High-stability blueberry anthocyanin polycystic liposome as well as preparation method and application thereof

Abstract

The invention discloses a high-stability blueberry anthocyanin polycystic liposome as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, preparing an internal water phase containing pure water, an organic acid buffer solution, blueberry anthocyanin and an osmotic pressure regulator; s2, preparing an oil phase, and mixing neutral lipoid, lipid, an antioxidant and an organic solvent; s3, preparing an external water phase; s4 / S5 mixing and homogenizing, and removing the solvent to obtain liposome; according to the invention, a multi-vesicular liposome preparation technology which is mostly applied in the pharmaceutical field is applied to the field of food, and meanwhile, an organic acid buffer solution is used as an acidity regulator and an anthocyanin protective agent, so that the stability of blueberry anthocyanin is greatly improved while the encapsulation stability of the multi-vesicular liposome is improved, and the application of the multi-vesicular liposome in the field of food is widened; the prepared blueberry anthocyanin polycystic liposome is applied to a health-care capsule shell, and the capsule shell which can change color along with time is obtained and can be used for indicating the storage time.

Description

Technical Field

[0001] The invention belongs to the field of anthocyanin preparation, and specifically discloses a high-stability blueberry anthocyanin multivesicular liposome and a preparation method and application thereof. Background Art

[0002] In recent years, anthocyanins have received extensive attention for their multiple physiological functions such as antioxidant, anti-tumor, vision protection, cardiovascular and liver health. This natural pigment belonging to the flavonoids is widely distributed in nature. They give the roots, stems, flowers, leaves and fruits of plants a variety of colors such as blue, purple, and red. In addition, anthocyanins have shown great application potential in the pharmaceutical, food and cosmetics industries, and are highly valued for their rich nutritional value and pharmacological effects. Blueberry anthocyanins are mainly extracted from the fruits of blueberries, a plant of the genus Vaccinium in the Ericaceae family, and are converted into powder form through enzymatic hydrolysis, water extraction, purification, concentration and drying. The substance was officially approved as a new food ingredient on May 7, 2023. However, due to the characteristics of its molecular structure, blueberry anthocyanins are extremely sensitive to light, temperature and oxygen, and are easily decomposed and inactivated. At present, microencapsulation technology is mainly used to enhance its stability by coating with cellulose and casein, but the effect still needs to be improved. The stability issue has become a key factor restricting the development of the blueberry anthocyanin market, and further research and solutions are urgently needed.

[0003] Liposomes are tiny spherical vesicles composed of phospholipid molecules, and their structure is highly similar to that of cell membranes. These phospholipid molecules can self-assemble in aqueous solutions to form a lipid bilayer structure. In the bilayer structure, the hydrophobic parts of the molecules are close to each other, while the hydrophilic heads are facing the outside of the bilayer. This unique structure gives liposomes the ability to encapsulate hydrophobic and hydrophilic substances, and can effectively encapsulate components of different polarities in the lipid bilayer to form a physical barrier to protect bioactive ingredients from damage by adverse external conditions. Multivesicular liposomes are a special type of liposomes with an internal structure similar to a honeycomb, with multiple aqueous chambers separated by multiple non-concentric lipid bilayer membranes. This structure gives multivesicular liposomes significant advantages in the field of drug delivery, especially in improving the stability of hydrophilic drugs. In the field of nutritional health products, liposomes are gradually expanding their application range due to their protective effects, but limited by the characteristics of phospholipids, most products are oil-soluble raw materials or oil-water insoluble raw materials converted into water-soluble ones. In contrast, multivesicular liposomes are more suitable for protecting water-soluble health product raw materials, but their application in this field is not yet widespread. Summary of the invention

[0004] To solve the above problems, the present invention discloses a high-stability blueberry anthocyanin multivesicular liposome and a preparation method and application thereof. The multivesicular liposome technology is used to encapsulate blueberry anthocyanin, greatly improve the light and thermal stability of blueberry anthocyanin, prepare high-stability blueberry anthocyanin multivesicular liposome and open up new application fields thereof.

[0005] The present invention includes the following technical solutions:

[0006] A high-stability blueberry anthocyanin multivesicular liposome, composed of the following raw materials in parts by weight:

[0007]

[0008] Furthermore, in the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, the organic acid buffer a or the organic acid buffer b is a citric acid-sodium citrate buffer with a pH value between 4.2 and 6.6.

[0009] Furthermore, in the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, the osmotic pressure regulator a or osmotic pressure regulator b is selected from one or a mixture of glucose, sucrose or sorbitol, preferably glucose.

[0010] Furthermore, in the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, the neutral lipid is selected from one or a mixture of caprylic decanoic acid glyceryl, triolein, and soybean oil, preferably caprylic decanoic acid glyceryl.

[0011] Furthermore, in the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, the lipid is selected from one or a mixture of egg yolk lecithin and modified soybean lecithin.

[0012] Furthermore, in the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, the antioxidant is selected from one or a mixture of tocopherol, ascorbyl palmitate, tert-butylhydroquinone, and tea polyphenol palmitate, preferably tocopherol or ascorbyl palmitate.

[0013] Furthermore, in the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, the emulsifier is one or a mixture of Tween 80, sodium alginate, and sucrose fatty acid esters, preferably sucrose fatty acid esters.

[0014] The present invention also discloses a method for preparing the above-mentioned high-stability blueberry anthocyanin multivesicular liposomes, comprising the following steps:

[0015] Add organic acid buffer a, blueberry anthocyanin, and osmotic pressure regulator a into pure water S1 to prepare the inner water phase;

[0016] S2: neutral lipids, lipids, and antioxidants are mixed and dissolved in 80 wt% edible ethanol to obtain an oil phase;

[0017] S3 mixes organic acid buffer b, emulsifier, osmotic pressure regulator b and pure water to prepare an external water phase;

[0018] S4 mixed the inner water phase and oil phase obtained by S1 and S2, and sheared and homogenized at 15000 rpm for 5 min to obtain a W / O emulsion;

[0019] S5: mixing the W / O emulsion and the external aqueous phase, shearing and homogenizing at 15000 rpm for 5 min to obtain a W / O / W double emulsion, and removing the organic solvent by rotary evaporation to obtain the high-stability blueberry anthocyanin multivesicular liposomes.

[0020] The present invention also discloses the use of the high-stability blueberry anthocyanin multivesicular liposomes in preparing a material that can change color over time. Preferably, the material is a capsule skin that can change color over time.

[0021] The present invention discloses a capsule skin capable of changing color over time, which is prepared by the following steps:

[0022] S1: weigh 300-800 parts of gelatin, 100-300 parts of glycerin and 300-800 parts of pure water, put them into a gelatinizing tank, heat at 50-70°C, and stir at 50-200rpm to dissolve the gelatin to make a gelatin solution;

[0023] Add 3-9 portions of the high-stability blueberry anthocyanin multivesicular liposomes described in any one of claims 1-7 to the S2 glue solution, stir evenly at 10-100 rpm, and keep warm at 45-55°C for 10-60 min;

[0024] S3 pours the glue solution into a capsule forming machine to obtain a soft capsule skin.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention realizes new applications of the technology beyond the medical field by encapsulating blueberry anthocyanins in multivesicular liposomes. Compared with conventional liposome technology, multivesicular liposome technology retains the original solubility characteristics of the raw materials, provides a new encapsulation and protection method for blueberry anthocyanins and other water-soluble nutritional health care raw materials, and does not limit their use in downstream applications. In addition, the raw materials used in the present invention are all food grade, which is conducive to large-scale production in the future.

[0027] (2) The organic acid buffer used in the present invention serves as both an anthocyanin protective agent and an acidity regulator for multivesicular liposomes. On the one hand, the organic acid undergoes an acylation reaction with anthocyanins, and the acylation site is mainly located at the sugar group at the C3 or C6 position of the anthocyanin. This reaction results in the formation of a hydrophobic interaction between the anthocyanin nucleus and the acyl residue, thereby constructing a "sandwich" sandwich structure. In this structure, the acyl group tightly surrounds the 2-phenylbenzopyran skeleton of the anthocyanin, providing protection for the anthocyanin nucleus through a steric hindrance effect. On the other hand, the acidic environment promotes the anthocyanin molecules to form non-covalent chiral supramolecular complexes, enhances the self-aggregation of anthocyanins, and thus inhibits hydration. In addition, the organic acid buffer helps to maintain the pH environment of the multivesicular liposomes stable, prevents excessively low pH values ​​from inducing phospholipid oxidation and hydrolysis, and thus protects the structural integrity of the multivesicular liposomes.

[0028] (3) The present invention cleverly applies the prepared high-stability blueberry anthocyanin multivesicular liposomes to the production of capsule skins. This innovative measure not only gives the capsule skins new functional properties, but also greatly enhances their application value. Specifically, due to the unique sustained-release properties and stable multivesicular structure of multivesicular liposomes, the capsule skin can maintain a complete multivesicular particle morphology in the initial stage of molding. At this time, the light transmittance is low and the capsule presents an elegant purple color. However, as the storage time gradually increases, the multivesicular particles begin to gradually break, and the blueberry anthocyanin molecules encapsulated inside are released and gradually penetrate into the surface of the capsule skin, causing the color of the capsule to gradually deepen. This characteristic of changing color over time provides consumers with an intuitive and convenient way to judge the storage time of the capsule. By simply observing the color change of the capsule skin, consumers can roughly estimate the freshness and shelf life of the capsule, thereby avoiding the health risks that may be caused by using expired capsules. In addition, this color-changing characteristic also provides an effective quality control method for the production, circulation and use of capsules, which helps to ensure the safety and effectiveness of capsule products.

[0029] Therefore, the application of high-stability blueberry anthocyanin multivesicular liposomes in the preparation of capsule skin not only realizes the innovation of capsule skin function, but also provides great convenience for the quality control of capsule products and consumer use. It has broad market application prospects and important social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an optical microscope photo of the multivesicular liposomes of Example 3;

[0031] Figure 2 The anthocyanin retention rate bar graph of blueberry anthocyanin in multivesicular liposomes of the embodiment and comparative example is tested for light stability and heat stability. DETAILED DESCRIPTION

[0032] The technical scheme in the embodiment of the present invention is clearly and completely described below, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The reagent or instrument used in the embodiment of the present invention do not indicate the manufacturer, and are all conventional reagent products that can be obtained by commercial purchase.

[0033] Test method:

[0034] (1) Determination of blueberry anthocyanin content: The total anthocyanin content was determined by the pH differential method. The product was dissolved in methanol containing 0.1% hydrochloric acid to prepare a solution with a concentration of 1 mg / mL. Subsequently, the solution was diluted with buffer solutions with pH values ​​of 1.0 and 4.5 at a ratio of 1:100 and equilibrated at 37°C for 60 minutes. After equilibration, the absorbance was measured at 518 nm using a UV spectrophotometer, and the absorbance at 700 nm was used as the calibration reference. The total anthocyanin calculation formula is:

[0035]

[0036] Wherein, ε is the molar extinction coefficient, 26900L / (mol*cm); L is the optical path, 1cm; MW is the relative molecular mass of cyanidin anthocyanin, 449.2g / mol; DF is the dilution factor; V is the total volume, L; M is the sample mass, g.

[0037] (2) Thermal stability test: The prepared blueberry anthocyanin multivesicular liposomes were placed in a constant temperature water bath at 85°C for 3 h. After cooling, the total anthocyanin content was determined. The anthocyanin preservation rate (%) was calculated by total anthocyanin content before determination (mg / g) / total anthocyanin content after determination (mg / g) × 100%.

[0038] (3) Photostability test: The prepared blueberry anthocyanin multivesicular liposomes were placed under a 40 W, 250 nm ultraviolet light source and irradiated continuously for 3 h. After the irradiation, the total anthocyanin content was measured. The anthocyanin preservation rate (%) was calculated by total anthocyanin content before measurement (mg / g) / total anthocyanin content after measurement (mg / g) × 100%.

[0039] Example 1

[0040] According to the formula, the mass ratios of the components of a high-stability blueberry anthocyanin multivesicular liposome are: 1 part of pH 5.3 citric acid-sodium citrate buffer a, 1 part of 36% blueberry anthocyanin, 1 part of glucose a, 5 parts of caprylic and capric acid glyceride, 5 parts of egg yolk lecithin, 1 part of tocopherol, 8 parts of pH 5.3 citric acid-sodium citrate buffer b, 8 parts of sucrose fatty acid ester, 16 parts of glucose b, and 110 parts of pure water.

[0041] The preparation steps include:

[0042] S1 adds pH 5.3 citric acid-sodium citrate buffer a, 36% blueberry anthocyanin, and glucose a to pure water according to the formula to prepare the inner water phase;

[0043] S2: caprylic and capric glyceride, egg yolk lecithin, and tocopherol are mixed according to the formula amount, and dissolved in 80wt% edible ethanol to obtain an oil phase;

[0044] S3: Mix pH 5.3 citric acid-sodium citrate buffer b, sucrose fatty acid ester, glucose b and pure water according to the formula to prepare an external water phase;

[0045] S4 mixed the inner water phase and oil phase obtained by S1 and S2, and sheared and homogenized at 15000 rpm for 5 min to obtain a W / O emulsion;

[0046] S5: mixing the W / O emulsion and the external aqueous phase, shearing and homogenizing at 15000 rpm for 5 min to obtain a W / O / W double emulsion, and removing the organic solvent by rotary evaporation to obtain the high-stability blueberry anthocyanin multivesicular liposomes.

[0047] Example 2

[0048] According to the formula, the mass ratio of each component of a high-stability blueberry anthocyanin multivesicular liposome is:

[0049] 3 parts of pH 5.3 citric acid-sodium citrate buffer a, 2 parts of 36% blueberry anthocyanins, 5 parts of glucose a, 7 parts of caprylic acid glyceride, 5 parts of egg yolk lecithin, 5 parts of tocopherol, 15 parts of pH 5.3 citric acid-sodium citrate buffer b, 10 parts of sucrose fatty acid ester, 20 parts of glucose b, and 130 parts of pure water.

[0050] The preparation steps are the same as in Example 1.

[0051] Example 3

[0052] According to the formula, the mass ratio of each component of a high-stability blueberry anthocyanin multivesicular liposome is:

[0053] 5 parts of pH 5.3 citric acid-sodium citrate buffer a, 15 parts of 36% blueberry anthocyanins, 10 parts of glucose a, 11 parts of caprylic acid glyceride, 19 parts of egg yolk lecithin, 12 parts of tocopherol, 30 parts of pH 5.3 citric acid-sodium citrate buffer b, 20 parts of sucrose fatty acid ester, 30 parts of glucose b, and 150 parts of pure water.

[0054] The preparation steps are the same as those in Example 1. The optical microscope photo of the prepared stable blueberry anthocyanin multivesicular liposomes is shown in FIG. Figure 1 shown.

[0055] Example 4

[0056] According to the formula, the mass ratios of the components of a high-stability blueberry anthocyanin multivesicular liposome are: 7 parts of pH 5.3 citric acid-sodium citrate buffer a, 15 parts of 36% blueberry anthocyanin, 15 parts of glucose a, 14 parts of caprylic and capric glyceride, 25 parts of egg yolk lecithin, 12 parts of tocopherol, 50 parts of pH 5.3 citric acid-sodium citrate buffer b, 50 parts of sucrose fatty acid ester, 50 parts of glucose b, and 180 parts of pure water.

[0057] The preparation steps are the same as in Example 1.

[0058] Example 5

[0059] According to the formula, the mass ratios of the components of a high-stability blueberry anthocyanin multivesicular liposome are: 9 parts of pH 5.3 citric acid-sodium citrate buffer a, 15 parts of 36% blueberry anthocyanin, 24 parts of glucose a, 30 parts of caprylic and capric glyceride, 30 parts of egg yolk lecithin, 12 parts of tocopherol, 80 parts of pH 5.3 citric acid-sodium citrate buffer b, 80 parts of sucrose fatty acid ester, 80 parts of glucose, and 210 parts of pure water.

[0060] The preparation steps are the same as in Example 1.

[0061] Comparative Example 1

[0062] The pH 5.3 citric acid-sodium citrate buffer a and the pH 5.3 citric acid-sodium citrate buffer b in Example 1 were replaced with pure water, and the other raw materials remained unchanged.

[0063] The preparation steps are the same as in Example 1.

[0064] Comparative Example 2

[0065] The pH 5.3 citric acid-sodium citrate buffer a and the pH 5.3 citric acid-sodium citrate buffer b in Example 2 were replaced with pure water, and the other raw materials remained unchanged.

[0066] The preparation steps are the same as in Example 1.

[0067] Comparative Example 3

[0068] The pH 5.3 citric acid-sodium citrate buffer a and the pH 5.3 citric acid-sodium citrate buffer b in Example 3 were replaced with pure water, and the other raw materials remained unchanged.

[0069] The preparation steps are the same as in Example 1.

[0070] Comparative Example 4

[0071] The pH 5.3 citric acid-sodium citrate buffer a and the pH 5.3 citric acid-sodium citrate buffer b in Example 4 were replaced with pure water, and the other raw materials remained unchanged.

[0072] The preparation steps are the same as in Example 1.

[0073] Comparative Example 5

[0074] The pH 5.3 citric acid-sodium citrate buffer a and the pH 5.3 citric acid-sodium citrate buffer b in Example 5 were replaced with pure water, and the other raw materials remained unchanged.

[0075] The preparation steps are the same as in Example 1.

[0076] Comparative Example 6

[0077] Unprocessed 36% blueberry anthocyanins raw material.

[0078] Test Case

[0079] The products in the above examples 1-5 and comparative examples 1-6 were subjected to light stability test and heat stability test. The results are as follows: Figure 2 As shown, it is shown that the low pH of blueberry anthocyanins in the comparative example formula will significantly affect the embedding stability of multivesicular liposomes because there is no organic acid buffer, and the structural damage leads to leakage of contents, but due to the presence of antioxidants, the anthocyanin retention rate is still significantly higher than that of untreated raw materials. The photothermal stability of the multivesicular liposome blueberry anthocyanin products in all example formulas is significantly higher than that of the untreated blueberry anthocyanin raw materials, and the multivesicular liposomes significantly improve the photothermal stability of blueberry anthocyanins.

[0080] Example 6

[0081] A method for preparing a color-changing capsule skin for indicating storage time comprises the following steps:

[0082] S1: weigh 500g of gelatin, 200g of glycerin and 500g of pure water and put them into a gelatinizing tank, heat at 60℃, and stir at 100rpm to dissolve the gelatin to make a gelatin solution;

[0083] Add 6 g of blueberry anthocyanin multivesicular liposomes prepared in Example 3 into the S2 gel solution, stir evenly at 20 rpm and keep warm at 50°C for 30 min;

[0084] S3 pours the glue solution into a capsule forming machine to obtain a soft capsule skin.

[0085] The prepared soft capsule skin was stored and the lab color difference value during the storage period was tested. The results are shown in Table 1.

[0086] Table 1 Lab color difference values ​​of soft capsule skin during storage period

[0087]

[0088]

[0089] As can be seen from the data in Table 1, due to the sustained-release properties of multivesicular liposomes and the stable large multivesicular particles, the multivesicular particles are intact at the beginning of capsule formation, and the capsules with low light transmittance are light purple; as the storage time increases, the multivesicular particles break, the blueberry anthocyanin molecules inside are released, and the color of the capsule gradually darkens. By simply observing the color change of the capsule skin, consumers can roughly estimate the freshness and shelf life of the capsule, thereby avoiding the health risks that may be caused by using expired capsules. In addition, this color change characteristic also provides an effective quality control method for the production, circulation and use of capsules, which helps to ensure the safety and effectiveness of capsule products.

[0090] The above embodiments show and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A high-stability blueberry anthocyanin multivesicular liposome, characterized in that: It is composed of the following raw materials in parts by weight:

2. The high-stability blueberry anthocyanin multivesicular liposome according to claim 1, characterized in that: The organic acid buffer a or the organic acid buffer b is a citric acid-sodium citrate buffer with a pH value between 4.2 and 6.

6.

3. The high-stability blueberry anthocyanin multivesicular liposome according to claim 1, characterized in that: The osmotic pressure regulator a or osmotic pressure regulator b is selected from one or a mixture of glucose, sucrose or sorbitol, preferably glucose.

4. The high-stability blueberry anthocyanin multivesicular liposome according to claim 1, characterized in that: The neutral lipid is selected from one or a mixture of caprylic acid decanoic acid glyceride, triolein, and soybean oil, preferably caprylic acid decanoic acid glyceride.

5. The high-stability blueberry anthocyanin multivesicular liposome according to claim 1, characterized in that: The lipid is selected from egg yolk lecithin, modified soybean lecithin or a mixture of the two.

6. The high-stability blueberry anthocyanin multivesicular liposome according to claim 1, characterized in that: The antioxidant is selected from tocopherol, ascorbyl palmitate, tert-butylhydroquinone, tea polyphenol palmitate or a mixture of several thereof, preferably tocopherol or ascorbyl palmitate.

7. The high-stability blueberry anthocyanin multivesicular liposome according to claim 1, characterized in that: The emulsifier is one or a mixture of Tween 80, sodium alginate, and sucrose fatty acid ester, preferably sucrose fatty acid ester.

8. The method for preparing high-stability blueberry anthocyanin multivesicular liposomes according to any one of claims 1 to 7, characterized in that: The following steps are involved: Add organic acid buffer a, blueberry anthocyanin, and osmotic pressure regulator a into pure water S1 to prepare the inner water phase; S2: neutral lipids, lipids, and antioxidants are mixed and dissolved in 80 wt% edible ethanol to obtain an oil phase; S3 mixes organic acid buffer b, emulsifier, osmotic pressure regulator b and pure water to prepare an external water phase; S4 mixed the inner water phase and the oil phase obtained by S1 and S2, and sheared and homogenized at 15000 rpm for 5 min to obtain a W / O emulsion; S5: mixing the W / O emulsion and the external aqueous phase, shearing and homogenizing at 15000 rpm for 5 min to obtain a W / O / W double emulsion, and removing the organic solvent by rotary evaporation to obtain the high-stability blueberry anthocyanin multivesicular liposomes.

9. Use of the high-stability blueberry anthocyanin multivesicular liposomes according to any one of claims 1 to 7 in preparing a material that can change color over time, preferably, the material is a capsule skin that can change color over time.

10. A capsule skin that can change color over time, characterized in that: Prepared by the following steps: S1: weigh 300-800 parts of gelatin, 100-300 parts of glycerin and 300-800 parts of pure water, put them into a gelatinizing tank, heat at 50-70°C, and stir at 50-200rpm to dissolve the gelatin to make a gelatin solution; Add 3-9 portions of the high-stability blueberry anthocyanin multivesicular liposomes described in any one of claims 1-7 to the S2 glue solution, stir evenly at 10-100 rpm, and keep warm at 45-55°C for 10-60 min; S3 pours the glue solution into a capsule forming machine to obtain a soft capsule skin.

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