A polypeptide-loaded seabuckthorn fruit oil microcapsule powder, its preparation method and application

Abstract

The present invention discloses a polypeptide-loaded seabuckthorn fruit oil microcapsule powder, its preparation method and application. The seabuckthorn fruit oil microcapsule powder comprises seabuckthorn fruit oil and polypeptide. The preparation method is to mix seabuckthorn fruit oil and polypeptide and then ball-mill them in a ball mill to obtain the seabuckthorn fruit oil microcapsule powder. The present invention forms a molecular mixture through the action of mechanical force, increases the dispersion uniformity of the drug and the carrier, increases the wettability, etc., to promote the dissolution of seabuckthorn fruit oil, and forms a complex through the inclusion effect of the carrier, thereby inhibiting the degradation of seabuckthorn fruit oil and the loss of its antioxidant activity; at the same time, the microcapsule powder can be assembled into a stable nano-microemulsion in water, with a refreshing taste and no greasiness. The antioxidant free radical scavenging ability of seabuckthorn fruit oil is increased, and the efficacy of seabuckthorn fruit oil in preventing non-alcoholic fatty liver is significantly improved. The preparation method is simple, easy to implement, low in cost, non-toxic and harmless.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to a polypeptide-loaded sea buckthorn fruit oil microcapsule powder, a preparation method thereof, and an application thereof. Background Art

[0002] With the improvement of people's living standards and the increase of life pressure, people's neglect of lifestyle and eating habits has led to an increasing number of obese and fatty liver patients. However, existing chemical drugs still cannot effectively treat them, and long-term medication is required with relatively large side effects. In recent years, the strategy of diet therapy and health preservation has been highly praised by consumers. Treating obesity and improving health problems such as fatty liver through diet regulation has a huge market prospect. However, most current functional foods only come from natural plants and are primary processed products or extracts, which need to be used in large doses to achieve better effects. Moreover, due to the lack of scientific and technological content, it is difficult to gain consumers' favor. Therefore, there is an urgent need for a high-tech, highly effective, and safe functional food for the treatment of fatty liver.

[0003] Sea buckthorn is a perennial deciduous shrub belonging to the genus Hippophae of the Elaeagnaceae family, with the Latin name Hippophae rhamnoides L., and is a traditional food. Sea buckthorn fruit oil is the most abundant functional oil in sea buckthorn, which is rich in a large amount of Omega-9 unsaturated fatty acids and has significant dietetic value. Scientific research has confirmed that sea buckthorn seed oil can help the skin repair damage and treat skin photoaging, such as repairing skin damage caused by radiotherapy, sunlight, and cosmetic lasers; it can also be used to treat some skin diseases such as eczema and burns. Oral administration of sea buckthorn fruit oil has the effect of significantly reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and triglyceride (TG) in hypercholesterolemic mice induced by CCl4 injection. Therefore, sea buckthorn fruit oil is a functional food with special efficacy for hyperlipidemia. However, the active ingredients of sea buckthorn fruit oil have poor water solubility, exist in the form of oil droplets in aqueous solutions, have a greasy taste and are prone to oxidative rancidity. Taking it directly has a very poor taste and ineffective efficacy, making it difficult to gain consumers' recognition and greatly limiting the further application of sea buckthorn fruit oil in foods. Therefore, developing sea buckthorn fruit oil products with good water solubility, strong stability, a refreshing taste, and better efficacy has practical significance for solving the above problems.

[0004] The main methods for water-solubilizing seabuckthorn fruit oil are the solvent method and the solvent spray drying method. These methods require the use of organic solvents, resulting in the problem of residual organic solvents. Moreover, there are problems such as complex preparation processes and increased loss rates of seabuckthorn fruit oil during the process. Therefore, how to avoid the above problems is the biggest obstacle in the development of water-soluble seabuckthorn fruit oil products. Using the mechanochemical method to prepare microcapsule powder does not require the use of organic solvents during the process, which can avoid environmental pollution caused by organic solvents during the reaction, and does not require heating, reducing the loss of active ingredients in the fruit oil. At the same time, by selecting natural sources and corresponding carriers that can promote the absorption of oil-soluble drugs in the body, nano-level emulsification can be achieved, improving the efficacy of seabuckthorn fruit oil. Therefore, this method is not only an innovation in the preparation process of traditional water-soluble functional health edible oil products, but also a major breakthrough in the preparation process of high-performance seabuckthorn fruit oil. Summary of the Invention

[0005] Aiming at the problems of poor water solubility, easy oxidation, poor taste and poor absorption in the body of seabuckthorn fruit oil in the existing technology, the purpose of the present invention is to provide a polypeptide-loaded seabuckthorn fruit oil microcapsule powder, its preparation method and application, which can improve the dissolution degree of seabuckthorn fruit oil in aqueous solution and reduce its volatility. The use of organic solvents is avoided in its preparation, meeting the requirements of environmental protection. At the same time, it further enhances the health care efficacy of seabuckthorn fruit oil, and develops new seabuckthorn fruit oil products with good taste, stability and good effects.

[0006] A polypeptide-loaded seabuckthorn fruit oil microcapsule powder, the components of which include seabuckthorn fruit oil and a polypeptide carrier material.

[0007] Furthermore, the seabuckthorn fruit oil is extracted from seabuckthorn fruits or seeds, and the extraction process adopts the supercritical extraction method or the solvent extraction method.

[0008] Furthermore, the polypeptide carrier material is selected from one of seabuckthorn peptide, whey protein peptide, soy protein peptide, wheat protein peptide, corn protein peptide, licorice polypeptide, astragalus polypeptide and millet polypeptide, and preferably seabuckthorn peptide.

[0009] Furthermore, the mass ratio of seabuckthorn fruit oil to the polypeptide carrier material is 1:1 - 1:100, and preferably 1:10.

[0010] A preparation method of a polypeptide-loaded seabuckthorn fruit oil microcapsule powder. According to the feeding ratio, add seabuckthorn fruit oil and the polypeptide carrier material into a ball mill tank lined with polytetrafluoroethylene and mix evenly. Add grinding media, then place the ball mill tank in a ball mill for ball milling, and then pass through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder.

[0011] Furthermore, the grinding media are stainless steel beads with a diameter of 5 - 25 mm, preferably 15 mm. The filling rate of the grinding media in the ball mill tank is 8% - 84%, which is controlled by the amount of steel beads added, and the preferred filling rate is 34%.

[0012] Furthermore, the ball mill is a drum - type ball mill with a rotation speed of 10 - 50 rpm, preferably 35 rpm.

[0013] Furthermore, the ball - milling time of the ball mill is 0.5 - 24 h, preferably 8 h.

[0014] The application of a polypeptide - loaded seabuckthorn fruit oil microcapsule powder in the preparation of foods, drugs, and health products for antioxidant use and improvement of non - alcoholic fatty liver.

[0015] By adopting the above - mentioned technology, compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1) The seabuckthorn fruit oil microcapsule powder of the present invention can be used as the main raw material to make various solid beverages and liquid beverages, and can also be other solid and liquid food raw materials, mainly functional foods. It is convenient to use, has good flexibility, is compatible with various food raw materials and additives, can be completely dispersed in the solution to form a nano - microemulsion, has a refreshing taste and no special smell, and its storage stability and antioxidant property are higher than those of the seabuckthorn fruit oil raw material, and it has the effects of antioxidant and improvement of non - alcoholic fatty liver.

[0017] 2) The microcapsule powder is prepared by the defined preparation method of the present invention. Its operation is simple. Compared with conventional preparation methods such as spray - drying method, it avoids the use of organic solvents, that is, it avoids the loss of seabuckthorn fruit oil that is extremely likely to occur during the process of removing the solvent. It has the advantages of simple operation, short preparation time, safe and reliable production, low production cost, and less pollution. It is a processing and preparation method of solid food raw materials with good popularization and application prospects. Description of the Drawings

[0018] Figure 1 It is the solubility of seabuckthorn fruit oil and the solubility of the active ingredient β - carotene in the microcapsule powder prepared by different natural protein peptide carriers;

[0019] Figure 2 It is the influence of the seabuckthorn fruit oil content on the solubility and dissolution rate of seabuckthorn fruit oil (A), the influence of the filling rate of the ball - milling tank (B), the rotation speed of the ball mill (C), and the ball - milling time (D) on the solubility of seabuckthorn fruit oil and the solubility of the active ingredient β - carotene in the prepared microcapsule powder;

[0020] Figure 3 It is the dissolution curve of seabuckthorn fruit oil / seabuckthorn peptide and seabuckthorn fruit oil in the microcapsule powder of Example 16;

[0021] Figure 4 It is the remaining content curve graph of total carotenoids in seabuckthorn fruit oil / seabuckthorn peptide and the microcapsule powder of Example 16 after being placed for different times;

[0022] Figure 5It is the transmission electron microscope image of the solution in Example 16;

[0023] Figure 6 It is the scavenging effects of four common free radicals of Example 16 and the physical mixture of seabuckthorn fruit oil / seabuckthorn peptides;

[0024] Figure 7 It is the therapeutic effect of Example 16 and the physical mixture of seabuckthorn fruit oil / seabuckthorn peptides on non-alcoholic liver injury in mice. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0026] Example 1: Determination of the solubility of seabuckthorn fruit oil

[0027] Accurately weigh 0.063 g of seabuckthorn fruit oil into a 25 ml volumetric flask and make up the volume with ethyl acetate. Respectively take 0.25, 0.50, 1.00, 1.25, 1.50 and 2.00 ml of the solution into 10 ml volumetric flasks, and make up the volume with ethyl acetate to obtain seabuckthorn fruit oil ethyl acetate solutions with concentration gradients of 0.063, 0.126, 0.252, 0.315, 0.378 and 0.504 mg / ml. After passing through a 0.45μm organic filter membrane, measure the absorbance value at 451nm. The standard curve equation obtained is: c 沙棘果油 (g / L) = 1.72Abs - 0.03 (R 2 = 0.9996). It shows that there is a good linear relationship within the detection concentration range.

[0028] Add an excessive amount of the sample to be measured into a conical flask containing distilled water, place it in a constant temperature oscillator for 24 h (37 °C, 150 rpm) to obtain a supersaturated solution, and perform three parallel experiments. Filter the obtained supersaturated solution through a 0.45 μm filter membrane, appropriately dilute it, and measure the content of seabuckthorn fruit oil in the aqueous solution by the above method.

[0029] Example 2: Determination of the content of β-carotene in seabuckthorn fruit oil

[0030] Liquid phase conditions: The chromatographic column is an ODS-3 C18 chromatographic column (4.6×250 mm, 5 μm); the flow rate is 1.0 mL / min; the injection volume is 10 μL; the column temperature is 30 °C; the mobile phase is methanol: water: glacial acetic acid = 75%:24.5%:0.5%; the detection time is 30 min; the detection wavelength is 440 nm. β-Carotene was diluted to solutions of 40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 3 μg / mL, and 1 μg / mL in a certain proportion. After the dilution solution was filtered through a 0.45 μm filter membrane, the above conditions were used for analysis. C 胡萝卜素 (mg / L) = 32679*PA - 10378, R 2 = 0.9991, n = 5. An excessive amount of the sample to be measured was added to a conical flask containing distilled water, and it was placed in a constant temperature oscillator for 24 h (37 °C, 150 rpm) to obtain a supersaturated solution. Three parallel experiments were conducted. The obtained supersaturated solution was filtered through a 0.45 μm filter membrane. After appropriate dilution, the content of β-carotene in the aqueous solution was measured by the above method.

[0031] Example 3:

[0032] In a 100 mL ball milling tank lined with polytetrafluoroethylene, seabuckthorn peptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, the extraction process uses supercritical extraction method, 0.6 g) were added. Then, 16 stainless steel ball milling beads with a diameter of 15 mm were added as the grinding medium. The filling rate was 34% ((ball volume × number of balls) / total volume of the ball milling tank). After mixing evenly, the ball milling tank was placed in a drum-type ball mill, and the set rotation speed was 30 rpm and the time was 4 hours. After ball milling, the powder was passed through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. An appropriate amount of this powder was placed in a conical flask, 10 mL of distilled water was added, and it was shaken at 37 °C and 150 rpm on a shaker for 24 h. After the supernatant was filtered through a 0.45 μm membrane, the solubility of seabuckthorn fruit oil and the solubility of β-carotene were detected by ultraviolet spectrophotometry and high performance liquid chromatography.

[0033] Example 4:

[0034] Add whey protein peptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37°C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil and the solubility of β-carotene therein.

[0035] Example 5:

[0036] Add wheat protein peptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37°C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil and the solubility of β-carotene therein.

[0037] Example 6:

[0038] Add soy protein peptides (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37°C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil and the solubility of β-carotene therein.

[0039] Example 7:

[0040] Add corn protein peptides (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder.

[0041] Example 8:

[0042] Add glycyrrhizin polypeptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37°C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil and the solubility of β-carotene therein.

[0043] Example 9:

[0044] Add astragalus polypeptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37 °C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil and the solubility of β-carotene therein.

[0045] Example 10:

[0046] Add millet polypeptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g) into a 100 mL ball milling jar, then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, place the ball milling jar into a drum-type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37 °C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil and the solubility of β-carotene therein.

[0047] Example 11:

[0048] To compare the solubilization effect, take seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g), place it in a conical flask, add 10 mL of distilled water, and shake it at 37 °C and 150 rpm on a shaker for 24 h. After passing the supernatant through a 0.45 μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect that the solubility of seabuckthorn fruit oil is 0.35 ± 0.07 g / L and the solubility of β-carotene is 0.75 ± 0.02 mg / L.

[0049] To compare the efficacy of peptide carriers, here, in a 100 mL ball milling jar, β-cyclodextrin (6.0 g), a common inclusion material, was added as a solubilizing carrier and seabuckthorn fruit oil (extracted from seabuckthorn fruit using the supercritical extraction method, 0.6 g). Then, 16 stainless steel ball milling beads with a diameter of 15 mm were added as grinding media. The filling rate was 34% ((ball volume × number of balls) / total volume of the ball milling jar). After mixing evenly, the ball milling jar was placed in a drum-type ball mill, and the rotation speed was set at 30 rpm for 4 hours. After ball milling, the powder was passed through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. After ball milling, the powder was passed through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. An appropriate amount of this powder was placed in a conical flask, 10 mL of distilled water was added, and it was shaken at 37 °C and 150 rpm on a shaker for 24 h. After the supernatant was passed through a 0.45 μm membrane, the solubility of seabuckthorn fruit oil was detected by ultraviolet spectrophotometry and high performance liquid chromatography to be 1.82 ± 0.02 g / L, and the solubility of β-carotene was 5.52 ± 0.03 mg / L.

[0050] The experimental results show that seabuckthorn fruit oil and the carotene in it are fat-soluble components, and their solubility in water is extremely low.

[0051] Examples 3 to 10 used natural polypeptides with different chemical structures as carriers to prepare seabuckthorn fruit oil-polypeptide microcapsule powder, and through the solubility of seabuckthorn fruit oil and β-carotene in it, as Figure 1 shown, the solubilizing effects of various plant protein peptides on seabuckthorn fruit oil and β-carotene in it vary greatly. Among them, most polypeptides have a better solubilizing effect on seabuckthorn fruit oil than seabuckthorn fruit oil itself and seabuckthorn fruit oil encapsulated with β-cyclodextrin. Further carrier optimization results show that seabuckthorn peptide has the best effect as a carrier, and the solubility of seabuckthorn fruit oil and β-carotene increases the most. Therefore, the optimal peptide carrier is seabuckthorn peptide.

[0052] Example 12: Optimization of carrier dosage

[0053] The process was optimized with a formula of seabuckthorn peptide loaded with seabuckthorn fruit oil. In a 100 mL ball milling tank, add seabuckthorn peptide (6g, 12g, 30g, 60g, 120g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 6.0g). Control the ratio of seabuckthorn fruit oil to seabuckthorn peptide as (1:1 - 1:20), add 16 stainless steel ball milling beads with a diameter of 15mm as the grinding medium, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling tank). After mixing evenly, place the ball milling tank into a drum type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, sieve the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. Take an appropriate amount of this powder and place it in a conical flask, add 10 mL of distilled water, and shake it at 37°C and 150 rpm on a shaker for 24 h. After filtering the supernatant through a 0.45μm membrane, use ultraviolet spectrophotometry and high performance liquid chromatography to detect the solubility of seabuckthorn fruit oil in it, and calculate the amount of solubilized seabuckthorn fruit oil accounting for the total added seabuckthorn fruit oil (dissolution rate of seabuckthorn fruit oil). The experimental results are shown in Figure 2 A shown in. The test results show that although the solubility of seabuckthorn fruit oil increases with the increase in the dosage of seabuckthorn fruit oil, the percentage of solubilized seabuckthorn fruit oil in the total seabuckthorn fruit oil decreases. By balancing the solubility and solubilization rate, the optimal content of seabuckthorn fruit oil in the prepared seabuckthorn fruit oil microcapsule powder is 10%.

[0054] Example 13: Optimization of the filling rate of the ball milling tank

[0055] The process was optimized with a formula of seabuckthorn peptide loaded with seabuckthorn fruit oil at a ratio of 10:1. In a 100 mL ball milling tank, add seabuckthorn peptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, extraction process using supercritical extraction method, 0.6 g), and add different numbers of ball milling beads as the grinding medium. The diameter of the ball milling beads is 15mm, and the filling rates are controlled at 8%, 16%, 25%, 34%, 58%, 67%, 75%, and 84% respectively. After mixing evenly, place the ball milling tank into a drum type ball mill, set the rotation speed to 30 rpm, and the time to 4 hours. After ball milling, sieve the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. The steps for detecting the solubility are the same as in Example 3. The solubility of seabuckthorn fruit oil and β-carotene in the prepared seabuckthorn fruit oil microcapsule powder is as shown in Figure 2 B shown in. The test results show that when the filling rate is too low, the grinding is insufficient and the solubilization effect is poor; while when the filling rate is too high, the running space of the grinding medium decreases and the solubilization effect becomes worse. Therefore, the preferred filling rate is 34%.

[0056] Example 14: Optimization of the rotation speed of the ball mill

[0057] The process was optimized with a formula of seabuckthorn peptide loaded with seabuckthorn fruit oil at a ratio of 10:1. In a 100 mL ball milling jar, add seabuckthorn peptide (6.0 g) and seabuckthorn fruit oil (0.6 g, extracted from seabuckthorn fruit or seeds using supercritical extraction method or solvent extraction method), then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm and the filling rate is 34%. After mixing evenly, place the ball milling jar into a drum-type ball mill, and control the rotation speed of the ball mill at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 rpm for 4 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. The steps for detecting solubility are the same as in Example 3. The solubility of seabuckthorn fruit oil and β-carotene in the prepared seabuckthorn fruit oil microcapsule powder is as shown in Figure 2 C in it. The test results show that too low rotation speed leads to insufficient grinding and dispersion, resulting in poor solubilization effect; while too high grinding rate increases the heat release of the system, causing oxidation and polymerization of the essential oil and a significant decrease in solubility. Therefore, the preferred rotation speed is 35 rpm.

[0058] Example 15: Optimization of ball milling time

[0059] In a 100 mL ball milling jar, add seabuckthorn peptide (6.0 g) and seabuckthorn fruit oil (0.6 g), then add 16 stainless steel ball milling beads as grinding media. The diameter of the ball milling beads is 15 mm and the filling rate is 34%. After mixing evenly, place the ball milling jar into a drum-type ball mill, control the rotation speed of the ball mill at 30 rpm, start ball milling, and take samples at 0.5, 1, 2, 4, 8, 16, 24 hours respectively. The steps for detecting solubility are the same as in Example 3. The solubility of seabuckthorn fruit oil and β-carotene in the prepared seabuckthorn fruit oil microcapsule powder is as shown in Figure 2 D in it. The test results show that when the ball milling time is short, seabuckthorn fruit oil fails to fully contact with the carrier, resulting in poor solubilization effect; while with the extension of ball milling time, the solubilization effect gradually decreases and the solubility tends to be stable. Therefore, considering energy conservation, the preferred ball milling time is 8 h.

[0060] Example 16: Optimal process and conditions for seabuckthorn fruit oil microcapsule powder

[0061] After formula and process optimization, the preparation method of seabuckthorn fruit oil microcapsule powder prepared with seabuckthorn peptide as the carrier is as follows. Add seabuckthorn polypeptide (6.0 g) and seabuckthorn fruit oil (extracted from seabuckthorn fruit, the extraction process uses supercritical extraction method, 0.6 g) into a 100 mL ball milling tank, then add 16 stainless steel ball milling beads as the grinding medium. The diameter of the ball milling beads is 15 mm, and the filling rate is 34% ((ball volume × number of balls) / total volume of the ball milling tank). After mixing evenly, put the ball milling tank into a drum type ball mill, set the rotation speed to 35 rpm, and the time to 8 hours. After ball milling, pass the powder through a 100-mesh sieve to obtain seabuckthorn fruit oil microcapsule powder. The solubility of seabuckthorn fruit oil in this microcapsule powder is 9.21 ± 0.11 g / L, and the solubility of β-carotene is 72.71 ± 4.72 mg / L.

[0062] Example 17: Dissolution experiment

[0063] Taking seabuckthorn fruit oil and its main component β-carotene as indicators, the dissolution rate of seabuckthorn fruit oil in the microcapsule powder was measured. The method is as follows: Accurately weigh Example 16 (0.55 g, equivalent to about 50 mg of seabuckthorn fruit oil) and the physical mixture of seabuckthorn fruit oil (50 mg) and seabuckthorn peptide (0.5 g) (abbreviated as seabuckthorn fruit oil / seabuckthorn peptide) respectively for the dissolution experiment. The paddle method is used: that is, 900 mL of purified water is used as the dissolution medium, the temperature is 37 o °C, the rotation speed is 100 rpm, and the dissolution time is 120 min; Take 5 mL of each solution at 5 min, 10 min, 20 min, 30 min, 60 min, 75 min, 90 min, and 120 min respectively. Immediately after sampling, add 5 mL of 37 o °C purified water to the dissolution tank. The sampled solution is filtered through a 0.45-μm microporous filter membrane and used as the test sample. The concentration of seabuckthorn fruit oil is measured by ultraviolet spectrophotometry. The dissolution curve of seabuckthorn fruit oil measured by solubility is shown in Figure 3 . From the solubilization effect on seabuckthorn fruit oil, it can be seen that the microcapsule powder shown in Example 16 not only significantly improves the solubility of seabuckthorn fruit oil and its key components, but also can effectively promote the rapid dissolution of seabuckthorn fruit oil, and is significantly superior to the physical mixture, indicating that seabuckthorn peptide has good surface activity and promotes the water dispersion of lipophilic seabuckthorn fruit oil.

[0064] Example 18: Accelerated aging assessment of seabuckthorn fruit oil microcapsule powder

[0065] Take the microcapsule powder of Example 16 (5.5 g, with a seabuckthorn fruit oil content of about 0.5 g) and the physical mixture of seabuckthorn fruit oil and seabuckthorn peptide at a ratio of 1:10 (5.5 g, with about 0.5 g of seabuckthorn fruit oil). Spread the samples flat to make the thickness uniform, seal them with an aluminum foil bag, and place them in an assessment box (set temperature 45 oPlace it continuously for 6 months at a humidity of C±2 and RH of 65%±2. Then, take 0.55 g of samples at 0.5, 1, 2, 3, 4, 5, and 6 months respectively, dissolve them with 95% ethanol, make the volume up to 100 mL, dilute to a certain concentration, record the dilution factor, and use HPLC to determine the mass m of β-carotene in the sampled samples. t , the remaining amount of β-carotene = (m t / m0) × 100%; The results are as Figure 4 shown. Example 16 was subjected to accelerated aging assessment, and the remaining amount of β-carotene could still reach more than 95%, indicating that all examples have good stability and can be stored at room temperature for a long time.

[0066] Example 19: Observation of the microscopic morphology of the solution

[0067] To further observe the structure of the nano-microcapsules formed in the aqueous solution of Example 16 ( Figure 5 ), a JEM 2100EX type transmission electron microscope was used to observe the morphology of the nano-microcapsules at an accelerating voltage of 100 kV. First, dilute the microcapsule powder of Example 16 in ultrapure water and sonicate for 5 min, then place it on a copper grid, and then stain it with uranyl acetate (2% w / w). The results show that there are a large number of microcapsules in the solution under the microscope. The microcapsules are in a regular spherical structure with uniform dispersion, and the color inside the sphere is significantly darker, which is significantly different from the light-colored part on the periphery, indicating that the sea buckthorn fruit oil has been successfully encapsulated by sea buckthorn peptides, and a micelle-like self-assembly behavior has occurred in the aqueous solution.

[0068] Furthermore, the particle size and ζ potential of Example 16 were measured by using a Malvern Nano ZS90 dynamic light scattering nanoparticle size analyzer. Dissolve Example 16 in distilled water (0.1 mg / ml) at 25°C, then take an appropriate amount of the solution and place it in the detector for measurement, and measure continuously for 20 times. The experimental results show that the particle size of the microcapsule powder solution of Example 16 is 131.30 nm, and the ζ value is (-25.2 ± 0.3). The microcapsule powder of Example 16 forms stable nano-scale microcapsules in the solution.

[0069] Example 20: Study on the free radical scavenging ability of sea buckthorn fruit oil microcapsule powder

[0070] Through experiments, the antioxidant capacities of different concentrations of Example 16 and the sea buckthorn fruit oil / peptide mixture were determined, with vitamin C as a control. As Figure 6 the results show, the conjugated structures such as carotenoids and unsaturated fatty acids in the sea buckthorn fruit oil determine its excellent antioxidant performance. Figure 6The scavenging rate results of DPPH free radicals, superoxide free radicals, hydroxyl free radicals and ABTS free radicals by Example 16, seabuckthorn fruit oil / peptide mixture and VC are respectively shown. The results indicate that when the concentration of the positive control vitamin C is 0.8 mg / mL, the scavenging rates for DPPH, superoxide anion, hydroxyl and ABTS free radicals are 85.2%, 73.2%, 65.32% and 97.10% respectively. Compared with the seabuckthorn fruit oil / peptide mixture, the ability of Example 16 to scavenge the above four types of free radicals has been significantly improved, with the scavenging rate increasing by 5%-10%. The scavenging effect on hydroxyl free radicals is even better than that of VC, fully demonstrating that Example 16 has more excellent free radical scavenging effect and better antioxidant ability.

[0071] Example 21: Study on the Therapeutic Effect on Non-alcoholic Fatty Liver

[0072] Forty healthy male ICR mice were randomly divided into a normal group, a model group, an SJY group, and an SJY-SJT group. The mice were given a high-fat diet, and at 10 am every day for 5 consecutive days, they were intraperitoneally injected with a tetracycline saline solution at a dose of 15 mg / 100 g to establish a non-alcoholic liver injury model. In the SJY group, seabuckthorn fruit oil and seabuckthorn peptide were suspended in pure water and administered by gavage at a dose of 30 mg / 100 g in a ratio of 1:10; in the SJY-SJT group, Example 16 was dissolved in pure water and administered by gavage at a dose of 30 mg / 100 g; the normal control group and the model group were given pure water by gavage according to body weight (the normal group was normal animals, and the model group was non-alcoholic liver injury model animals without treatment); the administration was continued for 15 days. After the end, the body weights of the mice in each group were weighed, and the livers were dissected, fixed, stained and sectioned ( Figure 7 ), and the corresponding kits were used to measure the biochemical index indexes such as total cholesterol, triglyceride, high-density lipoprotein detection, low-density lipoprotein, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase in serum and liver homogenate, as shown in Figure 7 .

[0073] The results of blood biochemical indexes of mice showed that compared with the normal control group, the serum total TG, TC, HDL-C, LDL-C, AST and ALT in the model group were significantly increased (*P<0.01), indicating the formation of liver injury; while both the SJY-SJT group and the SJY group could effectively reduce the related factors, and the serum TC, HDL-C, ALT and LDL-C indexes in the SJY-SJT group were significantly better than those in the SJY group (P<0.01). Further detection of liver-related factors and liver sections showed that the liver TC and LDL-C in the SJY-SJT group were decreased. Observation of liver sections showed that the hepatic cords in the SJY-SJT group were arranged neatly, and the hepatic sinusoids were not significantly dilated or compressed; there were more hepatocytes with slightly loose cytoplasm (black arrows) in the tissue; there were few lymphocyte and neutrophil infiltrations, which was close to the normal group, better than the SJY group, and significantly better than the model group. The above experimental results showed that sea buckthorn fruit oil could relieve fatty liver to a certain extent, and after being prepared into sea buckthorn fruit oil microcapsule powder - Example 16, its ability to relieve fatty liver was significantly increased, and it was more suitable for development as a functional food.

Claims

1. A preparation method of polypeptide-loaded sea buckthorn fruit oil microcapsule powder, characterized in that According to the feeding ratio, seabuckthorn fruit oil and seabuckthorn peptides are added into a ball milling tank lined with polytetrafluoroethylene and mixed evenly. Then, grinding media are added, and the ball milling tank is placed in a ball mill for ball milling. Subsequently, after passing through a 100-mesh sieve, seabuckthorn fruit oil microcapsule powder is obtained. The ball mill is a drum-type ball mill with a rotation speed of 10 - 50 rpm.

2. The preparation method of a polypeptide-loaded seabuckthorn fruit oil microcapsule powder according to claim 1, wherein Seabuckthorn fruit oil is extracted from seabuckthorn fruits or seeds, and the extraction process adopts supercritical extraction method or solvent extraction method.

3. The preparation method of a polypeptide-loaded seabuckthorn fruit oil microcapsule powder according to claim 1, characterized in that, The feeding ratio of seabuckthorn fruit oil and seabuckthorn peptides is 1:1 - 1:

100.

4. The preparation method of a polypeptide-loaded seabuckthorn fruit oil microcapsule powder according to claim 1, characterized in that, The grinding media are stainless steel beads with a diameter of 5 - 25 mm, and the filling rate of the grinding media in the ball milling tank is 8% - 84%, which is controlled by the addition amount of the steel beads.

5. The preparation method of a polypeptide-loaded seabuckthorn fruit oil microcapsule powder according to claim 1, characterized in that, The ball milling time of the ball mill is 0.5 - 24 h.

6. Use of the polypeptide-loaded seabuckthorn fruit oil microcapsule powder prepared by any one of the preparation methods of claims 1-5 in the preparation of drugs, characterized in that, Used for preparing drugs for antioxidant and improving non-alcoholic fatty liver.

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