unless otherwise specified, it is understood that the experiment was conducted at room temperature.

[0056] The sources of some reagents in the following examples and comparative examples are shown in Table 1:

[0057] Table 1

[0058]

[0059] In the following examples and comparative examples, the bovine pancreatic lipase used is based on the prior art, "Bovine pancreaticlipase. I. Isolation, homogeneity, and characterization" (KM Shahani et al., Journal of Dairy Science, 1976, 59(3): 369-375). The specific steps are as follows:

[0060] (1) Place fresh bovine pancreas in a 0℃, 0.01mol / L sucrose solution, remove fat and non-glandular tissues, cut into small pieces, and quick-freeze at -20℃ for later use.

[0061] (2) Thaw 500g of the bovine pancreas pieces prepared in (1), mix with 1000mL of 0.01mol / L sucrose aqueous solution at 0-5℃, and stir for 90s. Filter using 5 layers of gauze to obtain 1400mL of clear and homogeneous liquid. Centrifuge (15000g, 30min, -4℃). The liquid separates into 3 layers: the upper layer is fat, the lower layer is precipitate, and the middle layer is clear liquid. Use a dropper to remove the middle layer liquid, combine them, and the total is 1200mL. Add 0.22g of diisopropyl fluorophosphate to inactivate the protease to protect the esterase. Freeze-dry to obtain 24g of dry powder, and freeze at -20℃ for later use.

[0062] (3) Take 5g of the dry powder prepared in (2) and mix it with 75mL of 0.01mol / L cold sucrose solution. Stir magnetically at 4℃ (200rpm, 15min). Centrifuge the mixture at 4℃ (2000g, 15min) and collect the supernatant in a brown bottle. Resuspend the precipitate in 75mL of 0.01mol / L cold sucrose solution and repeat the above stirring and centrifugation steps. Combine the two supernatants to obtain crude bovine pancreatic lipase extract.

[0063] (4) Place the crude bovine pancreatic lipase extract obtained in (3) (approximately 150 mL) in a container, immerse the container in an acetone-dry ice bath, and cool it to -10°C. While stirring, add cold acetone to the crude extract (a total of 16.6 mL of cold acetone is added). After stirring for 25 min, centrifuge at -4°C (10000 g, 15 min). Collect the supernatant and pour it into a beaker, place it in an acetone-dry ice bath again, and discard the precipitate. Add cold acetone again to increase the acetone concentration in the supernatant to 45%. At this point, the precipitate contains enriched lipase and some impurities. Collect the precipitate and dissolve it in 20 mL of 0.01 mol / L sucrose solution. Adjust the pH to 8.5 using 1 mol / L sodium hydroxide aqueous solution for the next step of column chromatography separation.

[0064] (5) Column chromatography separation.

[0065] Separation was performed using Sephadex G-100 dextran gel. As an example of the present invention, the gel column used was 2 cm in diameter, 32 cm in length, and had an empty column volume of 35 mL. During separation, 5 mL of the crude enzyme solution obtained in (4) was loaded onto the column, eluted with 0.01 mol / L sucrose solution at a flow rate of 12 mL / h, the fraction was collected, and lyophilized to obtain bovine pancreatic lipase.

[0066] Example 1

[0067] A method for preparing diglyceride oil.

[0068] (1) Mix 500 parts by weight of vegetable oil, 90 parts by weight of glycerol, 1.5 parts by weight of water, 22.5 parts by weight of linoleic acid, 22.5 parts by weight of α-linolenic acid and 15 parts by weight of lipase thoroughly. The lipase is a mixture of Candida antarcticis lipase, Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 7:1.5:1.5.

[0069] (2) The mixture obtained in step (1) was enzymatically hydrolyzed at 60°C for 8 hours. After enzymatic hydrolysis, the mixture was allowed to stand, and the supernatant was retained for molecular distillation. The molecular distillation process included first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation. The temperature of the first-stage molecular distillation was 100-160°C, and the vacuum degree was less than 500 Pa; the temperature of the second-stage molecular distillation was 170-200°C, and the vacuum degree was less than 10 Pa; the temperature of the third-stage molecular distillation was 220-280°C, and the vacuum degree was less than 10 Pa; the condensation surface temperature of the molecular distillation was 50°C. The molecular distillation was repeated 4 times. The fractions at 170-200°C were collected and combined, and cooled to room temperature. 1% by weight of activated carbon was added, and the mixture was stirred for 0.5 h for decolorization and deodorization. The activated carbon was removed by filtration using a filter cartridge to obtain low-erucic acid rapeseed diester oil.

[0070] Example 2

[0071] Compared with Example 1, the difference is that the lipase used in step (1) is a mixture of Candida antarcticis lipase, Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 7:2:1, while the rest are the same.

[0072] Example 3

[0073] Compared with Example 1, the difference is that the lipase used in step (1) is a mixture of Candida antarcticis lipase, Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 7:1:2, while all other aspects are the same.

[0074] Example 4

[0075] Compared with Example 1, the difference is that in step (1), glycerol is replaced with 80 parts by weight, water with 2 parts by weight, linoleic acid with 15 parts by weight, α-linolenic acid with 15 parts by weight, and lipase with 20 parts by weight, while the rest are the same.

[0076] Example 5

[0077] Compared with Example 1, the difference is that in step (1), glycerol is replaced with 100 parts by weight, water is replaced with 1 part by weight, linoleic acid is replaced with 30 parts by weight, α-linolenic acid is replaced with 30 parts by weight, and lipase is replaced with 10 parts by weight, while the rest are the same.

[0078] Comparative Example 1

[0079] Compared with Example 1, the difference is that the lipase used in step (1) is a mixture of Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 8:2, and all other aspects are the same.

[0080] Comparative Example 2

[0081] Compared with Example 1, the difference is that the lipase used in step (1) is a mixture of Candida antarcticis lipase and Candida columnaris lipase in a weight ratio of 7:3, and all other aspects are the same.

[0082] Comparative Example 3

[0083] Compared with Example 1, the difference is that the lipase used in step (1) is a mixture of Candida antarcticis lipase, Candida columnaris lipase and Novozym 435 in a weight ratio of 7:2:1, while the rest are the same.

[0084] Comparative Example 4

[0085] Compared with Example 1, the difference is that in step (1), 1.5 parts by weight of water is not added and 91.5 parts by weight of glycerin is added instead, while the rest are the same.

[0086] Comparative Example 5

[0087] Compared with Example 1, the difference is that in step (1), glycerol is replaced with 130 parts by weight, α-linolenic acid is replaced with 35 parts by weight, linoleic acid is not used, and lipase is replaced with 5 parts by weight. All other steps are the same.

[0088] In Examples 1-5 and Comparative Examples 1-5, when the vegetable oil in step S1 is a low-erucic acid rapeseed oil with an erucic acid weight content ≤3%, the prepared diglyceride oil is a low-erucic acid rapeseed diglyceride oil; when the vegetable oil in step S1 is corn oil, the prepared diglyceride oil is corn diglyceride oil.

[0089] The yields of low-erucic acid rapeseed diglyceride oil and corn diglyceride oil prepared using the above method were calculated. The calculation formula is as follows:

[0090] Diglyceride oil yield = Diglyceride oil production × Diglyceride mass percentage in diglyceride oil ÷ Vegetable oil input amount × 100%.

[0091] The weight content of diglycerides in diglyceride oil was determined using the existing technology (Wang Yong et al., Study on determination of diglyceride content by reversed-phase high performance liquid chromatography, Journal of Chinese Cereals and Oils, 3(2010):5).

[0092] The iodine value of diglyceride oil was determined using the method described in standard GB / T5532-2022.

[0093] The weight content, yield, and iodine value of the low erucic acid rapeseed diglyceride oil and corn diglyceride oil prepared in Examples 1-5 and Comparative Examples 1-5 were determined. The results are shown in Tables 2 and 3.

[0094] Table 2:

[0095]

[0096] Table 3:

[0097]

[0098] It can be seen that the preparation methods provided in Examples 1-5 can prepare rapeseed diglyceride oil and corn diglyceride oil with higher diglyceride content and higher iodine value.

[0099] Examples 6-9 provide formulations for diglyceride oil compositions, as shown in Table 4.

[0100] Table 4:

[0101]

[0102] Among them, in the diglyceride oil compositions provided in Examples 6 to 9, the preparation methods of the low-erucic acid rapeseed diglyceride oil and the corn diglyceride oil are as follows:

[0103] In Example 1, when the vegetable oil in step S1 is corn oil, the obtained diglyceride oil is the corn diglyceride oil in Examples 6 to 9.

[0104] In Example 1, when the vegetable oil in step S1 is rapeseed oil with an erucic acid content less than or equal to 3%, the diglyceride oil obtained in Example 1 is the low-erucic acid rapeseed diglyceride oil in Examples 6 to 9.

[0105] The preparation method of the diglyceride oil composition provided in Examples 6 to 9 is: mix each component according to the formula amount.

[0106] Effect Example 1

[0107] Effect of the diglyceride oil composition on the blood flow velocity in the thrombus model zebrafish.

[0108] 1. Experimental conditions

[0109] 1.1. Experimental animals

[0110] The zebrafish are all raised in fish-raising water at 28°C (water quality: add 200 mg of instant sea salt to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; hardness is 50 - 100 mg / L in terms of CaCO3), provided by the fish-raising center of the Innovation Experiment Center of Huante Biotech, and the license number for the use of experimental animals is: SYXK(Zhe)2022 - 0004, and the feeding management meets the requirements of international AAALAC certification (certification number: 001458). IACUC ethical review number: IACUC - 2023 - 8002 - 01.

[0111] 1.2. Samples, instruments and consumables

[0112] The sample detected in this effect example is the diglyceride oil composition provided in Example 6.

[0113] Dissecting microscope (SZX7, OLYMPUS); precision electronic balance (CP214, OHAUS); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd.); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd.); cardiac blood flow analysis system (Zebra Blood 3.4, ViewPoint Life Sciences). Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma); methylcellulose (batch number C2004046, Shanghai Aladdin Biotechnology Co., Ltd.); isoproterenol hydrochloride (batch number VNVFK-QF, TCI (Shanghai) Chemical Industry Development Co., Ltd.).

[0114] 2. Evaluation Methods

[0115] 2.1 Determination of Maximum Detectable Concentration (MTC)

[0116] Zebrafish of the Albino strain, a melanin allele mutant, were randomly selected 3 days post-fertilization (3 dpf) and placed in 6-well plates, with 30 zebrafish treated in each well. Different concentrations of the diglyceride oil provided in Example 6 were administered, along with a normal control group and a model control group. Each well contained 3 mL of the oil. Except for the normal control group, all other experimental groups were given isoproterenol hydrochloride in water to establish a zebrafish model of qi stagnation, blood stasis, and thrombosis. After treatment at 28°C for 2 days, the MTC of the samples in the model zebrafish was measured.

[0117] 2.2 Evaluation of efficacy against qi stagnation and blood stasis type thrombosis

[0118] Zebrafish of the Albino strain (3dpf melanin allele mutant) were randomly selected and treated in 6-well plates, with 30 zebrafish per well. A certain concentration of sample was administered (Example 6 group received 2000 μg / mL of the diglyceride oil composition provided in Example 6, peanut oil group received 2000 μg / mL of peanut oil, and the model control group received an equal volume of zebrafish culture medium). A normal control group was also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups received isoproterenol hydrochloride in water to establish a zebrafish qi stagnation and blood stasis thrombosis model. After treatment at 28℃ for 2 days, 10 zebrafish from each experimental group were randomly selected and placed in a cardiac blood flow analysis system to record zebrafish blood flow videos. The zebrafish blood flow velocity was analyzed and statistically analyzed to evaluate the anti-qi stagnation and blood stasis thrombosis efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; P < 0.05 indicated statistical significance.

[0119] 3. Test Results

[0120] 3.1 Maximum Detection Concentration

[0121] The experimental results for determining the maximum detectable concentration are shown in Table 5 below.

[0122] Table 5:

[0123]

[0124] It can be seen that the maximum detectable concentration of the diglyceride oil composition in Example 6 for improving the effect of qi stagnation and blood stasis is 2000 μg / mL.

[0125] 3.2 Evaluation of efficacy against qi stagnation and blood stasis type thrombosis

[0126] The evaluation results of the efficacy against qi stagnation and blood stasis type thrombosis are shown in Table 6 below.

[0127] Table 6:

[0128]

[0129] Wherein, * represents a comparison with the model control group, p < 0.05, and *** represents a comparison with the model control group, p < 0.001. Compared with the model control group, the blood flow velocity of zebrafish using the diglyceride oil composition of Example 6 increased by 17%, which has a significant effect on improving hyperthrombosis, while no significant difference was observed in the peanut oil group.

[0130] It is evident that the diglyceride oil prepared by the process of this invention can yield a diglyceride oil composition, which has the effect of increasing blood flow velocity and improving thrombosis caused by qi stagnation and blood stasis.

[0131] Example 2

[0132] Evaluation of the effects of diglyceride oil composition on serum uric acid, triglyceride and cholesterol levels in Uox gene knockout mice.

[0133] SPF-grade C57BL / 6J mice were used in the experiment. The hyperuricemia mouse model was a Uox gene knockout male mouse model, provided by the Medical Research Center of the Affiliated Hospital of Qingdao University. Uox, also known as uricase, is responsible for oxidizing and degrading small uric acid molecules in the body, thereby maintaining the blood uric acid concentration at a normal level. In the Uox gene knockout male C57BL / 6J mice, the reduced expression of Uox led to uric acid metabolism disorder. The mice that successfully modeled the model had stable blood uric acid levels between 400-600 μmol / L and could survive for a long time. The weight and age of the experimental animals at the time of grouping were as follows: Uox gene knockout male mice: 18-22g, 8-12 weeks old; wild-type male mice: 20-25g, 8-12 weeks old.

[0134] Animal acclimatization period: 7 days. During the acclimatization period, the researchers conducted daily observations at the cage.

[0135] Mice were housed in individually ventilated cages (IVCs), with no more than 5 mice per cage. Environmental parameters of the animal housing were recorded during the rearing period, and no other species of animals were housed in the same room during the experiment. The rearing environment was as follows: temperature 20-26℃, relative humidity 40%-70%, ventilation via an individually ventilated cage system, and a 12-hour alternating light and dark cycle, with lights turned off at 7 PM and turned on at 7 AM the following morning.

[0136] SPF mouse maintenance diets sterilized by cobalt-60 irradiation were provided by Shanghai Southern Model Biotechnology Co., Ltd.; oil-substitute feeds were provided by Beijing Keao Feed Co., Ltd., using the diglyceride oil composition of Example 7 to replace the vegetable oil added to the feed. Quality inspection reports were provided for each batch of feed, including tests for conventional nutrients, heavy metals, pesticides, and microbiological indicators. No contaminants affecting the quality of this experiment were known in the qualified feeds. Animals had free access to self-prepared deionized water. Water quality analysis, including pH, conductivity, microorganisms, and heavy metals, was conducted quarterly; a comprehensive water quality test, including sensory, physicochemical, and microbiological indicators, was conducted annually. No contaminants affecting the quality of this experiment were known. The corn cob bedding used in this experiment was provided by Shanghai Southern Model Biotechnology Co., Ltd., and quality inspection reports, including tests for heavy metals and pesticides, were provided for each batch of bedding. No contaminants affecting the quality of this experiment were known in the qualified bedding.

[0137] Animals were randomly assigned to groups using Provantis' built-in grouping module. The selected Uox gene knockout mice were divided into four groups. The differences in body weight among the groups were all within ±20% of the mean body weight. After grouping, there was no statistically significant difference in the mean body weight of the animals in each group at the 5% significance level.

[0138] The experimental groups and drug dosages are shown in Table 7.

[0139] Table 7:

[0140]

[0141] The normal uric acid control group consisted of wild-type male C57BL / 6J mice, genotype HO-; the hyperuricemia model group and Example 7 group consisted of male C57BL / 6J mice with the Uox gene knocked out, genotype HO+. Mice in the normal uric acid control group and the hyperuricemia model group (Example 7 group) were 6-8 weeks old and weighed 20g ± 2g. The normal uric acid control group and the hyperuricemia model group were administered 200μL of distilled water by gavage once daily and fed a maintenance diet. The Example 7 group was administered 200μL of the diglyceride oil composition provided in Example 7 by gavage once daily and fed an oil-substitute diet.

[0142] All animals were observed once a day (3 days before grouping, including the day of grouping). Before the first gavage and after drug administration, animals were closely observed for changes in respiration, movement, and other behaviors, and then observed daily thereafter. All animals were weighed on the day of grouping (day 0), and on days 14, 28, and 42 after grouping. Blood was collected from the inner canthus, serum was separated, and blood biochemical indicators were tested once.

[0143] Body weight and blood biochemistry results are expressed as mean ± standard deviation. GraphPad Prism 9.5 software was used for statistical analysis and graph creation. One-way ANOVA was used for comparisons, with P < 0.05 considered statistically significant. If the one-way ANOVA results showed differences, Dunnett's or Tukey's correction was used for pairwise comparisons, with corrected P < 0.05 considered statistically significant. For comparisons involving gavage time and group as two factors, two-way ANOVA was used. If the two-way ANOVA results showed differences, Tukey's HSD correction was used for pairwise comparisons, with corrected P < 0.05 considered statistically significant.

[0144] The experimental results are as follows:

[0145] 1. General observation: There were no significant changes in respiration, defecation, movement, mental state, etc. of all animals before and after gavage.

[0146] 2. Changes in serum uric acid, triglycerides, and total cholesterol in mice.

[0147] The mean ± standard deviation of the three biochemical indicators of serum uric acid, serum triglycerides and serum total cholesterol concentration on days 0, 14, 28 and 42 of the experiment are shown in Table 8.

[0148] Table 8:

[0149]

[0150] On day 42 of the experiment, the results of serum uric acid testing in mice were as follows: Figure 1 As shown, the difference between the blood uric acid test results and those on day 0 of the experiment is as follows: Figure 2 As shown; the results of the mouse blood total cholesterol concentration test are as follows. Figure 3 As shown, the difference between the blood total cholesterol concentration test results and those on day 0 of the experiment is as follows: Figure 4 As shown; the results of the mouse blood triglyceride concentration test are as follows. Figure 5 As shown, the difference between the blood triglyceride concentration test results and those on day 0 of the experiment is as follows: Figure 6 As shown.

[0151] As can be seen, compared with the model control group, the diglyceride oil composition provided in Example 7 has the effect of reducing the blood uric acid level in mice (p<0.05) and significantly reducing the blood triglyceride and cholesterol levels in mice (p<0.001).

[0152] Effect Example 3

[0153] Effect of diglyceride oil composition on blood glucose in hyperglycemic zebrafish in a thrombosis model

[0154] 1. Experimental conditions

[0155] 1.1 Experimental animals

[0156] Zebrafish were all raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L calculated as CaCO3). They were provided by the fish-raising center of the Innovative Experiment Center of Huante Biotechnology. The license number for the use of experimental animals was: SYXK(Zhe)2022 - 0004. The feeding management met the requirements of international AAALAC accreditation (accreditation number: 001458). IACUC ethical review number: IACUC - 2024 - 10165 - 01

[0157] 1.2 Samples, instruments and consumables

[0158] The samples detected in this effect example were the diglyceride oil compositions provided in Example 6, Example 8, and Example 9

[0159] Dissecting microscope (SZX7, OLYMPUS); precision electronic balance (CP214, OHAUS); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd.); blood glucose meter (ACCU-CHEK Performa, Roche Diagnostic Products (Shanghai) Co., Ltd.); blood glucose meter test strips (batch number 670452, Roche Diagnostic Products (Shanghai) Co., Ltd.). Egg yolk powder (batch number 20230203, Zhejiang Aige Biotechnology Co., Ltd.); anhydrous glucose (batch number C15778026, Shanghai Macklin Biochemical Co., Ltd.); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma)

[0160] 2. Evaluation method

[0161] 2.1 Determination of the maximum detection concentration (MTC)

[0162] 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. Different concentrations of diglyceride oil composition were given respectively. At the same time, a normal control group and a model control group were set up, and the volume of each cup was 25 mL. Except for the normal control group, the other experimental groups were given a high-sugar and high-fat diet in water solution to establish a zebrafish hyperglycemic model. After treatment at 28 °C for 2 days, the MTC of the samples for model zebrafish was measured

[0163] 2.2 Evaluation of hypoglycemic efficacy

[0164] Wild-type AB strain zebrafish (5 dpf) were randomly selected and placed in beakers, with 30 zebrafish per beaker. Different concentrations of diglyceride oil mixtures (dissolved with DMSO) were administered. The positive control group received a 400 μg / mL metformin solution. Normal and model control groups were also established. Except for the normal control group, all experimental groups were fed a high-sugar, high-fat diet to establish a zebrafish thrombosis model. After treatment at 28℃ for 2 days, blood glucose levels were collected using a glucometer. The glucose levels of the zebrafish were analyzed and statistically evaluated to assess the hypoglycemic efficacy. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; P < 0.05 indicated statistical significance.

[0165] 3. Test Results

[0166] 3.1 Maximum Detection Concentration

[0167] The experimental results for determining the maximum detectable concentration are shown in Table 9 below.

[0168] Table 9:

[0169]

[0170] It can be seen that the maximum detection concentration for the hypoglycemic efficacy test of the diglyceride oil compositions in Examples 6, 8, and 9 is 2000 μg / mL.

[0171] 3.2 Evaluation of hypoglycemic efficacy

[0172] Under the evaluation conditions in 2.2, the hypoglycemic efficacy evaluation results of Examples 6, 8, and 9 are shown in Table 10 below. Figure 7 As shown.

[0173] Table 10:

[0174]

[0175] Where *** represents a comparison with the model control group, p < 0.001.

[0176] It is evident that the diglyceride oil composition prepared by the process of this invention has the effect of lowering blood sugar.

[0177] 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 diglyceride oil with five benefits for improving vascular health through enzymatic hydrolysis, characterized in that, Includes the following steps: Low-erucic acid rapeseed oil, glycerol, water, fatty acids and lipase are mixed and subjected to enzymatic hydrolysis, followed by molecular distillation to decolorize and deodorize, to obtain diglyceride oil. The fatty acids are selected from linoleic acid and α-linolenic acid; The lipase is a mixture of Antarctic Candida niger lipase, Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 7:1.5:1.5 or 7:1:

2. The weight ratio of the low-erucic acid rapeseed oil, glycerol, water, fatty acids and lipase is 500:90:1.5:45:15 or 500:100:1:60:

10. The temperature of the enzymatic hydrolysis reaction is 50-65℃.

2. The method according to claim 1, characterized in that, The lipase is a mixture of Antarctic Candida niger lipase, Candida columnaris lipase and bovine pancreatic lipase in a weight ratio of 7:1.5:1.

5.

3. The method according to claim 1, characterized in that, The enzymatic hydrolysis reaction takes 1-9 hours.

4. The method according to claim 1, characterized in that, The molecular distillation includes first-stage molecular distillation, second-stage molecular distillation, and third-stage molecular distillation; The temperature of the first-stage molecular distillation is 100-160℃, and the vacuum degree is less than 500Pa; The temperature of the second-stage molecular distillation is 170-200℃, and the vacuum degree is less than 10Pa; The temperature of the third-stage molecular distillation is 220-280℃, and the vacuum degree is less than 10Pa. The condensation surface temperature of the molecular distillation is 35-55℃, and the molecular distillation is repeated 3-5 times.

5. The method according to claim 1, characterized in that, The weight ratio of the low-erucic acid rapeseed oil, glycerol, water, fatty acids and lipase is 500:90:1.5:45:

15.

6. The diglyceride oil obtained by the method according to any one of claims 1-5.

7. The use of the diglyceride oil obtained by the method according to any one of claims 1-5 in the production of oil and fat products.

8. A diglyceride oil composition, characterized in that, This includes low-erucic acid rapeseed diglyceride oil, corn diglyceride oil, edible vegetable oils, and food additives; The low-erucic acid rapeseed diglyceride oil is the diglyceride oil obtained by the method described in any one of claims 1-5.

9. The diglyceride oil composition according to claim 8, characterized in that, The edible vegetable oil is selected from corn oil and / or peanut oil, and the food additive is vitamin E.

Images