Egg yolk phospholipid polypeptide-dihydromyricetin compound and preparation method thereof

Abstract

The invention discloses an egg yolk phospholipid polypeptide-dihydromyricetin compound and a preparation method thereof, and the preparation method comprises the following steps: (1) diluting egg yolk phospholipid protein powder with purified water, and then carrying out enzymolysis treatment to obtain egg yolk phospholipid polypeptide; (2) diluting the egg yolk phospholipid polypeptide obtained in the step (1) to prepare an egg yolk phospholipid polypeptide solution, then mixing the egg yolk phospholipid polypeptide solution with a dihydromyricetin aqueous solution, and stirring in a dark place to obtain a mixed solution; and (3) concentrating the mixed solution prepared in the step (2), and performing spray drying to obtain a powder product, namely the egg yolk phospholipid polypeptide-dihydromyricetin compound. The method has the advantages of simple process operation, no introduction of toxic organic reagents, safe and nontoxic carrier, simple operation and low cost, and has good economic benefit, environmental protection benefit and application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of functional food processing, and in particular to a method for preparing an egg yolk phospholipid polypeptide-dihydromyricetin complex. Background Art

[0002] Dihydromyricetin is an extract of the grape plant rattan tea, and is the main active ingredient in rattan tea. It has attracted widespread attention due to its various health benefits in vivo. Among them, the lipid-lowering effect is particularly significant. However, its poor water solubility limits its use in the development of lipid-lowering functional foods or health foods.

[0003] Egg yolk lecithin is a lecithin extracted from egg yolks, which can effectively reduce blood cholesterol, especially low-density lipoprotein (LDL) cholesterol, while increasing the level of high-density lipoprotein (HDL) cholesterol, thereby helping to prevent arteriosclerosis and cardiovascular diseases. At the same time, lecithin has also been effectively used in dihydromyricetin phospholipid complex preparations with high bioavailability, and is a potential dihydromyricetin synergist. However, conventional phospholipid complexes are relatively viscous and require a large amount of inactive excipients to prepare dispersants. In addition, a large amount of organic solvents are required during the preparation process, and safety and environmental protection are relatively low. Summary of the invention

[0004] [Technical issues]

[0005] The main problem in the preparation of traditional dihydromyricetin phospholipid complexes is the large amount of organic solvents used. The preparation process requires a large amount of inactive excipients to prepare the dispersant. Therefore, it is necessary to develop a phospholipid-rich component with good solubility properties for the preparation of dihydromyricetin phospholipid complexes, and no organic reagents are used in the preparation process to achieve its good application.

[0006] [Technical solution]

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

[0008] The technical solution of the present invention is as follows:

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

[0010] (1) diluting egg yolk phospholipid protein powder with purified water and performing enzymatic hydrolysis 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 it with the dihydromyricetin aqueous solution, stirring in the dark, and obtaining a mixed solution;

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

[0013] In one embodiment of the present invention, 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 one embodiment of the present invention, in step (1), the mass ratio of egg yolk phospholipid protein powder to purified water is 1:5-15.

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

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

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

[0018] In one embodiment of the present invention, in step (2), 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 carried out at 80-90°C.

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

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

[0021] In one embodiment of the present invention, in step (3), the concentration is performed by using a vacuum scraper rotary thin film evaporator for concentration and dehydration, with a vacuum degree of -0.06 to -0.08 MPa and a temperature of 40 to 60° C.; the concentration is performed to a solid concentration of 20 to 40%.

[0022] In one embodiment of the present invention, in step (3), spray drying adopts low-temperature spray drying, with an inlet temperature of 110-130°C and an outlet temperature of 55-65°C.

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

[0024] The third object of the present invention is to provide an application of the above-mentioned egg yolk phospholipid polypeptide-dihydromyricetin complex in the preparation of lipid-lowering functional foods or medicines.

[0025] The beneficial technical effects of the present invention are:

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

[0027] No organic reagent is used in the preparation process of the invention, thereby achieving good application thereof, significantly improving the stability of dihydromyricetin, and utilizing the membrane affinity of endogenous phospholipids to improve the permeability of the biomembrane barrier, thereby improving the bioaccessibility of the loaded hydrophobic polyphenols and thus improving the bioavailability thereof, which has important scientific significance and practical value.

[0028] The process of the invention is simple to operate, does not require the introduction of toxic organic reagents, has a safe and non-toxic carrier, is easy to operate, and has low cost. This feature makes the technology have good economic benefits, environmental benefits, and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0032] Figure 4 Serological index of animal experiment of the product obtained in Example 1;

[0033] Figure 5 It is the combined drug effect index of the animal experimental serological indicators of the product obtained in Example 1. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and examples. Egg yolk phospholipid protein powder was purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd. It is made from egg yolk powder through granulation, supercritical CO2 extraction, separation and other processes. The main components are protein (≥40.0g / 100g) and phospholipids (≥24.0g / 100g), which can be used as a food raw material (Weishi Xinshenzi [(2023) No. 0011]).

[0035] Detection method:

[0036] (1) Powder solubility

[0037] The egg yolk phospholipid polypeptide-dihydromyricetin complex powder was diluted with water at 5% solid content and mixed evenly, then centrifuged at 8000 rpm for 20 min, the supernatant was taken, and dried at 105° C. to constant weight, and the powder dissolution was obtained by dividing the solid content in the centrifuged 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% solid content, and then diluted with anhydrous ethanol. The absorbance of the dihydromyricetin-ethanol solution was measured at 292 nm using an ultraviolet spectrophotometer.

[0040] The standard curve of dihydromyricetin (y = 0.1773x + 0.0049, R 2 =0.9999) for quantification.

[0041]

[0042] (3) Determination of malondialdehyde content (thiobarbituric acid method)

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

[0044] Quantification was performed using a standard curve prepared with 1,1,3,3-tetraethoxypropane (y=0.7262x+0.0037).

[0045] (4) Evaluation of lipid-lowering function

[0046] Experimental design: The mice were randomly divided into 5 groups, 8 in the blank group and 10 in each of the other groups. They were blank group, model group and experimental group (60 mg / kg dihydromyricetin, 600 mg / kg egg yolk phospholipid polypeptide, 600 mg / kg egg yolk phospholipid polypeptide-dihydromyricetin complex). The blank group was male C57BL / 6J mice fed with ordinary feed, and the other groups were male C57BL / 6JGpt DIO mice fed with high-fat feed. After a 1-week adaptation period, the mice were used for the experiment. Days 8 to 38: The blank group and the model group were gavaged with 0.2 ml of normal saline, and the other groups were gavaged with the corresponding amount (once a day). Day 39: The mice were fasted overnight, anesthetized with isoflurane, and blood was collected from the eyeballs and the mice were killed by cervical dislocation. The blood of the mice was collected for determination. Index determination method: Serum TC (total cholesterol), TG (triglycerides), LDL-C (low-density lipoprotein cholesterol) and HDL-C (high-density lipoprotein cholesterol) were determined using corresponding commercial kits (Jiancheng, Nanjing, China).

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

[0048]

[0049] E(A+B) is the index reduction rate when the two drugs are used together;

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

[0051] q>=1 indicates that the two drugs have a synergistic effect when used together.

[0052] Example 1

[0053] A method for preparing an egg yolk phospholipid polypeptide-dihydromyricetin complex comprises the following steps:

[0054] (1) diluting the egg yolk phospholipid protein powder with purified water at a ratio of 1:9 (w / w) and performing enzymatic hydrolysis (alkaline protease addition amount 9000 U / g, enzymatic hydrolysis temperature 55° C., enzymatic hydrolysis time 6 h) to obtain the egg yolk phospholipid polypeptide;

[0055] (2) flash-distilling the egg yolk phospholipid polypeptide obtained in step (1) to inactivate the enzyme, and then diluting with purified water to a solid concentration of 6%;

[0056] (3) preheating the egg yolk phospholipid polypeptide solution prepared in step (2) to 90° C., and preparing a 12 mg / mL dihydromyricetin aqueous solution, heating at 90° C. for 20 min until the dihydromyricetin is completely dissolved, and mixing the egg yolk phospholipid polypeptide solution and the dihydromyricetin solution at 90° C. in a ratio of 2:1 (v / v), stirring in the dark, to obtain a complex mixture;

[0057] (4) The egg yolk phospholipid polypeptide-dihydromyricetin complex prepared in step (3) was concentrated and desolvated using a vacuum scraper rotary thin film evaporator at a vacuum degree of -0.08 MPa and a temperature of 40° C. to a solid concentration of 20%;

[0058] (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 110° C. and an outlet temperature of 55° C.

[0059] Example 2

[0060] A method for preparing an egg yolk phospholipid polypeptide-dihydromyricetin complex comprises the following steps:

[0061] (1) diluting the egg yolk phospholipid protein powder with purified water at a ratio of 1:9 (w / w) and performing enzymatic hydrolysis (addition amount of alkaline protease 7000 U / g, enzymatic hydrolysis temperature 52° C., enzymatic hydrolysis time 9 h) to obtain the egg yolk phospholipid polypeptide;

[0062] (2) flash-distilling the egg yolk phospholipid polypeptide obtained in step (1) to inactivate the enzyme, and then diluting with purified water to a solid concentration of 4%;

[0063] (3) preheating the egg yolk phospholipid polypeptide solution prepared in step (2) to 85° C., and preparing an 8 mg / mL dihydromyricetin solution, heating at 80° C. for 20 min until the dihydromyricetin is completely dissolved, and mixing the egg yolk phospholipid polypeptide solution and the dihydromyricetin solution at 80° C. in a ratio of 3:1 (v / v), stirring in the dark, to obtain a complex mixture;

[0064] (4) The egg yolk phospholipid polypeptide-dihydromyricetin complex prepared in step (3) was concentrated and desolvated using a vacuum scraper rotary thin film evaporator at a vacuum degree of -0.07 MPa and a temperature of 50° C. to a solid concentration of 30%;

[0065] (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 120° C. and an outlet temperature of 60° C.

[0066] Example 3

[0067] A method for preparing an egg yolk phospholipid polypeptide-dihydromyricetin complex comprises the following steps:

[0068] (1) diluting the egg yolk phospholipid protein powder with purified water at a ratio of 1:9 (w / w) and performing enzymatic hydrolysis (addition amount of alkaline protease 6000 U / g, enzymatic hydrolysis temperature 48° C., enzymatic hydrolysis time 12 h) to obtain the egg yolk phospholipid polypeptide;

[0069] (2) flash-distilling the egg yolk phospholipid polypeptide obtained in step (1) to inactivate the enzyme, and then diluting with purified water to a solid concentration of 3%;

[0070] (3) preheating the egg yolk phospholipid polypeptide solution prepared in step (2) to 80° C., and preparing a 4 mg / mL dihydromyricetin aqueous solution, heating at 80° C. for 20 min until the dihydromyricetin is completely dissolved, and mixing the egg yolk phospholipid polypeptide solution and the dihydromyricetin solution at 80° C. in a ratio of 4:1 (v / v), stirring in the dark, to obtain a complex mixture;

[0071] (4) The egg yolk phospholipid polypeptide-dihydromyricetin complex prepared in step (3) was concentrated and desolvated using a vacuum scraper rotary thin film evaporator at 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] Refer to Example 1, the only difference is that the addition of dihydromyricetin solution is omitted in step (3), and the other steps and parameters remain unchanged.

[0075] Comparative Example 2

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

[0077] Comparative Example 3

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

[0079] Comparative Example 4

[0080] Refer to Example 1, the only difference is that the alkaline protease in step (1) is replaced by neutral protease, and the other steps and parameters remain unchanged.

[0081] Comparative Example 5

[0082] Refer 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] Refer 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 5:1, and the other steps and parameters remain unchanged.

[0085] Comparative Example 7

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

[0087] Test Example: (1) The test data of the solubility, dihydromyricetin retention rate and malondialdehyde content of the products obtained in Examples 1 to 3 and Comparative Examples 1 to 4 are as follows: Figures 1 to 3 As shown, it can be seen from the figure:

[0088] In Examples 1-3, the powder solubility reached more than 80%, and the storage retention rate of dihydromyricetin in the corresponding composite was also at a relatively high level.

[0089] By comparing Example 1 and Comparative Example 1, it can be seen that the solubility of the powder in Comparative Example 1 is relatively high, indicating that the solubility of the egg yolk phospholipid polypeptide and dihydromyricetin will be reduced to a certain extent after being compounded. 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] By comparing Example 1 and Comparative Example 2, it can be seen that the solubility of the powder obtained in Comparative Example 2 is extremely low (about 0.0008%), but its retention rate is relatively high, indicating that the compounding of egg yolk phospholipid polypeptide is beneficial to greatly improve the solubility of dihydromyricetin, but the stability of dihydromyricetin cannot be further improved in the powder state.

[0091] By comparing Example 1 and Comparative Example 3, it can be seen that when egg yolk lecithin and dihydromyricetin are compounded in Comparative Example 3, the powder solubility is low and the malondialdehyde content is significantly increased, indicating that the solubility of egg yolk lecithin polypeptide is significantly better than that of egg yolk lecithin, and the polypeptide in the egg yolk lecithin polypeptide dihydromyricetin complex exerts a certain antioxidant activity, inhibiting the oxidation of lipids in the powder.

[0092] By comparing Example 1 and Comparative Example 4, it can be seen that when the egg yolk phospholipid polypeptide obtained by hydrolysis with neutral protease in Comparative Example 4 is compounded with dihydromyricetin, 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] By comparing Example 1 and Comparative Example 5, it can be seen that the ratio of egg yolk phospholipid polypeptide to dihydromyricetin in Comparative Example 5 is 1 times smaller than that in Example 1, and the powder solubility is significantly reduced, which indicates that an excessively high ratio of dihydromyricetin cannot be effectively loaded by the egg yolk phospholipid polypeptide.

[0094] By comparing 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 the increase in the ratio of egg yolk phospholipid polypeptide will reduce the retention rate of dihydromyricetin, and there are more lipid oxidation products malondialdehyde.

[0095] By comparing Example 1 and Comparative Example 7, it can be seen that when the egg yolk phospholipid polypeptide and dihydromyricetin are mixed at room temperature in Comparative Example 7, the solubility is significantly reduced, indicating that the compounding 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 in the model group were significantly higher than those in the control group, while the TC, TG, and LDL-C in the egg yolk phospholipid polypeptide group, the dihydromyricetin group and the Example 1 group were significantly lower than those in the model group, among which the lipid-lowering effect of the sample in Example 1 was the most obvious.

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

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

Claims

1. A method for preparing an egg yolk phospholipid polypeptide-dihydromyricetin complex, characterized in that: The preparation method comprises the following steps: (1) diluting egg yolk phospholipid protein powder with purified water and performing enzymatic hydrolysis to obtain egg yolk phospholipid polypeptide; (2) diluting the egg yolk phospholipid polypeptide obtained in step (1) to obtain an egg yolk phospholipid polypeptide solution, and then mixing it with the dihydromyricetin aqueous solution, stirring in the dark, and obtaining a mixed solution; (3) Concentrating and spray-drying the mixed solution obtained in step (2) to obtain a powder product, namely the egg yolk phospholipid polypeptide-dihydromyricetin complex.

2. The preparation method according to claim 1, characterized in that: In step (1), the protein in the egg yolk phospholipid protein powder is ≥40.0g / 100g, and the phospholipid is ≥24.0g / 100g.

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

4. The preparation method according to claim 1, characterized in that: In step (1), the enzymatic hydrolysis conditions are: alkaline protease 6000-9000 U / g, enzymatic hydrolysis temperature 48-55° C., and enzymatic hydrolysis time 6-12 h.

5. The preparation method according to claim 1, characterized in that: In step (2), the egg yolk phospholipid polypeptide is diluted with purified water to a solid content of 3-6%.

6. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the dihydromyricetin aqueous solution is 4 to 12 mg / mL.

7. The preparation method according to claim 1, characterized in that: In step (2), 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 carried out at 80-90°C.

8. The preparation method according to claim 1, characterized in that: In step (3), the concentration is carried out by using a vacuum scraper rotary thin film evaporator for concentration and dehydration, with a vacuum degree of -0.06 to -0.08 MPa and a temperature of 40 to 60° C.; the concentration is carried out to a solid concentration of 20 to 40%.

9. The preparation method according to claim 1, characterized in that: In step (3), spray drying adopts low-temperature spray drying, with an inlet temperature of 110-130°C and an outlet temperature of 55-65°C.

10. An egg yolk phospholipid polypeptide-dihydromyricetin complex prepared by the preparation method according to any one of claims 1 to 9.

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