Egg yolk phospholipid polypeptide-dihydromyricetin complex and preparation method thereof

Abstract

The application discloses an egg yolk phospholipid polypeptide-dihydro myricetin compound and a preparation method thereof, and comprises the following steps: (1) egg yolk phospholipid protein powder is diluted with purified water and then subjected to enzymolysis treatment to obtain egg yolk phospholipid polypeptide; (2) the egg yolk phospholipid polypeptide obtained in the step (1) is diluted to obtain an egg yolk phospholipid polypeptide solution, then mixed with a dihydro myricetin aqueous solution, stirred in the dark to obtain a mixed solution; and (3) the mixed solution obtained in the step (2) is concentrated and spray dried to obtain a powder product, namely the egg yolk phospholipid polypeptide-dihydro myricetin compound. The application has the advantages of simple process operation, no introduction of toxic organic reagents, safe and non-toxic carrier, simple operation, low cost, good economic benefit, environmental protection benefit and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional food processing, in particular to a preparation method of egg yolk phospholipid polypeptide-dihydromyricetin complex. BACKGROUND

[0002] Dihydromyricetin is an extract from grapevine tea, which is the main active ingredient in grapevine tea. It has been widely concerned due to its various health benefits in the body. Among them, the effect of lowering blood lipids is particularly significant, but its poor water solubility limits its use in the development of functional foods or health foods for lowering blood lipids.

[0003] Egg yolk phospholipid is an egg phospholipid extracted from egg yolk, which can effectively reduce the cholesterol in the blood, especially low-density lipoprotein (LDL) cholesterol, while increasing the level of high-density lipoprotein (HDL) cholesterol, thereby helping to prevent the occurrence of arteriosclerosis and cardiovascular diseases. At the same time, phospholipid is also effectively applied in dihydromyricetin phospholipid complex preparation with high bioavailability, which is a potential dihydromyricetin synergist, but the conventional phospholipid complex is relatively viscous, and a large amount of inactive excipients are needed to prepare a dispersing agent, and a large amount of organic solvents are needed in the preparation process, which has low safety and environmental protection. SUMMARY

[0004] [Technical problem]

[0005] The main problem of traditional dihydromyricetin phospholipid complex preparation is that a large amount of organic solvent is used, and a large amount of inactive excipients are needed in the preparation process to prepare a dispersing agent, so it is necessary to develop a phospholipid-rich component with good solubility for the preparation of dihydromyricetin phospholipid complex, and no organic reagent is used in the preparation process, so as to realize its good application.

[0006] [Technical scheme]

[0007] In view of the above problems existing in the prior art, the present application provides an egg yolk phospholipid polypeptide-dihydromyricetin complex and a preparation method thereof.

[0008] The technical scheme of the present application is as follows:

[0009] The first object of the present application is to provide a preparation method of egg yolk phospholipid polypeptide-dihydromyricetin complex, comprising the following steps:

[0010] (1) diluting egg yolk phospholipid protein powder with purified water and then performing enzymatic treatment to obtain egg yolk phospholipid polypeptide;

[0011] (2) diluting the egg yolk phospholipid polypeptide obtained in step (1) to obtain an egg yolk phospholipid polypeptide solution, and then mixing with a dihydromyricetin aqueous solution, stirring in the dark to obtain a mixed solution;

[0012] (3) concentrating and spray drying the mixed solution prepared in step (2) to obtain a powder product, i.e., the egg yolk phospholipid polypeptide-dihydromyricetin complex.

[0013] In an embodiment of the present application, in step (1), the protein content of the egg yolk phospholipid protein powder is ≥40.0 g / 100 g, and the phospholipid content is ≥24.0 g / 100 g.

[0014] In an embodiment of the present application, in step (1), the mass ratio of the egg yolk phospholipid protein powder to purified water is 1:5-15.

[0015] In an embodiment of the present application, in step (1), the enzymolysis conditions are as follows: 6000-9000 U / g of alkaline protease, an enzymolysis temperature of 48-55°C, and an enzymolysis time of 6-12 h.

[0016] In an embodiment of the present application, in step (2), the egg yolk phospholipid polypeptide is diluted to a solid content of 3-6% using purified water.

[0017] In an embodiment of the present application, in step (2), the concentration of the dihydromyricetin aqueous solution is 4-12 mg / mL.

[0018] In an embodiment of the present application, in step (2), the volume ratio of the egg yolk phospholipid polypeptide solution to the dihydromyricetin aqueous solution is 2-4:1, and the mixing and dark stirring are performed at 80-90°C.

[0019] In an embodiment of the present application, in step (2), the dihydromyricetin aqueous solution is prepared by mixing dihydromyricetin with water and then heating at 80-90°C for 20 min.

[0020] In an embodiment of the present application, in step (2), the egg yolk phospholipid polypeptide solution is added to 80-90°C, and the egg yolk phospholipid polypeptide solution is mixed with the dihydromyricetin aqueous solution at 80-90°C, and dark stirring is performed to obtain a mixed solution.

[0021] In an embodiment of the present application, in step (3), the concentration is performed by using a vacuum scraped-rotating thin film evaporator to concentrate and dehydrate, the vacuum degree is -0.06 to -0.08 MPa, and the temperature is 40-60°C; and the concentration is performed to a solid content concentration of 20-40%.

[0022] In an embodiment of the present application, in step (3), the spray drying is performed by using low-temperature spray drying, the inlet temperature is 110-130°C, and the outlet temperature is 55-65°C.

[0023] The second object of the present application is to provide an egg yolk phospholipid polypeptide-dihydromyricetin complex prepared by the preparation method.

[0024] The third object of the present application is to provide an application of the egg yolk phospholipid polypeptide-dihydromyricetin complex in the preparation of a blood lipid-lowering functional food or medicine.

[0025] The present application has the beneficial technical effects of:

[0026] The egg yolk phospholipid polypeptide-dihydromyricetin complex has good solubility and synergistic blood lipid-lowering effect.

[0027] The present application does not use organic reagents in the preparation process, thereby realizing its good application, significantly improving the stability of dihydromyricetin, and also improving the biological membrane barrier permeability of endogenous phospholipids, improving the bioavailability of the loaded hydrophobic polyphenols, thereby improving the bioavailability, and having important scientific significance and practical value.

[0028] The present application has the characteristics of simple process operation, no introduction of toxic organic reagents, safe and non-toxic carrier, simple operation, low cost, good economic benefit, environmental benefit and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The solubility of the products obtained in Examples 1-3 and Comparative Examples 1-4;

[0030] Figure 2 The dihydromyricetin retention rate of the products obtained in Examples 1-3 and Comparative Examples 1-4;

[0031] Figure 3 The malondialdehyde content of the products obtained in Examples 1-3 and Comparative Examples 1-4;

[0032] Figure 4 The serological indexes of the animal experiment of the product obtained in Example 1;

[0033] Figure 5 The combined drug effect index of the serological indexes of the animal experiment of the product obtained in Example 1. DETAILED DESCRIPTION

[0034] The present application will be specifically described below in combination with the drawings and examples. The egg yolk phospholipid protein powder is purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd. and is prepared from egg yolk powder by granulation, supercritical CO2 extraction, separation and other processes. The main components are protein (≥40.0 g / 100 g) and phospholipid (≥24.0 g / 100 g) and can be used as a food raw material (Wei Shi Xin Shen Zi

(2023) No. 0011

[0035] Detection method:

[0036] (1) Solubility of powder

[0037] The egg yolk phospholipid polypeptide-dihydromyricetin complex powder was diluted with water at 5% solids, mixed uniformly, and then centrifuged at 8000 rpm for 20 min. The supernatant was taken and dried at 105°C to constant weight. The powder dissolution was obtained by dividing the solid content in the centrifugal supernatant by the solid content before centrifugation.

[0038] (2) Dihydromyricetin retention rate

[0039] The egg yolk phospholipid polypeptide-dihydromyricetin complex was placed in a sealed bag and stored at 60°C for 12 days, then diluted with water at 5% solids, and then diluted with anhydrous ethanol. The absorbance of the dihydromyricetin-ethanol solution was measured at 292 nm using a UV spectrophotometer.

[0040] The dihydromyricetin standard curve (y = 0.1773x + 0.0049, R 2 = 0.9999) was used for quantitative determination.

[0041]

[0042] (3) Malondialdehyde content determination (thiobarbituric acid method)

[0043] The secondary oxidation product malondialdehyde of the sample stored at 60°C for 12 days was determined. The method is as follows: 2 mL of thiobarbituric acid solution (consisting of 15% trichloroacetic acid, 0.375% thiobarbituric acid and 1.76% HCl) was added to a stoppered test tube, 1 mL of sample (or x amount of sample and 1-x mL of water) was added, and deionized water was used as a blank sample. Mix well, place in a boiling water bath for 15 min, then immediately place in a cold water bath for 10 min, centrifuge at 3000 rpm for 15 min at 25°C, then place in the dark for 10 min, pass through a 0.22 μm water membrane, take the supernatant and measure the absorbance at 532 nm wavelength.

[0044] The standard curve (y = 0.7262x + 0.0037) prepared with 1,1,3,3-tetraethoxypropane was used for quantitative determination.

[0045] (4) Evaluation of hypolipidemic function

[0046] Experimental design: the mice were randomly divided into 5 groups, 8 in the blank group, 10 in each of the other groups. The blank group was fed with ordinary feed, and the other groups were fed with high-fat feed. After 1 week of adaptation, the mice were used for experiments. From the 8th to the 38th day, the blank and model groups were given 0.2 ml of normal saline by gavage, and the other groups were given corresponding amounts by gavage (once a day). On the 39th day, the mice were fasted overnight, anesthetized with isoflurane, and then sacrificed by cervical dislocation. The blood of the mice was collected for determination. The determination method of the indexes: serum TC (total cholesterol), TG (triglyceride), LDL-C (low-density lipoprotein cholesterol) and HDL-C (high-density lipoprotein cholesterol) were determined by using the corresponding commercial kit (Nanjing Jiancheng, China).

[0047] The combined drug effect index was calculated by the King's formula q value:

[0048]

[0049] E(A+B) is the index reduction rate of the combination of the two drugs;

[0050] EA and EB are the index reduction rates of each drug alone;

[0051] q>=1 indicates that the combination of the two drugs has a synergistic effect.

[0052] Example 1

[0053] A preparation method of a yolk phospholipid polypeptide-dihydromyricetin complex, comprising the following steps:

[0054] (1) Dilute the yolk phospholipid protein powder with purified water at a ratio of 1:9 (w / w) and perform enzyme treatment (add 9000 U / g of alkaline protease, enzyme treatment temperature is 55℃, enzyme treatment time is 6h) to obtain yolk phospholipid polypeptide;

[0055] (2) Flash the yolk phospholipid polypeptide obtained in step (1) to remove the enzyme, and then dilute it with purified water to a solid concentration of 6%;

[0056] (3) Preheat the yolk phospholipid polypeptide solution prepared in step (2) to 90℃, and prepare a 12mg / mL dihydromyricetin aqueous solution, heat at 90℃ for 20min until the dihydromyricetin is completely dissolved, mix the yolk phospholipid polypeptide solution and the dihydromyricetin solution at a ratio of 2:1 (v / v) at 90℃, stir in the dark to obtain a complex mixture;

[0057] (4) The egg yolk phospholipid polypeptide-dihydroquercetin complex prepared in step (3) is concentrated and desolventized by a vacuum scraper rotary thin film evaporator, the vacuum degree is -0.08 MPa, the temperature is 40°C, and the concentration is performed until the solid concentration is 20%;

[0058] (5) The egg yolk phospholipid polypeptide-dihydroquercetin complex prepared in step (4) is subjected to low-temperature spray drying to obtain a powder product, the inlet temperature is 110°C, and the outlet temperature is 55°C.

[0059] Example 2

[0060] A method for preparing an egg yolk phospholipid polypeptide-dihydroquercetin complex, comprising the following steps:

[0061] (1) Egg yolk phospholipid protein powder is diluted with purified water at a ratio of 1:9 (w / w) and subjected to enzymatic treatment (alkaline protease is added at a dosage of 7000 U / g, the enzymatic treatment temperature is 52°C, and the enzymatic treatment time is 9 h) to obtain egg yolk phospholipid polypeptides;

[0062] (2) The egg yolk phospholipid polypeptides obtained in step (1) are subjected to flash enzyme removal, and then diluted with purified water to a solid concentration of 4%;

[0063] (3) The egg yolk phospholipid polypeptide solution prepared in step (2) is preheated to 85°C, and an 8 mg / mL dihydroquercetin solution is prepared, heated at 80°C for 20 min until the dihydroquercetin is completely dissolved, and the egg yolk phospholipid polypeptide solution and the dihydroquercetin solution are mixed at a ratio of 3:1 (v / v) at 80°C, stirred in the dark to obtain a complex mixture;

[0064] (4) The egg yolk phospholipid polypeptide-dihydroquercetin complex prepared in step (3) is concentrated and desolventized by a vacuum scraper rotary thin film evaporator, the vacuum degree is -0.07 MPa, the temperature is 50°C, and the concentration is performed until the solid concentration is 30%;

[0065] (5) The egg yolk phospholipid polypeptide-dihydroquercetin complex prepared in step (4) is subjected to low-temperature spray drying to obtain a powder product, the inlet temperature is 120°C, and the outlet temperature is 60°C.

[0066] Example 3

[0067] A method for preparing an egg yolk phospholipid polypeptide-dihydroquercetin complex, comprising the following steps:

[0068] (1) Egg yolk phospholipid protein powder is diluted with purified water at a ratio of 1:9 (w / w) and subjected to enzymatic treatment (alkaline protease is added at a dosage of 7000 U / g, the enzymatic treatment temperature is 52°C, and the enzymatic treatment time is 9 h) to obtain egg yolk phospholipid polypeptides;

[0069] (2) The egg yolk phospholipid polypeptide obtained in step (1) is subjected to flash enzyme removal, and then purified water is diluted to a solid concentration of 3%;

[0070] (3) The egg yolk phospholipid polypeptide solution prepared in step (2) is preheated to 80°C, and a 4 mg / mL aqueous solution of dihydromyricetin is prepared, heated at 80°C for 20 min until dihydromyricetin is completely dissolved, and the egg yolk phospholipid polypeptide solution and the dihydromyricetin solution are mixed at a ratio of 4:1 (v / v) at 80°C, stirred in the dark to obtain a compound mixture;

[0071] (4) The egg yolk phospholipid polypeptide-dihydromyricetin complex prepared in step (3) is concentrated and desolvated using a vacuum scraper rotary film evaporator, with a vacuum degree of -0.06 MPa and a temperature of 60°C, to a solid concentration of 40%;

[0072] (5) The egg yolk phospholipid polypeptide-dihydromyricetin complex prepared in step (4) is subjected to low-temperature spray drying to obtain a powder product, with an inlet temperature of 130°C and an outlet temperature of 65°C.

[0073] Comparative Example 1

[0074] Referring to Example 1, the only difference is that in step (3), the dihydromyricetin solution is omitted, and the other steps and parameters remain unchanged.

[0075] Comparative Example 2

[0076] Referring to Example 1, the only difference is that in step (3), an equal volume of water is used instead of the egg yolk phospholipid polypeptide solution, and the other steps and parameters remain unchanged.

[0077] Comparative Example 3

[0078] Referring to Example 1, the only difference is that in step (3), an equal concentration of egg yolk phospholipid solution is used instead of the egg yolk phospholipid polypeptide solution, and the other steps and parameters remain unchanged.

[0079] Comparative Example 4

[0080] Referring to Example 1, the only difference is that in step (1), neutral protease is used instead of alkaline protease, and the other steps and parameters remain unchanged.

[0081] Comparative Example 5

[0082] Referring to Example 1, the only difference is that in step (3), the volume ratio of the egg yolk phospholipid polypeptide solution to the dihydromyricetin aqueous solution is 1:1, and the other steps and parameters remain unchanged.

[0083] Comparative Example 6

[0084] Referring to Example 1, the only difference is that in step (3), the volume ratio of the egg yolk phospholipid polypeptide solution to the aqueous solution of dihydromyricetin is 5:1, and other steps and parameters remain unchanged.

[0085] Comparative Example 7

[0086] Referring to Example 1, the only difference is that in step (3), the egg yolk phospholipid polypeptide solution and the aqueous solution of dihydromyricetin are mixed at room temperature, and other steps and parameters remain unchanged.

[0087] The test data of the solubility, dihydromyricetin retention rate, and malondialdehyde content of the products obtained in Examples 1-3 and Comparative Examples 1-4 are shown in Table 1. Figures 1 to 3 As can be seen from the figure:

[0088] In Examples 1-3, the powder solubility reaches more than 80%, and the corresponding dihydromyricetin storage retention rate in the complex is also at a high level.

[0089] Comparing Comparative Example 1 with Example 1, it can be seen that the powder solubility in Comparative Example 1 is relatively high, indicating that the solubility of the egg yolk phospholipid polypeptide and dihydromyricetin complex will decrease to a certain extent. From the malondialdehyde content, it can be seen that the powder in Comparative Example 1 does not contain dihydromyricetin, and the malondialdehyde content is significantly higher than that in Example 1, indicating that the presence of dihydromyricetin is beneficial to the storage stability of the powder.

[0090] Comparing Comparative Example 2 with Example 1, it can be seen that the powder solubility in Comparative Example 2 is extremely low (about 0.0008%), but its retention rate is relatively high, indicating that the complex of egg yolk phospholipid polypeptide can greatly improve the solubility of dihydromyricetin, but it cannot further improve the stability of dihydromyricetin in the powder state.

[0091] Comparing Comparative Example 3 with Example 1, it can be seen that when egg yolk phospholipid and dihydromyricetin are used for complexing in Comparative Example 3, the powder solubility is low, and the malondialdehyde content increases significantly, indicating that the solubility of egg yolk phospholipid polypeptide is significantly better than that of egg yolk phospholipid, and the polypeptide in the egg yolk phospholipid polypeptide dihydromyricetin complex plays a certain antioxidant role, inhibiting the oxidation of lipids in the powder.

[0092] Comparing Comparative Example 4 with Example 1, it can be seen that when the egg yolk phospholipid polypeptide prepared by hydrolysis of neutral protease is used for complexing with dihydromyricetin in Comparative Example 4, the powder solubility is relatively low, which indicates that the hydrolysis ability of neutral protease on egg yolk phospholipid protein is lower than that of alkaline protease.

[0093] Comparing Comparative Example 5 with Example 1, it can be seen that the ratio of egg yolk phospholipid polypeptide to dihydromyricetin in Comparative Example 5 is 1 / 2 of Example 1, and the powder solubility decreases significantly, which indicates that too high a proportion of dihydromyricetin cannot be effectively loaded by egg yolk phospholipid polypeptide.

[0094] From Comparative Example 1 and Comparative Example 6, it can be seen that the ratio of egg yolk phospholipid polypeptide to dihydromyricetin in Comparative Example 6 is greater than that in Example 1, the retention rate of dihydromyricetin is further reduced, and the content of malondialdehyde is increased, indicating that increasing the ratio of egg yolk phospholipid polypeptide will reduce the retention rate of dihydromyricetin, and there will be more lipid oxidation products, malondialdehyde.

[0095] From Comparative Example 1 and Comparative Example 7, it can be seen that the solubility of egg yolk phospholipid polypeptide and dihydromyricetin mixed at room temperature in Comparative Example 7 is significantly reduced, indicating that complexing at room temperature cannot form an effective complex between the two.

[0096] (2) From Figure 4 It can be seen that the TC, TG and LDL-C of the model group are significantly higher than those of the control group, while the TC, TG and LDL-C of the egg yolk phospholipid polypeptide group, the dihydromyricetin group and the Example 1 group are significantly lower than those of the model group, and the sample in Example 1 has the most obvious effect on reducing blood lipids.

[0097] (3) From Figure 5 It can be seen from the combined drug effect index that the TC index combined drug effect index is significantly greater than 1, indicating that dihydromyricetin and egg yolk phospholipid polypeptide have significant synergistic effect on reducing TC. From the TG and LDL-C combined drug index, the combined drug effect index is about 1, indicating that dihydromyricetin and egg yolk phospholipid polypeptide have certain synergistic effect on reducing TG and LDL-C, but it is not obvious.

[0098] The examples provided above are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order. Those skilled in the art can make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing a yolk phospholipid polypeptide-dihydroquercetin complex, characterized in that, The preparation method comprises the following steps: (1) egg yolk phospholipid protein powder is diluted with purified water and then subjected to enzymatic treatment to obtain egg yolk phospholipid polypeptide; the enzymatic treatment is performed under the following conditions: 6000-9000 U / g of alkaline protease, 48-55 ℃ of enzymatic treatment temperature, and 6-12 h of enzymatic treatment time; (2) the egg yolk phospholipid polypeptide obtained in step (1) is diluted to obtain an egg yolk phospholipid polypeptide solution, which is then mixed with a dihydromyricetin aqueous solution, stirred in the dark to obtain a mixed solution; the dihydromyricetin aqueous solution has a concentration of 4-12 mg / mL; the volume ratio of the egg yolk phospholipid polypeptide solution to the dihydromyricetin aqueous solution is 2-4:1; the mixing and stirring in the dark are performed at 80-90 ℃; (3) the mixed solution obtained in step (2) is concentrated and spray-dried to obtain a powder product, i.e., the egg yolk phospholipid polypeptide-dihydromyricetin complex.

2. The production method according to claim 1, characterized by, In step (1), the egg yolk phospholipid protein powder contains ≥40.0 g / 100 g of protein and ≥24.0 g / 100 g of phospholipid.

3. The production method according to claim 1, characterized by, In step (1), the mass ratio of the egg yolk phospholipid protein powder to purified water is 1:5-15.

4. The method of claim 1, wherein, In step (2), the egg yolk phospholipid polypeptide is diluted to 3-6% of solid content with purified water.

5. The production method according to claim 1, characterized by, In step (3), the concentration is performed by using a vacuum scraper rotary thin film evaporator to concentrate and dehydrate under a vacuum degree of -0.06 to -0.08 MPa and a temperature of 40-60 ℃; the concentration is performed to a solid content concentration of 20-40%.

6. The production method according to claim 1, characterized by, In step (3), the spray drying is performed by using low-temperature spray drying under an inlet temperature of 110-130 ℃ and an outlet temperature of 55-65 ℃.

7. An egg yolk phospholipid polypeptide-dihydromyricetin complex prepared by the preparation method of any one of claims 1-6.

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