Multilayer microencapsulated small molecule peptide liposome, preparation method and application

Abstract

The invention discloses a multilayer microencapsulated small molecule peptide liposome as well as a preparation method and application thereof. According to the invention, a'liposome-wall material 'double-embedding structure is formed by optimizing the phospholipid purity and compounding the hydrophilic wall material and the stabilizer, and a high-pressure homogenization and low-temperature spray drying combined process is adopted, so that the preparation process is green and environment-friendly. The obtained lipidosome has the characteristics of good fluidity, excellent sensory characteristics, high stability and the like, the solvent residue is far lower than the industrial standard, and the lipidosome can be widely applied to dosage forms such as tablets, capsules, solid beverages and the like. According to the technology, the production process is simplified, the use of toxic solvents is avoided, the bioavailability of the small molecule peptide is remarkably improved, and the problems of stability and production cost in industrial application are solved.

Description

Technical Field

[0001] The present invention belongs to the field of food nutrition and health, and in particular relates to a multi-layer microencapsulated small molecule peptide liposome and a preparation method and application thereof. Background Art

[0002] Small molecule peptides are short-chain compounds formed by connecting 2-10 amino acids through peptide bonds. They have the characteristics of high biological activity, easy absorption, and strong targeting. They play an important role in anti-oxidation, immunomodulation, metabolic regulation, anti-aging and disease treatment. For example, glutathione protects cells from oxidative damage by neutralizing free radicals through sulfhydryl groups, alanine can promote muscle synthesis, and carnosine has an anti-fatigue effect. However, the active groups of small molecule peptides (such as sulfhydryl and amino groups) are easily affected by temperature, light, pH value and digestive enzymes, resulting in poor stability and low bioavailability, which limits their application in food, medicine and cosmetics.

[0003] Traditional small molecule peptide formulations often face the following challenges: 1) they are susceptible to environmental factors such as temperature, light, and pH, leading to decomposition and inactivation; 2) they are easily enzymatically hydrolyzed in the gastrointestinal tract, reducing their bioavailability; and 3) they have difficulty passing through cell membranes, limiting their ability to function within cells. To address these issues, liposome technology has been introduced into small molecule peptide delivery systems.

[0004] Liposomes are vesicles composed of a phospholipid bilayer membrane encapsulating either hydrophilic or hydrophobic components. Their unique structure holds great promise for improving drug stability, enhancing drug targeting, prolonging drug action, and reducing side effects. The successful application of liposome technology in medicine has provided new insights into improving the delivery efficiency of functional ingredients in the food industry.

[0005] CN115245558A discloses a method for preparing immunoreactive hexapeptide liposomes from thick-shelled mussels, using a thin-film evaporation method. This method is only suitable for laboratory pilot trials and is not suitable for scale-up production. Patent CN117679369A discloses a glutathione liposome preparation and its preparation method. The formula uses materials such as sulfobutyl-β-cyclodextrin sodium (SBECD), β-cyclodextrin sodium, and hydroxypropyl-β-cyclodextrin sodium. There are safety concerns about their addition to food, and chloroform is used in the preparation process, which poses a potential risk of solvent poisoning. Patent CN114950289A discloses a glutathione liposome preparation device and its preparation method. This method uses supercritical carbon dioxide as a medium, and the equipment and method are expensive, making it difficult to apply to large-scale production. Patent CN102697728A discloses a method for preparing water-soluble drug liposomes. The preparation method uses 10-300 mesh glass microspheres as a carrier for phospholipid film formation, which is also not suitable for industrialization and has food safety risks due to the introduction of foreign matter.

[0006] Existing methods for preparing small-molecule peptide liposomes often rely on single processes, such as thin-film hydration and reverse evaporation. These methods have limitations in particle size control, stability, and production efficiency. Furthermore, some preparation methods require the use of organic solvents, which can lead to environmental pollution and product residue issues. Therefore, developing small-molecule peptide liposomes with a pleasant flavor, excellent stability, high bioavailability, and environmentally friendly preparation processes is of great significance.

[0007] In summary, although existing technologies have attempted to improve the active protection and delivery efficiency of small molecule peptides through liposome technology, process limitations and formulation defects have led to an inability to balance multiple contradictions, namely: a single embedding structure (such as a phospholipid monolayer or a conventional wall material) is difficult to simultaneously improve stability and absorption efficiency. For example, although conventional liposomes can delay the environmental degradation of peptides, the phospholipid layer without secondary encapsulation is susceptible to gastric acid erosion (disintegration rate of more than 60%), and a single phospholipid material is prone to membrane leakage under high-frequency homogenization (the permeability measured in the literature is >15% / month). In addition, existing liposome solutions often regard "particle size control" and "solvent safety" as contradictory. In many liposome patents, thin film hydration or reverse phase evaporation methods are used, which rely on large amounts of organic solvents (such as chloroform), posing a food safety hazard (chloroform residues were detected in CN117679369A). Although supercritical technology avoids solvents, it also loses the possibility of industrialization due to its high cost (equipment investment >3 million yuan). More importantly, existing technical routes mostly focus on a single performance indicator (such as encapsulation rate or particle size) in isolation, and fail to recognize the dynamic synergistic mechanism of "double-layer microcapsule structure-component adaptation-low-temperature gradient drying", resulting in the final product showing fragmented performance in antioxidant properties, bioavailability and sensory properties (such as taste masking and absorption efficiency cannot be achieved at the same time, and the encapsulation rate drops by more than 20% after acceleration).

[0008] The decentralized nature of the above-mentioned technical paths determines that the existing technical solutions are all difficult to form a comprehensive solution framework, which is a difficult problem that technical personnel in this field urgently need to overcome. Summary of the Invention

[0009] In response to the problems raised in the above background technology, such as the easy degradation, low bioavailability of small molecule peptides and the difficulty in industrialization of traditional liposome preparation processes, the present invention provides a multi-layer microencapsulated small molecule peptide liposome, its preparation method and application. By selecting high-purity phospholipids, hydrophilic wall materials and membrane stabilizers, combined with high-pressure homogenization and low-temperature spray drying processes, a "liposome-wall material" double-layer embedding structure is constructed. This process breaks through the traditional technology's dependence on toxic solvents and high-temperature processing, realizes clean production, and has high loading capacity, excellent physical stability and taste-masking effects. The obtained liposomes have strong fluidity and uniform particle size, and can be adapted to dosage forms such as tablets, capsules, and solid beverages, significantly improving bioavailability while reducing industrial production costs, meeting the needs of the food and pharmaceutical fields for functional ingredient delivery carriers.

[0010] The first aspect of the present invention is to protect a multi-layer microencapsulated small molecule peptide liposome, comprising the following raw materials, by weight: 30-70 parts of a small molecule peptide, 15-50 parts of a phospholipid, 0.2-2 parts of an antioxidant, 2-10 parts of a membrane stabilizer, and 5-30 parts of a wall material. A more preferred composition range is: 30-50 parts by weight of a small molecule peptide, 25-45 parts by weight of a phospholipid, 1.0-2 parts by weight of an antioxidant, 10-25% by weight of a membrane stabilizer based on the mass of phosphatidylcholine in the phospholipid, and 15-30 parts by weight of a wall material.

[0011] For the technical solution described above, preferably, the small molecule peptide is a water-soluble peptide selected from glutathione, acetyl glutathione, carnosine, decarboxylated carnosine, alanine dipeptide, anserine, acetyl carnosine, enkephalin, homocarnosine, thymopentin, collagen tripeptide, blue copper peptide, glycyl glycine, alanyl glutamyl, lysyl alanine, arginyl glycine, aspartyl serine, glutamyl serine, histidyl glycine, threonyl threonine, galanine tripeptide, lysine-glutamic acid-serine, arginine-glycine The water-soluble peptides include, for example, aspartic acid-aspartic acid, asparagine-glycine-serine, histidine-glycine-histidine, serine-serine-serine, threonine-aspartic acid-lysine, cysteine-glycine-glutamic acid, and other water-soluble dipeptides and modified compounds thereof, tripeptides and modified compounds thereof, tetrapeptides and modified compounds thereof, pentapeptides and modified compounds thereof, hexapeptides and modified compounds thereof, heptapeptides and modified compounds thereof, octapeptides and modified compounds thereof, nonapeptides and modified compounds thereof, decapeptides and modified compounds thereof. Specifically, in a specific embodiment of the present invention, unmodified water-soluble dipeptides or tripeptides such as alanine-glutathione and glutathione are most preferred.

[0012] For the technical solution described above, preferably, the modification methods of the modified compound include acetylation, phosphorylation, and sulfonation.

[0013] For the technical solution described above, preferably, the phospholipid is selected from one or more of soybean lecithin, sunflower lecithin, and rapeseed lecithin.

[0014] For the technical solution described above, preferably, the content of acetone insoluble matter in the phospholipid is required to be greater than 60wt%, more preferably, the content of acetone insoluble matter is required to be greater than 90wt%, and more preferably, the content of acetone insoluble matter is required to be greater than 95wt%.

[0015] The phospholipids described above are a mixture comprising phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and other components. The technical solution of the present invention limits the phosphatidylcholine (PC) content in the phospholipids. Preferably, the phosphatidylcholine content in the phospholipids is required to be greater than 50wt%, more preferably, greater than 70wt%, and more preferably, greater than 90wt%. Specifically, in a specific embodiment of the present invention, sunflower lecithin or soybean lecithin having a phosphatidylcholine (PC) content of ≥70wt% is most preferred.

[0016] For the technical solution described above, preferably, the film stabilizer includes one or more of plant sterols, mono- and di-glycerol fatty acid esters, fatty acids, and vegetable oils.

[0017] For the technical solution described above, preferably, the weight ratio of the membrane stabilizer is 2 to 6 parts; more preferably, the weight ratio is 2 to 4.5 parts.

[0018] For the technical solution described above, preferably, the weight ratio of the membrane stabilizer to the phosphatidylcholine component in the phospholipid is 1:3-15, further preferably, the weight ratio of the membrane stabilizer to the phosphatidylcholine component in the phospholipid is 1:3-12, and more preferably 1:4-10.

[0019] For the technical solution described above, preferably, the phytosterol is selected from one or more of sitosterol, stigmasterol, brassicasterol, ergosterol, and microalgae sterol.

[0020] For the technical solution described above, preferably, in order to meet the stability of liposomes, the melting points of the above-mentioned mono- and diglycerol fatty acid esters, fatty acids, and vegetable oils are required to be 40-90°C, preferably 40-80°C, and more preferably 45-65°C.

[0021] For the technical solution described above, preferably, the antioxidant is selected from one or more of ascorbic acid, sodium ascorbate, ascorbyl palmitate, mixed tocopherols, citrus flavonoids, proanthocyanidins, tea polyphenols, and carotenoids.

[0022] For the technical solution described above, preferably, the wall material includes one or more of modified starch, gum arabic, maltodextrin, resistant dextrin, inulin, maltooligosaccharide, and isomalt-oligosaccharide.

[0023] Another aspect of the present invention is to provide a method for preparing multi-layer microencapsulated small molecule peptide liposomes, which comprises the following steps:

[0024] S1. Aqueous phase preparation: Prepare an aqueous solution containing small molecule peptides;

[0025] S2. Preparation of alcohol phase: Dissolve phospholipids and membrane stabilizer in anhydrous ethanol and mix thoroughly until completely dissolved;

[0026] S3. Wall material solution preparation: preparation of an aqueous solution of wall material;

[0027] S4. The alcohol phase is slowly injected into the aqueous phase and stirred thoroughly to form an initial liposome dispersion solution, which is concentrated under reduced pressure to obtain a concentrate which is slowly added to the aqueous solution of the wall material and stirred thoroughly to form a multilayer microencapsulated liposome dispersion solution;

[0028] S5. The obtained multi-layer microencapsulated liposome dispersion solution is subjected to high-speed shearing, high-pressure homogenization, and drying.

[0029] For the technical solution described above, preferably, the dissolution temperature of the small molecule peptide in S1 is 40-50°C.

[0030] Further preferably, in order to reduce the degradation of the small molecule peptide content during the sample preparation process, the dissolution process in S1 needs to be in a light-proof and inert gas environment.

[0031] For the technical solution described above, preferably, the dissolution temperature of the phospholipid and membrane stabilizer in S2 is 50-65°C.

[0032] For the technical solution described above, the weight ratio of the amount of deionized water in S1 to the amount of anhydrous ethanol in S2 should be within a certain range. Preferably, the weight ratio of water to ethanol is (1-8):1, further preferably, the weight ratio of water to ethanol is (1-5):1, and more preferably, it is (2-4):1.

[0033] For the technical solution described above, preferably, the solid content of the wall material solution in S3 is 30-50 wt%.

[0034] For the technical solution described above, preferably, the temperature of the reduced pressure concentration process in S4 is 40-55°C, and more preferably, the temperature is 45-50°C.

[0035] For the technical solution described above, preferably, the vacuum degree of the reduced pressure concentration process in S4 is -0.1 to -0.08 MPa, and more preferably, the vacuum degree is -0.09 to -0.085 MPa.

[0036] For the technical solution described above, preferably, the stirring temperature after adding the wall material aqueous solution in S4 is 40-60°C, and more preferably, the temperature is 40-50°C.

[0037] For the technical solution described above, preferably, the homogenization pressure in S5 is 20-45 MPa, and the number of homogenization times is ≥2 times; further preferably, the homogenization pressure is 30-40 MPa, and the number of homogenization times is 2-3 times.

[0038] For the technical solution described above, preferably, the drying method in S5 can be selected from one of spray drying, low-temperature spray drying, freeze drying, and freeze spray drying. Preferably, low-temperature spray drying or freeze drying is selected. Further preferably, in order to improve production efficiency and achieve a better product form, the drying method is low-temperature spray drying.

[0039] For the preferred low-temperature spray drying in S5, the inlet air temperature is selected to be 50-90°C, and the outlet air temperature is controlled at 30-40°C. Preferably, the inlet air temperature is selected to be 60-80°C, and more preferably, the inlet air temperature is selected to be 60-70°C.

[0040] The present invention also protects the application of the above-mentioned multi-layer microencapsulated small molecule peptide liposomes, and the application fields include the preparation of compressed tablet preparations, hard shell capsules, solid beverages, etc., among which solid beverages include fruit and vegetable solid beverages, protein solid beverages, tea solid beverages, plant solid beverages, etc.

[0041] Beneficial effects of the present invention

[0042] 1. The prepared multi-layer microencapsulated small molecule peptide liposomes have good fluidity, uniform particle size, and excellent taste masking effect. The rehydrated particle size is in the range of 200-500nm and has good sensory properties.

[0043] 2. After secondary encapsulation, the liposomes have very good physical and chemical stability. After accelerated storage at 60°C for 20 days, no problems such as agglomeration, browning, or deterioration of flavor occurred, and the content degradation rate was less than 10%;

[0044] 3. The phospholipids in the raw materials are all natural plant-derived ingredients, and no cholesterol is added in the formula, which better meets consumer needs;

[0045] 4. The preparation method uses clean solvent ethanol as a processing aid, which is removed and recovered during the process. The residual solvent in the product is less than 50ppm, which meets the regulatory requirements of the food processing industry and does not cause environmental pollution.

[0046] 5. The preparation method has very good industrial adaptability and can meet the large-scale production of products;

[0047] 6. The prepared multi-layer microencapsulated small molecule peptide liposomes have a wide range of applications, including various compressed tablet preparations, hard shell capsules, solid beverages, and other dietary supplements and functional health foods;

[0048] 7. Through the phospholipid-wall material double-layer embedding structure design, the protective masking and delivery efficiency of small molecule peptides are synergistically optimized. The rehydrated particle size is controlled in the range of 200-500nm, which not only effectively blocks the release of bitter molecules, but also improves intestinal absorption efficiency and significantly enhances bioavailability.

[0049] 8. The formula uses natural high-purity phospholipids (PC content 50-90%) and introduces plant-derived membrane stabilizers (such as stigmasterol) to replace traditional cholesterol ingredients. The liposome phase stability is regulated through hydrophobic interactions to meet the safety and natural ingredient requirements of food-grade formulas.

[0050] 9. The process integrates low-temperature vacuum concentration and high-pressure homogenization technology to achieve efficient removal of ethanol solvent (residual amount ≤ 50ppm) and liposome particle size control (screening rate > 99%), avoiding activity loss, structural damage and high energy consumption defects caused by high temperature, and is suitable for industrial production. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the examples, but it should be understood that the scope of the present invention is not limited by the examples. Unless otherwise stated, the chemical reagents and biological materials used in the present invention were obtained from commercial sources and processed and used in accordance with standard operating procedures. All experimental steps were carried out under conventional laboratory conditions to ensure the repeatability and reliability of the technical solutions.

[0052] Solvent residues: The detection method refers to the Chinese Pharmacopoeia, 0861 residual solvents;

[0053] Entrapment efficiency: The entrapment efficiency of liposomes was determined using ultrafiltration centrifugation. The sample solution was added to an ultrafiltration tube equipped with an ultrafiltration membrane (molecular cutoff 3 kDa) and centrifuged at 8000 r / min. The unentrapped free peptide passed through the ultrafiltration membrane under the action of centrifugal force, while the liposomes were retained, thus achieving separation of the two.

[0054] Example 1

[0055] Aqueous phase: Weigh 162 parts of deionized water, heat to 40°C, add 30 parts of glutathione and 1.5 parts of ascorbic acid, and stir to completely dissolve.

[0056] Alcohol phase: Weigh 100 parts of anhydrous ethanol, add 50 parts of rapeseed lecithin PC50 (PC50: refers to phosphatidylcholine content in phospholipids ≥50%, the same below) and 2.5 parts of mono- and diglycerol fatty acid esters, heat to 60°C, and stir until completely dissolved.

[0057] Wall material solution: Add 16 parts of resistant dextrin to 38 parts of deionized water, heat and stir to completely dissolve.

[0058] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly to mix, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 50° C. to obtain a glutathione liposome concentrate.

[0059] The glutathione liposome concentrate was added to the wall material solution and stirred at 45°C for 1 hour. The solution was sheared at 5500 rpm for 2 minutes and homogenized twice at 35 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 80°C and the outlet air temperature at 40°C, to obtain glutathione liposome microcapsule powder.

[0060] Example 2

[0061] Water phase: Weigh 130 parts of deionized water, heat to 45°C, add 40 parts of propylglutathione and 2 parts of tea polyphenols, and stir to completely dissolve them.

[0062] Alcohol phase: Weigh 60 parts of anhydrous ethanol, add 25 parts of sunflower lecithin PC70 and 3 parts of stigmasterol, heat to 65°C, and stir until completely dissolved.

[0063] Wall material solution: Add 30 parts of gum arabic to 50 parts of deionized water, heat and stir to completely dissolve.

[0064] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 45° C. to obtain a glutathione liposome concentrate.

[0065] The glutathione liposome concentrate was added to the wall material solution and stirred at 50°C for 1 hour. The solution was sheared at 4000 rpm for 3 minutes and homogenized twice at 40 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 70°C and the outlet air temperature at 35°C, to obtain glutathione liposome microcapsule powder.

[0066] Example 3

[0067] Water phase: Weigh 160 parts of deionized water, heat to 48°C, add 50 parts of carnosine, and stir to completely dissolve it.

[0068] Alcohol phase: Weigh 80 parts of anhydrous ethanol, add 30 parts of soybean lecithin PC70, 2.5 parts of palm oil and 0.2 parts of ascorbyl palmitate, heat to 55°C, and stir until completely dissolved.

[0069] Wall material solution: Add 7.3 parts of gum arabic and 10 parts of inulin to 30 parts of deionized water, heat and stir to completely dissolve.

[0070] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.085 MPa and 50° C. to obtain a carnosine liposome concentrate.

[0071] The carnosine liposome concentrate was added to the wall material solution and stirred at 40°C for 1 hour. The solution was sheared at 4500 rpm for 3 minutes and homogenized at 30 MPa three times. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 60°C and the outlet air temperature at 30°C, to obtain carnosine liposome microcapsule powder.

[0072] Example 4

[0073] Aqueous phase: Weigh 180 parts of deionized water, heat to 50°C, add 60 parts of histidine-glycine-histidine and 2 parts of citrus flavonoids, and stir to completely dissolve.

[0074] Alcohol phase: Weigh 45 parts of anhydrous ethanol, add 15 parts of sunflower lecithin PC90 and 2 parts of stearic acid, heat to 55°C, and stir until completely dissolved.

[0075] Wall material solution: Add 10 parts of modified starch and 11 parts of oligomaltose into 21 parts of deionized water, heat and stir to completely dissolve them.

[0076] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.088 MPa and 45° C. to obtain a histidine-glycine-histidine liposome concentrate.

[0077] The histidine-glycine-histidine liposome concentrate was added to the wall material solution and stirred at 45°C for 1.5 hours. The solution was sheared at 6000 rpm for 2 minutes and homogenized twice at 35 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 65°C and the outlet air temperature at 32°C, to obtain histidine-glycine-histidine liposome microcapsule powder.

[0078] Example 5

[0079] Aqueous phase: Weigh 270 parts of deionized water, heat to 40°C, add 70 parts of glutathione, and stir to completely dissolve.

[0080] Alcohol phase: Weigh 80 parts of anhydrous ethanol, add 20 parts of soybean lecithin PC90, 4.5 parts of sitosterol and 0.5 parts of mixed tocopherols, heat to 50°C, and stir until completely dissolved.

[0081] Wall material solution: Add 5 parts of modified starch to 10 parts of deionized water, heat and stir to completely dissolve it.

[0082] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 50° C. to obtain a glutathione liposome concentrate.

[0083] The glutathione liposome concentrate was added to the wall material solution and stirred at 50°C for 1 hour. The solution was sheared at 5000 rpm for 2 minutes and homogenized twice at 40 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 70°C and the outlet air temperature at 33°C, to obtain glutathione liposome microcapsule powder.

[0084] Example 6

[0085] Aqueous phase: Weigh 162 parts of deionized water, heat to 40°C, add 30 parts of collagen tripeptide and 1.5 parts of ascorbic acid, and stir to completely dissolve.

[0086] Alcohol phase: Weigh 100 parts of anhydrous ethanol, add 45 parts of soybean lecithin PC50 and 2.5 parts of mono- and diglycerol fatty acid esters, heat to 60°C, and stir until completely dissolved.

[0087] Wall material solution: Add 16 parts of maltodextrin to 38 parts of deionized water, heat and stir to completely dissolve.

[0088] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 50° C. to obtain a collagen tripeptide liposome concentrate.

[0089] The collagen tripeptide liposome concentrate was added to the wall material solution and stirred at 45°C for 1 hour. The solution was sheared at 5500 rpm for 2 minutes and homogenized twice at 35 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 80°C and the outlet air temperature at 40°C, to obtain collagen tripeptide liposome microcapsule powder.

[0090] Example 7

[0091] Aqueous phase: Add 180 kg of deionized water to the stirred tank, turn on the jacket heating, raise the temperature to 50°C, turn on the stirring paddle, add 60 kg of glutathione and 2 kg of citrus flavonoids, and continue stirring to completely dissolve them.

[0092] Alcohol phase: Add 45 kg of anhydrous ethanol to another stirred tank, add 15 kg of sunflower lecithin PC90 and 2 kg of stearic acid, turn on the jacket heating, raise the temperature to 50-55 ° C, and stir until completely dissolved.

[0093] Wall material solution: put 10kg modified starch and 11kg maltooligosaccharide into 21kg deionized water, heat and stir to completely dissolve them.

[0094] The alcohol phase was slowly injected into the aqueous phase kettle, and the mixture was thoroughly stirred. Then, the vacuum pump was turned on, and the pressure in the kettle was adjusted to -0.09 MPa, maintained at 45°C, and concentrated under reduced pressure to obtain a glutathione liposome concentrate.

[0095] The glutathione liposome concentrate was added to the wall material solution, maintained at 45°C, and stirred continuously for 1.5 hours. The shearing machine was then activated to transfer the liquid in the kettle to a buffer tank via the shearing machine. The homogenizer was activated and homogenized twice at a homogenization pressure of 35 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 65-70°C and the outlet air temperature at 30-35°C, to obtain glutathione liposome microcapsule powder.

[0096] In the above Examples 1 to 7, the following parameters were screened, as shown in the following table:

[0097]

[0098] The phosphatidylcholine (PC) content in Examples 1 to 7 was detected according to the detection method according to GB509.272. The measured results are as follows:

[0099] Example phospholipids Phosphatidylcholine (PC) content Example 1 Rapeseed Lecithin PC50 51.2％ Example 2 Sunflower Lecithin PC70 70.8％ Example 3 Soy lecithin PC70 71.3％ Example 4 Sunflower Lecithin PC90 90.2％ Example 5 Soy lecithin PC90 90.7％ Example 6 Soy lecithin PC50 50.4％ Example 7 Sunflower Lecithin PC90 91.0％

[0100] For the film stabilizers using mono- and diglycerol fatty acid esters, fatty acids, and vegetable oils in Examples 1 to 7, their melting points were tested using the method described in GB5009.272. The measured results are as follows:

[0101] Example Film stabilizer Melting point Example 1 Mono- and diglycerides of fatty acids 79.1℃ Example 2 Stigmasterol Not tested Example 3 palm oil 51.2℃ Example 4 stearic acid 69.4℃ Example 5 Sitosterol Not tested Example 6 Mono- and diglycerides of fatty acids 79.3℃ Example 7 stearic acid 69.7℃

[0102] Comparative Example 1 (adding low-specification lecithin)

[0103] On the basis of Example 1, the effects of lecithin specifications on product quality were compared. In this comparative example, low-specification lecithin was used.

[0104] Aqueous phase: Weigh 162 parts of deionized water, heat to 40°C, add 30 parts of glutathione and 1.5 parts of ascorbic acid, and stir to completely dissolve.

[0105] Alcohol phase: Weigh 100 parts of anhydrous ethanol, add 50 parts of powdered sunflower lecithin (phosphatidylcholine content ≈ 20%, as well as other phospholipid components such as phosphatidylinositol, phosphatidylethanolamine) and 2.5 parts of mono- and diglycerol fatty acid esters, heat to 60°C and stir. Since there are a large number of ethanol-insoluble substances in the powdered lecithin, add chloroform and keep stirring until it is completely dissolved.

[0106] Wall material solution: Add 16 parts of resistant dextrin to 38 parts of deionized water, heat and stir to completely dissolve.

[0107] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly to mix, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 50° C. to obtain a glutathione liposome concentrate.

[0108] The glutathione liposome concentrate was added to the wall material solution and stirred at 45°C for 1 hour. The solution was sheared at 5500 rpm for 2 minutes and homogenized twice at 35 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 80°C and the outlet air temperature at 40°C, to obtain glutathione liposome microcapsule powder.

[0109] Comparative Example 2 (no wall material added)

[0110] On the basis of Example 2, the influence of the wall material on the product properties was compared. In this comparative example, no wall material component was added.

[0111] Water phase: Weigh 130 parts of deionized water, heat to 45°C, add 40 parts of propylglutathione and 2 parts of tea polyphenols, and stir to completely dissolve them.

[0112] Alcohol phase: Weigh 60 parts of anhydrous ethanol, add 25 parts of sunflower lecithin PC70 and 3 parts of stigmasterol, heat to 65°C, and stir until completely dissolved.

[0113] The alcohol phase was slowly injected into the aqueous phase, thoroughly stirred and mixed, and then transferred to a rotary evaporator for vacuum concentration at -0.09 MPa and 45°C. The solid content was adjusted to 35%, and the mixture was stirred at 50°C for 1 hour. The liquid was sheared at 4000 rpm for 3 minutes and homogenized at 40 MPa twice. Subsequently, low-temperature spray drying was performed, with the inlet air temperature controlled at 70°C and the outlet air temperature at 35°C to obtain glutathione liposome microcapsule powder.

[0114] Comparative Example 3 (without adding film stabilizer)

[0115] On the basis of Example 3, the effect of the film stabilizer on the product quality was compared. In this comparative example, no film stabilizer was added.

[0116] Water phase: Weigh 160 parts of deionized water, heat to 48°C, add 50 parts of carnosine, and stir to completely dissolve it.

[0117] Alcohol phase: Weigh 80 parts of anhydrous ethanol, add 30 parts of soybean lecithin PC70 and 0.2 parts of ascorbyl palmitate, heat to 55°C, and stir until completely dissolved.

[0118] Wall material solution: Add 7.3 parts of gum arabic and 10 parts of inulin to 30 parts of deionized water, heat and stir to completely dissolve.

[0119] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.085 MPa and 50° C. to obtain a carnosine liposome concentrate.

[0120] The carnosine liposome concentrate was added to the wall material solution and stirred at 40°C for 1 hour. The solution was sheared at 4500 rpm for 3 minutes and homogenized at 30 MPa three times. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 60°C and the outlet air temperature at 30°C, to obtain carnosine liposome microcapsule powder.

[0121] Comparative Example 4 (Drying Method Investigation)

[0122] On the basis of Example 4, the effect of drying method on product quality was compared. In this comparative example, conventional spray drying was used.

[0123] Aqueous phase: Weigh 180 parts of deionized water, heat to 50°C, add 60 parts of histidine-glycine-histidine and 2 parts of citrus flavonoids, and stir to completely dissolve.

[0124] Alcohol phase: Weigh 45 parts of anhydrous ethanol, add 15 parts of sunflower lecithin PC90 and 2 parts of stearic acid, heat to 55°C, and stir until completely dissolved.

[0125] Wall material solution: Add 10 parts of modified starch and 11 parts of oligomaltose into 21 parts of deionized water, heat and stir to completely dissolve them.

[0126] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly to mix, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.088 MPa and 45° C. to obtain a histidine-glycine-histidine liposome concentrate.

[0127] The histidine-glycine-histidine liposome concentrate was added to the wall material solution and stirred at 45°C for 1.5 hours. The solution was sheared at 6000 rpm for 2 minutes and homogenized twice at 35 MPa. The solution was then spray-dried at an inlet temperature of 180°C and an outlet temperature of 80°C to obtain histidine-glycine-histidine liposome microcapsule powder.

[0128] Comparative Example 5 (increasing the proportion of small molecule peptides)

[0129] On the basis of Example 5, the proportion of glutathione in the formula was further increased.

[0130] Aqueous phase: Weigh 300 parts of deionized water, heat to 40°C, add 80 parts of glutathione, and stir to completely dissolve.

[0131] Alcohol phase: Weigh 80 parts of anhydrous ethanol, add 15 parts of soybean lecithin PC90, 3 parts of sitosterol and 0.5 parts of mixed tocopherols, heat to 50°C, and stir until completely dissolved.

[0132] Wall material solution: Add 1.5 parts of modified starch to 10 parts of deionized water, heat and stir to completely dissolve it.

[0133] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly to mix, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 50° C. to obtain a glutathione liposome concentrate.

[0134] The glutathione liposome concentrate was added to the wall material solution and stirred at 50°C for 1 hour. The solution was sheared at 5000 rpm for 2 minutes and homogenized twice at 40 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 70°C and the outlet air temperature at 33°C, to obtain glutathione liposome microcapsule powder.

[0135] Comparative Example 6 (reducing the proportion of small molecule peptides)

[0136] On the basis of Example 1, the proportion of glutathione in the formula was further reduced.

[0137] Aqueous phase: Weigh 162 parts of deionized water, heat to 40°C, add 20 parts of glutathione and 1.5 parts of ascorbic acid, and stir to completely dissolve.

[0138] Alcohol phase: Weigh 100 parts of anhydrous ethanol, add 60 parts of rapeseed lecithin PC50 and 2.5 parts of mono- and diglycerol fatty acid esters, heat to 60°C, and stir until completely dissolved.

[0139] Wall material solution: Add 16 parts of resistant dextrin to 38 parts of deionized water, heat and stir to completely dissolve.

[0140] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly to mix, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 50° C. to obtain a glutathione liposome concentrate.

[0141] The glutathione liposome concentrate was added to the wall material solution and stirred at 45°C for 1 hour. The solution was sheared at 5500 rpm for 2 minutes and homogenized twice at 35 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 80°C and the outlet air temperature at 40°C, to obtain glutathione liposome microcapsule powder.

[0142] Comparative Example 7 (reducing the proportion of phospholipids and increasing the proportion of wall materials)

[0143] On the basis of Example 2, the effects of the amounts of phospholipids and wall materials on the product properties were compared. In this comparative example, the proportion of phospholipids was reduced and the proportion of wall materials was increased.

[0144] Water phase: Weigh 130 parts of deionized water, heat to 45°C, add 40 parts of propylglutathione and 2 parts of tea polyphenols, and stir to completely dissolve them.

[0145] Alcohol phase: Weigh 60 parts of anhydrous ethanol, add 10 parts of sunflower lecithin PC70 and 1 part of stigmasterol, heat to 65°C, and stir until completely dissolved.

[0146] Wall material solution: Add 47 parts of gum arabic to 80 parts of deionized water, heat and stir to completely dissolve.

[0147] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 45° C. to obtain a glutathione liposome concentrate.

[0148] The glutathione liposome concentrate was added to the wall material solution and stirred at 50°C for 1 hour. The solution was sheared at 4000 rpm for 3 minutes and homogenized twice at 40 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 70°C and the outlet air temperature at 35°C, to obtain glutathione liposome microcapsule powder.

[0149] Comparative Example 8 (Increasing the proportion of phospholipids and reducing the proportion of wall materials)

[0150] On the basis of Example 2, the effects of the amounts of phospholipids and wall materials on the product properties were compared. In this comparative example, the proportion of phospholipids was increased and the proportion of wall materials was reduced.

[0151] Water phase: Weigh 130 parts of deionized water, heat to 45°C, add 40 parts of propylglutathione and 2 parts of tea polyphenols, and stir to completely dissolve them.

[0152] Alcohol phase: Weigh 80 parts of anhydrous ethanol, add 40 parts of sunflower lecithin PC70 and 3 parts of stigmasterol, heat to 65°C, and stir until completely dissolved.

[0153] Wall material solution: Add 15 parts of gum arabic to 50 parts of deionized water, heat and stir to completely dissolve.

[0154] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.09 MPa and 45° C. to obtain a glutathione liposome concentrate.

[0155] The glutathione liposome concentrate was added to the wall material solution and stirred at 50°C for 1 hour. The solution was sheared at 4000 rpm for 3 minutes and homogenized twice at 40 MPa. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 70°C and the outlet air temperature at 35°C, to obtain glutathione liposome microcapsule powder.

[0156] Comparative Example 9 (Increasing the amount of film stabilizer used)

[0157] On the basis of Example 3, the effect of the film stabilizer on the product quality was compared. In this comparative example, the film stabilizer was used in excess.

[0158] Water phase: Weigh 160 parts of deionized water, heat to 48°C, add 44.5 parts of carnosine, and stir to completely dissolve.

[0159] Alcohol phase: Weigh 80 parts of anhydrous ethanol, add 30 parts of soybean lecithin PC70, 8 parts of palm oil and 0.2 parts of ascorbyl palmitate, heat to 55°C, and stir until completely dissolved.

[0160] Wall material solution: Add 7.3 parts of gum arabic and 10 parts of inulin to 30 parts of deionized water, heat and stir to completely dissolve.

[0161] The alcohol phase was slowly injected into the aqueous phase, stirred thoroughly, and then transferred to a rotary evaporator for concentration under reduced pressure at -0.085 MPa and 50° C. to obtain a carnosine liposome concentrate.

[0162] The carnosine liposome concentrate was added to the wall material solution and stirred at 40°C for 1 hour. The solution was sheared at 4500 rpm for 3 minutes and homogenized at 30 MPa three times. Low-temperature spray drying was then performed, with the inlet air temperature controlled at 60°C and the outlet air temperature at 30°C, to obtain carnosine liposome microcapsule powder.

[0163] Application Example 1 (Preparation of Tablet Preparations)

[0164] 60 parts of the liposome sample of the above embodiment or comparative example, 23.5 parts of sorbitol, 15 parts of microcrystalline cellulose, and 1.5 parts of magnesium stearate were mixed evenly and then subjected to direct compression tableting to test the tablet indicators.

[0165] Application Example 2 (Making Hard Shell Capsules)

[0166] The liposome samples of the above examples or comparative examples were directly poured into capsule shells to prepare hard shell capsules, and the tablet indicators were tested.

[0167] Application Example 3 (Making Solid Beverages)

[0168] Take 35 parts of the liposome sample of the above embodiment or comparative example, 30 parts of erythritol, 25 parts of maltodextrin, 9.4 parts of steviol glycoside, 0.5 parts of citric acid, and 0.1 parts of edible flavor, mix them evenly, and then pack them into granules with a net content of 3g / pack to prepare a solid beverage.

[0169] Effect comparison group 1. Index evaluation

[0170] The liposome samples prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were evaluated on indicators including product state, color, flavor, particle size, embedding efficiency, angle of repose, solvent residue, and rehydrated particle size.

[0171] Status: Visual.

[0172] Color: visually.

[0173] Smell: smell.

[0174] Particle size: Weigh about 10g of sample, place it in a 40-mesh standard sieve, shake for more than 3 minutes, and calculate the 40-mesh sieving rate.

[0175] Embedment efficiency: The free peptide content was determined by ultrafiltration method, the ultrafiltration membrane molecular cutoff was 3kDa, and the embedding efficiency = (total content - free content) / total content * 100%

[0176] Angle of repose: measured using an angle of repose meter.

[0177] Residual solvents were determined using a headspace sampler coupled with gas chromatography.

[0178] Rehydrated particle size: After dissolving the liposomes at a concentration of 1%, the rehydrated particle size was measured using a nanoparticle size analyzer and the average particle size was taken.

[0179] Loss rate: Determine the actual content of peptide in the final sample and calculate the process loss rate = (theoretical content - actual content) / theoretical content * 100%.

[0180]

[0181] According to the liposome index evaluation results, the test results of product state, color, flavor, embedding rate, particle size, angle of repose and other indicators of different embodiments and comparative examples can be compared.

[0182] State: The liposomes of Examples 1 to 7 were all fluid powders, indicating that the powders had very good fluidity. However, the product of Comparative Example 2 was sticky and agglomerated. This was because no wall material was added to the formula. During the spray drying process, the drying temperature was much higher than the phase transition temperature of the phospholipid, resulting in the product sticking and agglomerating. The yield was also greatly reduced, making it inconvenient for industrial production.

[0183] Color: The color of the liposome samples is affected by the specifications and dosage of the phospholipids and the amount of wall material used in the formulation. The liposomes in Examples 1-7 ranged from off-white to light yellow. Comparative Example 1 used low-purity phospholipids, resulting in a yellowish color. Comparative Example 2, which did not include the addition of wall material, exhibited a light yellow color. In fact, the addition of high-purity lecithin and wall material can lighten the liposome color.

[0184] Odor: The liposomes in Examples 1 to 7 have no obvious odor, while Comparative Examples 1 and 2 have a slight phospholipid odor, which is also caused by the use of low-purity lecithin in the formula and the lack of added wall materials. The higher the purity of the lecithin, the lighter the odor, making the liposomes more soluble in various terminal dosage forms and more easily accepted by consumers. Comparative Example 8 has a slight phospholipid odor because the amount of phospholipids is increased while the amount of wall materials is reduced, resulting in poor secondary encapsulation effect and making the phospholipids easily oxidized. The addition of wall materials allows the liposomes to be encapsulated in a second layer, which can further mask the odor of the phospholipids and protect the small molecule peptides from damage. In Comparative Example 4, conventional spray drying is used, and the inlet and outlet air temperatures are high, which causes the small molecule peptides to decompose and the bitterness to increase.

[0185] Embedding Efficiency: The liposome embedding efficiencies of Examples 1-7 were all above 70%. This efficiencies were significantly affected by the product formulation. For example, reducing the small molecule peptide loading, increasing the phospholipid specification, using membrane stabilizers, and adding wall materials all resulted in higher embedding efficiencies. However, excessive use of membrane stabilizers had the opposite effect, resulting in lower embedding efficiencies. Comparing the embedding efficiencies of the products in the comparative examples with those in the corresponding examples reveals significant differences in embedding efficiencies due to formulation composition.

[0186] Particle size: The 40-mesh sieving rates of the liposomes in Examples 1 to 7 were all >99%, while the sieving rate of Comparative Example 2 was 90%, indicating that the product had serious adhesion, which was also caused by the lack of added wall material.

[0187] Angle of repose: A powder's angle of repose is an important parameter that describes its stacking properties at rest and reflects its flowability. A smaller angle of repose indicates better powder flowability. The liposomes in Examples 1-7 have an angle of repose of approximately 40°, indicating excellent flowability. However, the angles of repose in Comparative Examples 1 and 2 are significantly higher, indicating relatively poor flowability.

[0188] Solvent residue: This process can effectively remove the organic solvent used in the process. The solvent residue of the products in each embodiment and comparative example is less than 50 ppm.

[0189] Rehydrated Particle Size: After rehydration and dissolution, the average particle size of the liposomes in Examples 1-7 was less than 500 nm. However, the particle size of the liposomes in Comparative Examples 1-4 increased to a certain extent compared to the corresponding examples, also indicating that the formulation was affected. The rehydrated particle size distributions in Comparative Examples 7 and 9 were non-normal, indicating that reducing the phospholipid dosage and using excessive amounts of membrane stabilizers can both deteriorate the rehydration state of the liposomes.

[0190] Loss rate: The loss rate of the small molecule peptide in Examples 1-7 was less than 5%, indicating good process control. However, the loss rate of the small molecule peptide in Comparative Examples 1-4 increased, especially in Comparative Example 4, where the loss rate reached 16%. Because small molecule peptides are chemically unstable and extremely sensitive to heat, the high-temperature drying environment in Comparative Example 4 led to significant degradation of the small molecule peptide.

[0191] In addition, powdered sunflower lecithin (PC20) was used in Comparative Example 1. Since there is a large amount of ethanol-insoluble matter in the powdered lecithin, chloroform was added during the dissolution process. This is not a processing aid permitted by GB2760, and therefore does not meet the conditions for industrialization in the food industry.

[0192] The present invention also examines the effect of further increasing or decreasing the amount of small molecule peptide formula on product quality. Increasing the proportion of small molecule peptide will reduce the embedding effect, reduce the embedding rate, and worsen the taste masking effect. Although reducing the proportion of small molecule peptide can further improve the encapsulation effect, the product will experience pseudo-agglomeration and the yield will be reduced during the preparation process. At the same time, the low small molecule peptide content in the liposome product will also lead to the need to add more liposomes to achieve the expected content during the back-end application process, which greatly reduces the application performance. Therefore, the more suitable small molecule peptide formula amount is 30 to 70 parts.

[0193] In addition to laboratory sample preparation, the present invention further investigates pilot-scale scale-up production. Example 7 is a liposome product prepared in a pilot workshop, and its quality is comparable to that of laboratory samples, indicating that the patent has industrial utility and is suitable for scale-up production, solving the problem that some existing preparation methods cannot be used for large-scale production.

[0194] The evaluation results of some of the above indicators illustrate the necessity of the technical solution of the present invention for the content of small molecule peptides, the selection of phospholipid specifications, the addition of wall materials and the selection of appropriate drying conditions.

[0195] The analysis of the above indicators only reflects the initial level of the product, so further comparison of the accelerated stability of each group is needed.

[0196] Effect comparison group 2. Accelerated stability study

[0197] The liposome samples prepared in Examples 1 to 7 and Comparative Examples 1 to 4 were subjected to accelerated testing at 60° C. to observe the product status and determine the retention rate of the small molecule peptide.

[0198] Evaluation results of indicators after 20 days of liposome acceleration

[0199] Test items state Color Smell Embedding rate Peptide retention rate Example 1 Flowable powder light yellow No obvious odor 76％ 96.1％ Example 2 Flowable powder light yellow No obvious odor 74％ 95.7％ Example 3 Flowable powder light yellow No obvious odor 71％ 93.8％ Example 4 Flowable powder light yellow No obvious odor 68％ 94.3％ Example 5 Flowable powder light yellow No obvious odor 65％ 93.1％ Example 6 Flowable powder light yellow No obvious odor 76％ 95.1％ Example 7 Flowable powder light yellow No obvious odor 71％ 93.7％ Comparative Example 1 Flowable powder tan Lecithin oxidation taste, bitter taste 56％ 92.7％ Comparative Example 2 caking tan Lecithin oxidation taste, bitter taste 51％ 85.2％ Comparative Example 3 Slightly clumping yellow bitterness 48％ 83.2％ Comparative Example 4 Flowable powder tan bitterness 51％ 92.5％

[0200] Sensory indicators: After accelerated testing at 60°C for 20 days, the state, color, and taste of the liposomes of Examples 1 to 7 did not change significantly. The color of the liposomes of Comparative Examples 1 to 4 deepened, and a clear phospholipid oxidation smell and bitterness appeared. The reason is: low-purity lecithin was used in Comparative Example 1, and the large amount of phospholipids and glycolipids of other components would aggravate oxidation. In Comparative Example 2, no wall material was used for secondary encapsulation, and the phospholipids were directly exposed to the outermost layer, which aggravated the oxidative browning process. The free radicals produced by the oxidation of phospholipids will further lead to the oxidation or decomposition of small molecule peptides, resulting in a bitter taste. No stabilizer was added in Comparative Example 3, resulting in the rupture of the liposome structure and leakage of contents during the acceleration process, resulting in the destruction of the small molecule peptides. Comparative Example 4 had a bitter taste at the beginning, and the acceleration process aggravated the production of bitterness.

[0201] Embedding efficiency: The embedding efficiency of each group of products decreased to some extent after 20 days of acceleration. The degree of reduction in the liposome embedding efficiency of Examples 1-7 was all within 10%, while the embedding efficiency of the samples in Comparative Examples 1-3 decreased significantly. The reasons are: the use of low-purity lecithin in Comparative Example 1 increased the risk of liposome leakage during the acceleration process; the lack of wall material for secondary embedding in Comparative Example 2 accelerated phospholipid oxidation and reduced encapsulation efficiency; and the lack of membrane stabilizer in Comparative Example 3 accelerated the leakage of contents.

[0202] Small molecule peptide retention rate: After accelerated storage at 60°C for 20 days, the small molecule peptide retention rates of Examples 1-7 were all >90%. However, the small molecule peptide retention rates of Comparative Examples 1-4 were significantly lower than those of the corresponding Examples, with Comparative Example 2 having a retention rate of 85.2% and Comparative Example 3 having a retention rate of 83.2%.

[0203] The above accelerated stability investigation results further illustrate the necessity of selecting phospholipid specifications, adding wall materials, adding stabilizers, and choosing appropriate drying conditions.

[0204] Effect comparison group 3. Application performance inspection

[0205] The liposome samples prepared in Examples 1 to 4 and Comparative Examples 1 to 4 were used to prepare tablet preparations, hard shell capsules, and solid beverages, respectively, and their application performance was investigated.

[0206] Tablet compression performance inspection: inspect the state and stability of tablets.

[0207] Hard shell capsule inspection: Investigate the process feasibility and capsule stability.

[0208] Investigation of dissolution performance: Investigate the dissolution rate and solution uniformity.

[0209] Results of the performance test of liposome tableting

[0210]

[0211]

[0212] Results of performance investigation of liposome hard shell capsules

[0213]

[0214] Results of the investigation on the dissolution performance of liposomes

[0215] Preparation status Let stand for 12 hours Example 1 Quick dispersion and uniform dissolution Uniform Example 2 Quick dispersion and uniform dissolution Uniform Example 3 Quick dispersion and uniform dissolution Uniform Example 4 Quick dispersion and uniform dissolution Uniform Comparative Example 1 Slow dispersion, uniform dissolution Uniform Comparative Example 2 Slow dispersion, uniform dissolution Slight flocculation Comparative Example 3 Quick dispersion and uniform dissolution Slight flocculation Comparative Example 4 Quick dispersion and uniform dissolution Uniform

[0216] Based on the results of the liposome application performance investigation, its applicability in tableting, hard shell capsule and solid beverage applications was analyzed.

[0217] Tablet application investigation: The liposomes of Examples 1 to 4 were tested for tableting and stability, and the process was feasible and the stability passed. The liposome process of Comparative Examples 1 to 4 was feasible, but the retention rate of the tablet products prepared in Comparative Examples 2 and 3 decreased significantly after 20 days of acceleration. This is because without the protection of wall material and membrane stabilizer, the mechanical force during the tableting process destroyed its structure to a certain extent, resulting in a decrease in the retention rate of small molecule peptides.

[0218] Application in hard shell capsules: All examples passed the test. However, the liposomes in Comparative Example 2 could not be used in hard shell capsules due to their poor fluidity and easy clogging of the discharge port.

[0219] Solid Beverage Application: The liposomes in Examples 1-4 dispersed quickly and dissolved uniformly after dissolution, and remained uniform even after 12 hours of standing. However, the liposomes in Comparative Examples 1 and 2 dispersed more slowly, and Comparative Examples 2 and 3 exhibited slight flocculation after 12 hours of standing. This was also due to the lack of added wall material and membrane stabilizer. The liposomes, upon dissolution in water, experienced fusion of their phospholipid membranes, leading to flocculation and poor application in solid beverages.

[0220] Effect comparison group 4. Bioavailability study

[0221] A rat model was used to investigate the differences in bioavailability between different liposome products and their corresponding raw materials.

[0222] 18 rats were randomly divided into 3 groups, 6 in each group, namely raw material group, embodiment group and comparative example group. Alanine Glucose Dipeptide (raw material group), Alanine Glucose Dipeptide Liposome (Example 2) and Alanine Glucose Dipeptide Liposome (Comparative Example 7) were dissolved in deionized water to form a solution with the same Alanine Glucose Dipeptide concentration. Rats were fasted for 12 hours before gavage and resumed eating after gavage. All rats were drawn blood 0.2 ml at 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0h, 12.0h, 18.0h and 24.0h after administration. The concentration of Alanine Glucose Dipeptide in plasma at each time point was detected, and the area under the plasma concentration curve (AUC) was calculated.

[0223] Taking the relative bioavailability of the glutathione raw material as 100%, the difference in bioavailability between Example 2 and Comparative Example 7 is shown in the following table, showing the difference in relative bioavailability of different products.

[0224] Product Name Relative bioavailability (%) Alanylglutathione 100 Alanylglutamine liposomes (Example 2) 315 Alanylglutamine liposomes (Comparative Example 7) 174

[0225] Compared with the glutathione raw material, the glutathione liposomes (Example 2) can significantly improve the bioavailability. The bioavailability of the glutathione liposomes in Comparative Example 7 is higher than that of the raw material group and lower than that of Example 2 because the proportion of phospholipids is reduced. The specifications and proportion of phospholipids in liposomes can significantly affect the bioavailability of the product. When other indicators are consistent, increasing the amount of phospholipids can improve the bioavailability effect.

[0226] The comprehensive effect comparison results of groups 1 to 4 were analyzed to further summarize the importance of the formulation composition and preparation method of multilayer microencapsulated small molecule peptide liposomes:

[0227] The importance of lecithin purity: High-purity lecithins help improve the chemical stability of the product, especially in high-temperature accelerated stability testing. High-purity lecithins have a lighter color and odor, resulting in liposomes with better sensory quality. While low-purity lecithins can achieve good results in some cases, they require more organic solvents during preparation. Considering a comprehensive range of product performance indicators, including sensory and stability, high-purity lecithins are the preferred choice.

[0228] The importance of wall materials: Appropriate addition of wall materials can achieve secondary microencapsulation of liposomes, delaying the oxidation of phospholipids, further protecting the stability of small molecule peptides, and improving the entrapment efficiency and content retention rate of liposome products. It also enhances the fluidity of liposome products, improves the physical stability of the product, and prevents clumping and poor fluidity.

[0229] The role of membrane stabilizers: Membrane stabilizers enhance the structural stability of liposomes, preventing leakage of contents, thereby improving the retention rate of small molecule peptides. The lack of membrane stabilizers will slightly reduce the stability of the product.

[0230] Importance of Preparation Methods: Small molecule peptides are chemically unstable ingredients that are very sensitive to moisture, oxygen, light, and temperature. Therefore, stringent preparation methods can achieve higher product quality. Choosing low-temperature spray drying not only improves production efficiency, protects small molecule peptides from high temperature damage, reduces content loss during the preparation process, but also achieves better sensory quality.

[0231] It should be understood that the above embodiments are intended only to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection thereof. Persons skilled in the art may make various modifications and variations to the above embodiments without departing from the spirit and basic principles of the present invention, but such modifications and variations shall still fall within the scope of protection of the claims of the present invention and their equivalents. The scope of protection of the present invention shall be based on the appended claims and shall not be limited to the details of the above embodiments.

Claims

1. A multi-layer microencapsulated small molecule peptide liposome, characterized by: The composition comprises the following raw materials in parts by weight: 30-70 parts of small molecule peptide, 15-50 parts of phospholipid, 0.2-2 parts of antioxidant, 2-10 parts of membrane stabilizer, and 5-30 parts of wall material.

2. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The small molecule peptide is a water-soluble peptide selected from glutathione, acetyl glutathione, carnosine, decarboxylated carnosine, alanine dipeptide, anserine, acetyl carnosine, enkephalin, homocarnosine, thymopentin, collagen tripeptide, blue copper peptide, glycyl glycine, alanyl glutamyl, lysyl alanine, arginyl glycine, aspartyl serine, glutamyl serine, histidyl glycine, threonyl threonine, galanine tripeptide, lysine-glutamic acid-serine, arginine-glycine-aspartic acid Acid, asparagine-glycine-serine, histidine-glycine-histidine, serine-serine-serine, threonine-aspartic acid-lysine, cysteine-glycine-glutamic acid, as well as water-soluble dipeptides and modified compounds thereof, tripeptides and modified compounds thereof, tetrapeptides and modified compounds thereof, pentapeptides and modified compounds thereof, hexapeptides and modified compounds thereof, heptapeptides and modified compounds thereof, octapeptides and modified compounds thereof, nonapeptides and modified compounds thereof, decapeptides and modified compounds thereof.

3. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The phospholipid is selected from one or more of soybean lecithin, sunflower lecithin and rapeseed lecithin.

4. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The content of acetone insoluble matter in the phospholipid is required to be greater than 60wt%; the content of phosphatidylcholine in the phospholipid is required to be greater than 50wt%.

5. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The membrane stabilizer includes one or more of phytosterols, mono- and diglycerol fatty acid esters, fatty acids, and vegetable oils.

6. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The weight ratio of the membrane stabilizer to the phosphatidylcholine component in the phospholipid is 1:3-15.

7. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The antioxidant is selected from one or more of ascorbic acid, sodium ascorbate, ascorbyl palmitate, mixed tocopherols, citrus flavonoids, proanthocyanidins, tea polyphenols, and carotenoids.

8. The multi-layer microencapsulated small molecule peptide liposome according to claim 1, characterized in that: The wall material comprises one or more of modified starch, gum arabic, maltodextrin, resistant dextrin, inulin, maltooligosaccharide and isomaltooligosaccharide.

9. The method for preparing multi-layer microencapsulated small molecule peptide liposomes according to claim 1, wherein: The steps include: S1. Aqueous phase preparation: Prepare an aqueous solution containing small molecule peptides; S2. Preparation of alcohol phase: Dissolve phospholipids and membrane stabilizer in anhydrous ethanol and mix thoroughly until completely dissolved; S3. Wall material solution preparation: preparation of an aqueous solution of wall material; S4. The alcohol phase is slowly injected into the aqueous phase and stirred thoroughly to form an initial liposome dispersion solution, which is concentrated under reduced pressure to obtain a concentrate which is slowly added to the aqueous solution of the wall material and stirred thoroughly to form a multilayer microencapsulated liposome dispersion solution; S5. The obtained multi-layer microencapsulated liposome dispersion solution is subjected to high-speed shearing, high-pressure homogenization, and drying.

10. The method according to claim 9, characterized in that: The drying method in S5 can be selected from spray drying, low-temperature spray drying, freeze drying, and freeze spray drying.

11. The method according to claim 9, wherein: For the preferred low-temperature spray drying in S5, the inlet air temperature is selected to be 50-90°C, and the outlet air temperature is controlled at 30-40°C.

12. Use of the multi-layer microencapsulated small molecule peptide liposomes as claimed in claim 1, including the preparation of compressed tablet preparations, hard shell capsules, and solid beverages.

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