Milk-derived exosome lozenge and preparation method therefor

Abstract

A preparation method for a milk-derived exosome lozenge, comprising: mixing a milk-derived exosome with a matrix agent, a binder, and an excipient to prepare a dispersion solution; and placing the dispersion solution in an environment of -30°C for vacuumizing, and then carrying out freeze-drying according to a freeze-drying program setting to obtain a milk-derived exosome lozenge. By means of the preparation method, the activity of the milk-derived exosome in the product can be maintained to the maximum extent.

Description

Milk-derived exosome buccal sugar and preparation method thereof TECHNICAL FIELD

[0001] The present disclosure relates to the field of health food, in particular to a milk exosome freeze-dried buccal tablet and a preparation method thereof. BACKGROUND

[0002] Milk-derived exosomes or milk-derived extracellular vesicles (mEV) exhibit unique characteristics distinct from cell-derived exosomes as a unique biological nanocarrier, and its potential as an oral drug delivery system has attracted widespread research interest in recent years. Milk exosomes, as an important branch of milk-derived exosomes, are derived from milk, a natural resource, and are rich in various cell-derived exosomes. Studies have shown that milk exosomes can maintain their stability in the strong acidic environment of the stomach and the complex degradation conditions of the intestinal tract, and their unique ability to cross the gastrointestinal barrier provides a broad application prospect for their use as an oral drug delivery tool.

[0003] However, the extraction process of milk exosomes faces significant technical challenges. Due to the complexity of milk components, a large number of protein components such as casein and whey protein do not directly participate in the formation of exosomes, and these proteins do not exist on the surface of exosomes or in vesicles, so these impurities need to be effectively removed during the extraction process.

[0004] Currently, although the invention patent CN114790439B discloses a method for removing impurities by salt precipitation and tangential flow ultrafiltration technology, in actual application, high-concentration salt precipitation consumes too much and has no specificity for protein precipitation. The most important problem is that the addition of high-concentration salt (such as ammonium sulfate, sodium citrate or disodium ethylenediaminetetraacetate) conflicts with the food raw material application direction of milk exosomes, and an additional desalting and purification step is required to remove the early added salt, resulting in increased cost and limited scale. Moreover, the method of precipitating casein by adding salt acid to prepare exosomes causes greater damage to the activity of exosomes. Therefore, the current preparation of milk exosomes still faces problems such as low impurity removal efficiency, large reagent consumption, insufficient yield and purity, etc.

[0005] In addition, milk-derived exosomes not only have strong immunomodulatory potential, but are also rich in key nutrients such as phospholipids, nucleic acids, and whey proteins. These characteristics open up new avenues for their application in the food industry, not only providing important supplements of the above nutrients, but also exhibiting significant advantages in promoting gastrointestinal health (such as relieving symptoms such as gastric ulcers, intestinal inflammation, colitis, and constipation). Milk-derived exosomes have significant gastrointestinal health benefits when taken orally. However, although the development of milk-derived exosomes in the medical field (such as drug delivery systems and pharmaceutical excipients) has been relatively in-depth, their application in the food industry is still in its infancy. Existing mEV oral products are usually oral solutions obtained by reconstituting frozen solutions or lyophilized powders prepared by conventional lyophilization techniques, and still face a series of technical bottlenecks.

[0006] On the one hand, liquid formulations of mEVs have poor stability and are difficult to store. They are extremely unstable at room temperature, and even if stored at low temperatures (below -40 degrees), their shelf life is still limited. The effective ingredients will decay after 1 month of storage, and the storage and transportation conditions are demanding, resulting in high costs.

[0007] On the other hand, solid formulations of mEVs are mainly freeze-dried powders. To overcome physical stress damage during the lyophilization process, a large amount of lyoprotectant or excipient is often added during processing. These additives can change the natural composition of exosomes and affect their biological activity. Freeze-dried powders often have a bitter taste and are difficult to dissolve when taken orally, and the original "milk fat taste" may not be acceptable to all consumers, limiting the market penetration of the product.

[0008] In summary, there is an urgent need to develop a new technology for extracting milk exosomes that can significantly improve the yield and purity of milk exosomes while reducing the consumption of reagents during impurity removal, thereby promoting the application of milk exosomes in oral drug delivery systems and other biomedical fields. At the same time, there is a lack of a low-cost storage and transportation, easy-to-take, long-shelf-life, and pleasant-taste milk exosome food. SUMMARY

[0009] Therefore, the purpose of the present disclosure is to solve at least one of the problems in the prior art. More specifically, the present invention aims to further exploit the application potential of milk-derived exosomes in the food industry through technical innovation, formula optimization, and other aspects of research and development, and to provide more diversified solutions for human health. The present disclosure aims to provide a method for preparing milk-derived exosome oral sugar (lyophilized oral disintegrating tablets) and its application, to obtain milk exosomes with high unit yield, high vesicle purity, and high biological activity, as well as oral sugar with far superior taste, flavor, and effective ingredient release speed to lyophilized powder, and far superior storage stability and storage and transportation costs to frozen oral solution.

[0010] To achieve the above-mentioned object of the application, the present disclosure provides the following technical solutions:

[0011] Specifically, the present disclosure provides a preparation method of milk-derived exosome oral sugar, comprising the following steps:

[0012] The milk-derived exosomes are mixed with a skeleton agent, a binder, and an auxiliary material to form a dispersion solution;

[0013] The skeleton agent is selected from mannitol, glycine, or dextran 70;

[0014] The binder includes pullulan and / or hydroxypropyl methyl cellulose;

[0015] The solution is placed in a -30°C freezer, vacuumed, and then freeze-dried according to the following freeze-drying program settings to obtain the milk-derived exosome oral sugar:

[0016] In stage 1, the temperature is raised from -30°C to -10°C, the temperature rising time is 20 min, and the temperature is maintained at -10°C for 60 min;

[0017] In stage 2, the temperature is raised from -10°C to 0°C, the temperature rising time is 10 min, and the temperature is maintained at 0°C for 60 min to 90 min;

[0018] In stage 3, the temperature is raised from 0°C to 25°C, the temperature rising time is 25 min, and the temperature is maintained at 25°C for 60 min.

[0019] In the aforementioned stages, stage 1 is a primary drying stage, mainly controlling the temperature and pressure below the product's eutectic point to remove water in the form of ice crystals, and maintaining a high vacuum degree in this stage, so that the water vapor has enough power to escape the product due to the water vapor pressure difference. This stage can remove most of the water;

[0020] Stage 2 is a secondary drying stage, which is used to remove part of the adsorbed water and bound water that is not frozen and cannot be removed in the primary drying stage. To avoid microbial proliferation and stabilize the product quality, secondary drying, i.e., stage 2, is adopted;

[0021] Stage 3 is the final finishing part, which adjusts the product temperature close to room temperature to achieve ordinary conditions for storing the product and stabilizing the product state.

[0022] Optionally, stage 1 specifically includes:

[0023] In stage 1-1, the temperature is raised from -30°C to -25°C, the temperature rising time is 5 min, and the temperature is maintained at -25°C for 30 min;

[0024] Stage 1-2, the temperature is raised from -25℃ to -10℃, the temperature rising time is 15 min, and maintained at -10℃ for 60 min.

[0025] Optionally, stage 3 specifically comprises:

[0026] Stage 3-1: the temperature is raised from 0℃ to 10℃, the temperature rising time is 10 min, and maintained at 10℃ for 40 min;

[0027] Stage 3-2: the temperature is raised from 10℃ to 25℃, the temperature rising time is 15 min, and maintained at 25℃ for 60 min.

[0028] Preferably, in stage 2, the temperature is set to be raised from -10℃ to 0℃, the temperature rising time is 10 min, and the maintaining time is 60 min.

[0029] More preferably, in stage 2, the temperature is set to be raised from -10℃ to 0℃, the temperature rising time is 10 min, and the maintaining time is 90 min.

[0030] The freeze-drying process is the core process in the production process of the milk-derived exosome oral sugar, and through the reasonable setting of the above freeze-drying curve, the mEV activity is directly maximized, the product quality is improved, and the production cost is reduced. On the one hand, the freeze-drying curve removes most of the water in the product in the sublimation stage, and the reasonable setting of the freeze-drying curve directly affects the sublimation time, avoiding the melting collapse of the product; on the other hand, the freeze-drying curve affects the analytical drying temperature, avoiding the potential impact on the related substances of the product, such as product collapse caused by too high drying temperature, too long drying time caused by low drying temperature, etc., ultimately avoiding the waste of energy. In addition, the freeze-drying curve also directly affects the water content of the product, improves the high and low water content, and improves the stability of the product and the active ingredients therein. The technical solution of the present disclosure not only improves the product quality, but also greatly shortens the freeze-drying time. Further, the milk-derived exosome is mixed with a matrix agent, a binder, and an excipient, and purified water is added to 400 mg to form the dispersion solution. Preferably, the amount of milk-derived exosome (raw material mEV) is 50 mg. In the present disclosure, the active component (raw material) of the milk-derived exosome oral sugar is only milk-derived exosome, without other pharmaceutical ingredients, and the role of the milk-derived exosome is to provide key nutrients and promote gastrointestinal health, and it is not used as a drug delivery carrier.

[0031] Preferably, the matrix agent is any one of mannitol, glycine, and dextran 70, and preferably the matrix agent is mannitol; preferably, the amount is 8-12 mg, and more preferably, the amount is 8 mg.

[0032] Preferably, mannitol is used as a skeleton agent, and mannitol also has the function of a freeze-drying protective agent. Compared with the prior art, the ordinary freeze-dried powder injection 10 mg preparation generally needs a freeze-drying protective agent or a skeleton agent of 50 mg or more (for example, ginkgo leaf freeze-dried powder injection), and the amount of the freeze-drying protective agent can be reduced from 50 mg or more to 8 mg. Obviously, the technical solution of the present disclosure greatly reduces the amount of the freeze-drying protective agent. Not only does it solve the problem of poor solubility, but it also further improves the serious impact on the taste and smell of the oral administration.

[0033] In the oral freeze-dried tablet preparation, the skeleton agent is used as a carrier to form a hard and uniform skeleton to improve the appearance of the oral freeze-dried tablet and provide a certain hardness. The amount of mannitol in the present disclosure has no significant effect on the appearance, sticking wall and disintegration time of the tablet. The appearance, sticking wall and disintegration time of the sample are good, and the sample can be quickly disintegrated within 1 s after being put into the disintegration tester.

[0034] Preferably, the binder includes pullulan and hydroxypropyl methyl cellulose; more preferably, the amount of pullulan is 4 mg, and more preferably, the amount of hydroxypropyl methyl cellulose is 3 mg.

[0035] In the oral freeze-dried tablet preparation, the binder plays a role in shaping, providing tablet strength and toughness, and the amount of the binder affects the shape, sticking wall, disintegration and dissolution of the freeze-dried tablet. The binder and its amount in the present disclosure avoid edge and tablet cracking, and improve the disintegration rate, which in turn affects the dissolution effect. In addition, the use of the binder improves the sticking wall effect.

[0036] Pullulan replaces the traditional gelatin auxiliary material of Zydis technology, solving the safety problem of the source of the auxiliary material. Compared with gelatin, pullulan is a natural polysaccharide amylopectin with excellent adhesion and adhesion. The aqueous solution is more stable, and there is no safety problem such as "mad cow disease" and "foot-and-mouth disease" that may exist in animal-derived gelatin. In addition, animal-derived gelatin is difficult to be recognized by some people with religious beliefs, so the audience of the milk-derived exosome oral sugar obtained by the technical solution of the present disclosure is wider.

[0037] Preferably, the auxiliary material includes polysorbate 80, xanthan gum, sucralose, sweet orange powder essence, and purified water. Preferably, the amount is: 0.4 mg of polysorbate 80, 0.2 mg of xanthan gum, 0.5 mg of sucralose, 0.5 mg of sweet orange powder essence, and the rest is purified water filled to 400 mg.

[0038] Among them, polysorbate 80 can also be used as a solubilizing agent, a co-solvent, a dispersant; xanthan gum is mainly a suspending agent and a stabilizer; sucralose is a flavoring agent, and powder essence is a taste masking agent; water is a solvent.

[0039] Further, the adjuvant also includes a flavoring additive (sweetener), preferably, the flavoring additive accounts for 5% (w / v) of the mass percentage of the dispersion solution, more preferably, the flavoring additive is sucrose or xylitol. Through the above technical solution, milk exosome lozenges can be prepared, and the milk exosome lozenges added with sucrose and xylitol have a more suitable taste and a milk aroma and sweetness. Compared with conventional dosage forms, the lozenges have the best taste, retain the milk aroma, and are easy to disintegrate in the oral cavity.

[0040] The lozenge particles prepared by the flavoring formula, the lozenge adjuvant formula, the freeze-drying process technology and parameters provided in the technical solution have the best stability, can be stored at -20°C after freeze-drying, and the particle recovery rate is still 85% after being placed for 3 months. Through natural flavoring and improvement: the milk exosome product is flavored with natural ingredients to mask the undesirable flavor while ensuring that the health attributes of the product are not affected. Compound formula design: design a compound formula by combining other functional ingredients (such as probiotics and dietary fiber) to further enhance the benefits of the product to the gastrointestinal health and improve consumer acceptance.

[0041] The technical solution of the present disclosure wraps the drug raw material in a water-soluble matrix material, then injects the drug suspension into a bubble cap, rapidly freezes it in liquid nitrogen, and removes the water by sublimation to obtain a loose and porous preparation product by freeze-drying technology. The preparation product disintegrates quickly, dissolves in the mouth immediately, has no sand feeling, and is convenient to take. The freeze-dried buccal tablets obtained at the same time do not need to be taken with water and can be quickly disintegrated or dissolved in the mouth by saliva; the inactive ingredients and adjuvant ingredients used in the preparation process of the buccal tablets have a low proportion and no effect on taste and flavor.

[0042] Further, the second aspect of the present disclosure provides a preparation method of milk exosome, comprising the following steps:

[0043] 1) Take milk, adjust the pH to 5.5, add 0.8 mg / ml rennet, heat to 42-43°C, remove the precipitate, and obtain supernatant A;

[0044] 2) Take supernatant A, wash and filter by 300-750 KD pore size tangential flow hollow fiber ultrafiltration to obtain supernatant B;

[0045] 3) Take supernatant B, column purification to obtain milk exosome.

[0046] In some embodiments, the temperature of the milk is adjusted, preferably, the temperature is 13°C.

[0047] In some embodiments, the pH of the above-mentioned milk is adjusted by citric acid, and after adjusting the pH, the temperature is adjusted, preferably, the temperature is 35°C.

[0048] In some embodiments, the above-mentioned rennet includes rennet golden drops and bovine-derived rennet.

[0049] Although chymosin has been widely recognized in the industrial application of coagulating casein protein, its direct application in the preparation of milk exosomes still lacks sufficient theoretical basis and experimental verification. The present disclosure innovatively explores the application of chymosin, a common additive in cheese production, in the preparation of milk exosomes without affecting the surface proteins of milk exosomes. However, it was found in preliminary practice that chymosin can only coagulate proteins in a suitable pH environment, and the protease activity of chymosin may have a potential impact on the integrity of the surface proteins of milk exosomes, which may affect their biological activity. To solve this problem, the present disclosure explores the applicability of chymosin in the production of milk exosomes through systematic research, screening and experimental verification. The experimental results show that under specific addition conditions and processing methods, chymosin can effectively precipitate casein protein while maintaining high purity, high yield and high biological activity of milk exosomes. This discovery provides a new approach and method for the preparation of milk exosomes, which has important academic value and application prospects.

[0050] The present disclosure uses less chymosin and can specifically precipitate casein protein. Food-grade chymosin meets the regulatory requirements for food raw material production. Chymosin specifically hydrolyzes casein protein without affecting the functional proteins on the surface of exosomes, which can maximize the biological activity of milk exosomes.

[0051] In some embodiments, the precipitate is removed by filtration with a 400-mesh mesh cloth to obtain supernatant A.

[0052] In some embodiments, the supernatant A is micro-filtered with a 0.45 μm capsule filter to further remove large particles and large particle impurities. The supernatant after microfiltration is washed with 300 KD-750 kD tangential flow hollow fiber ultrafiltration to obtain supernatant B.

[0053] In some embodiments, the tangential flow hollow fiber ultrafiltration washing frequency is 5-8 times.

[0054] The present disclosure prepares milk exosomes by chymosin and specific tangential flow hollow fiber ultrafiltration. Compared with traditional filtration methods, it can avoid blockage caused by large protein volume. Even at high flow rates, it can still achieve high filtration efficiency. In addition, the preparation method described above does not require chromatography, which is simpler, lower in cost, and safer for food raw material production without considering chromatography filler resin composition residues and contamination.

[0055] In some embodiments, the above-mentioned milk includes skim fresh milk.

[0056] In some embodiments, the above-mentioned purification uses a Capto core 700 chromatographic column.

[0057] The third aspect of the present disclosure provides a milk exosome prepared by the following steps:

[0058] 1) Take milk, adjust the pH to 5.5 with citric acid, add 0.8 mg / ml rennet, heat to 42°C, remove the precipitate, and obtain supernatant A.

[0059] 2) Take supernatant A, wash and filter by tangential flow ultrafiltration with a pore size of 300-750 KD hollow fiber to obtain supernatant B.

[0060] 3) Take supernatant B, purify with a Capto core 700 chromatographic column to obtain milk exosomes.

[0061] The present disclosure provides milk exosomes for preparing a drug for treating gastric ulcer.

[0062] In some embodiments, the above-mentioned drug comprises milk exosomes and a pharmaceutically acceptable excipient or adjuvant.

[0063] The fourth aspect of the present disclosure provides milk exosomes for an oral drug delivery system.

[0064] The fifth aspect of the present disclosure provides milk exosomes for preparing food.

[0065] In some embodiments, the above-mentioned food comprises milk exosomes and a food acceptable adjuvant.

[0066] The foregoing technical solution combines the process of removing casein by rennet precipitation and the process of removing whey protein by tangential flow washing and filtering, which does not affect the surface proteins of milk exosomes, and can significantly improve the purity of exosomes. Using the technology in the present disclosure, 1 x 10 16 ~ 2 x 10 16 particles can be obtained from 50 L of milk.

[0067] The milk exosomes with specific surface proteins obtained by the technical solution of the present disclosure significantly improve the biological activity of milk exosomes, which is more conducive to improving the effect of preventing and treating gastric ulcer. At the same time, the present disclosure applies rennet to the production and preparation of milk exosomes, which can efficiently remove precipitates and impurities. The technical solution of the present disclosure can prepare high-yield, high-biological-activity milk exosomes through fewer preparation steps, and a large-scale, low-cost, high-quality production process centered on rennet has been developed.

[0068] Further, the sixth aspect of the present disclosure provides a milk exosome oral sugar, comprising the following raw materials by mass: milk exosomes 50 mg, matrix agent 8-12 mg, and adhesive 8 mg.

[0069] Preferably, the matrix agent is mannitol 8 mg.

[0070] Preferably, the binder is pullulan 4 mg and hypromellose 3 mg.

[0071] Preferably, the adjuvant further comprises sucrose 5% (w / v) or xylitol 5% (w / v). BRIEF DESCRIPTION OF DRAWINGS

[0072] The above and other features, aspects and advantages of various embodiments of the present disclosure will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings, in which like reference numerals refer to like elements, in which:

[0073] FIG. 1 is a flow chart of a process for preparing milk exosomes according to an embodiment of the present disclosure;

[0074] FIG. 2 is a graph showing the particle size distribution of milk exosomes prepared according to an embodiment of the present disclosure;

[0075] FIG. 3 is a graph showing the electron microscopy (negative staining) of milk exosomes according to an embodiment of the present disclosure;

[0076] FIG. 4 is a graph showing the SEC-HPLC chromatogram of milk exosomes according to an embodiment of the present disclosure;

[0077] FIG. 5 is a bar graph showing the stability of particles of different dosage forms according to an embodiment of the present disclosure;

[0078] FIG. 6 is a schematic diagram of a packaging box for milk exosome lozenges according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0079] The present disclosure will be described more fully hereinafter with reference to the accompanying drawings, in which various embodiments are shown. The present disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art.

[0080] The terms "comprise", "have" or "contain", including their grammatical conjugations, are generally used to indicate that the elements or steps listed thereafter are open-ended and non-limiting, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0081] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present disclosure remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0082] The use of any and all examples, or exemplary language (e.g., "such as" or "including") provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0083] Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms, such as those defined in commonly utilized dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined in the specification.

[0084] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are used merely to convey general understanding of the application. Consequently, unless otherwise indicated herein, the numerical ranges and parameters are approximations and are understood to have a value, and are not to be construed as being limited to the specific values recited herein. Consequently, unless otherwise indicated herein, all ranges, numbers, values and percentages are to be read as "about" and are understood to have a value, and are not to be construed as being limited to the specific values recited herein. In this context, "about" is understood to refer to a value that is nearly the same as a true value.

[0085] The various reagents, raw materials, instruments or equipment involved as follows can be purchased and obtained from the market.

[0086] Example 1: Preparation of milk exosomes from cheese whey

[0087] Objective: To prepare milk exosomes with high concentration, high purity and good stability.

[0088] Process flow:

[0089] Chymosin precipitation of casein protein: 50 L of fresh skimmed milk was taken out from the refrigerator at 4°C, heated to 13°C, and then citric acid was added to adjust the pH to 5.5. Then the milk was heated to 35°C, and 0.8 mg / ml of bovine chymosin was added. After stirring evenly, it was placed at room temperature for 40 min, and then heated to 42°C. The coagulated solids were gently dispersed, and then placed at room temperature for 1 h.

[0090] Exosome preparation: The milk after chymosin pre-treatment precipitation was filtered with a 400 mesh screen cloth to remove the precipitate. The supernatant was further micro-filtered with a 0.45 μm capsule filter to remove large particles and impurities. The micro-filtered supernatant was subjected to tangential flow hollow fiber ultrafiltration with a 750 kD cut-off, and then washed with 50 L of purified water each time for a total of 8 times. Finally, the exosomes were obtained by purification on a Capto core 700 chromatography column.

[0091] Characterization methods: NanoFCM was used to detect particle number, particle size distribution and vesicle purity; negative staining electron microscopy was used to verify the morphology of exosomes; SEC-HPLC was used to detect the size purity of exosomes; proteomics results were used to characterize the main constituent proteins in exosomes.

[0092] Results: Comparing the exosome products of the same process of 3 batches, the exosome yield, vesicle purity, particle size distribution and size purity are relatively stable, and are consistent with the electron microscopy results; the protein types of the Top50 proteins with high abundance in the proteomics results are not much different.

[0093] The exosome yield and purity of different batches are shown in Table 1:

[0094] Table 1

[0095] According to the results in Table 1 and Figures 2-4 in Example 1, it can be seen that the exosome preparation technology in the present application has higher unit yield and higher purity.

[0096] The proteomics results of 3 batches of milk exosomes (top 50 proteins with the highest content) are shown in Tables 2-4:

[0097] Table 2 Milk exosome (batch 1) proteomics results of milk exosomes

[0098] Table 3 Milk exosome (batch 2) proteomics results of milk exosomes

[0099] Table 4 Milk exosome (batch 3) proteomics results of milk exosomes

[0100] Example 2: Preparation of milk exosome oral sugar

[0101] 1. Selection of matrix agent types

[0102] The matrix agent types are: mannitol, glycine, dextran 70. According to the set preparation process, mEV oral sugar was prepared, and appearance, wall sticking, disintegration time limit were used as evaluation indexes to screen the matrix agent types. The specific test design is shown in Table 5 Matrix agent type screening prescription information.

[0103] Table 5 Note: Purified water is used and removed during preparation. The following prescriptions are the same.

[0104] Table 6

[0105] Experimental conclusion:

[0106] From the evaluation results of the skeleton agent type screening in Table 6, it can be seen that the 240129-3-1 batch (skeleton agent: dextran 70) has irregular surface, uneven color, protrusions, and poor tablet shape; the 240129-1-1 batch (skeleton agent: mannitol) and the 240129-2-1 batch (skeleton agent: glycine) have smooth surface, uniform color, and good tablet shape, but the 240129-2-1 batch (skeleton agent: glycine) has serious sticking to the wall, with weight loss of 3.84% due to sticking to the wall. In summary, mannitol is selected as the skeleton agent of the product.

[0107] 2. Investigation of the amount of skeleton agent

[0108] The prescription uses mannitol as the skeleton agent, and the rosaxatan oral lyophilized tablets are prepared according to the set preparation process. The amount of the skeleton agent is investigated by taking the properties, sticking to the wall, and disintegration time as the evaluation indexes. The specific test design is shown in Table 7.

[0109] Table 7

[0110] Table 8

[0111] Experimental conclusion:

[0112] From the evaluation results of the skeleton agent type screening in Table 8, it can be seen that the 240218-1-1 batch (mannitol: 6 mg) has uneven color and poor tablet shape; the 240129-1-1 batch (mannitol: 8 mg), the 240218-2-1 batch (mannitol: 10 mg), and the 240218-3-1 batch (mannitol: 12 mg) all have uniform color, smooth surface, and good tablet shape, and there is no significant difference in the weight loss ratio due to sticking to the wall and the disintegration time, indicating that the amount of the skeleton agent mannitol within the range of 8-12 mg / tablet has no significant effect on the appearance, sticking to the wall, and disintegration time, and therefore the amount of mannitol is 8 mg / tablet.

[0113] 3. Investigation of the manufacturers and types of the binding agent

[0114] The prescription uses pullulan and hydroxypropyl methylcellulose as the binding agent, and the mEV oral sugar is prepared according to the set preparation process. The manufacturers and types of the binding agent are investigated. The specific test design is shown in Table 9.

[0115] Table 9

[0116] Table 10

[0117] Experimental conclusion:

[0118] From the results of adhesive manufacturer and type screening evaluation in Table 10, 240220-1-1 batch (adhesive: pullulan), 240220-2-1 batch (adhesive: hydroxypropyl methyl cellulose E50) and 240220-3-1 batch (adhesive: pullulan + hydroxypropyl methyl cellulose E50) all have uniform color, smooth surface and no obvious difference in tablet shape; 240220-1-1 batch has more serious sticking wall phenomenon compared with 240220-2-1 batch and 240220-3-1 batch. Through observation of disintegration phenomenon, 240220-1-1 batch has better disintegration time limit than 240220-2-1 batch and 240220-3-1 batch, and 240220-3-1 batch has less residual tablets than 240220-2-1 batch after 60s. It shows that the viscosity of hydroxypropyl methyl cellulose E50 is larger than that of pullulan, using hydroxypropyl methyl cellulose E50 alone as adhesive can improve the sticking wall phenomenon, but will prolong the disintegration time limit; using pullulan alone as adhesive, the sticking wall phenomenon is serious, but the disintegration is faster. In summary, the adhesive type is tentatively determined as pullulan and hydroxypropyl methyl cellulose E50, and the amount of hydroxypropyl methyl cellulose E50 will be adjusted in the future to further optimize the tablet shape, sticking wall and disintegration time limit.

[0119] 4. Adhesive dosage investigation

[0120] According to the set preparation process, the amount of pullulan is fixed as 4mg / tablet, and the amount of hydroxypropyl methyl cellulose E50 is reduced to prepare mEV buccal tablets. The specific experimental design is shown in Table 11 adhesive dosage screening prescription information.

[0121] Table 11

[0122] Table 12

[0123] Experimental conclusion:

[0124] From the results of the adhesive dosage screening evaluation in Table 12, it can be seen that the three batches of 240220-3-1 (hydroxypropyl methylcellulose E50: 4 mg), 240222-1-1 (hydroxypropyl methylcellulose E50: 3 mg), and 240222-2-1 (hydroxypropyl methylcellulose E50: 2 mg) are all uniform in color, smooth and clean in surface, and have no significant differences in tablet shape. With the decrease of the amount of hydroxypropyl methylcellulose E50, the weight loss ratio of sticking wall slightly increases, and the sticking wall phenomenon slightly worsens, but the difference is small. There is no significant difference in disintegration time, but with the decrease of the amount of hydroxypropyl methylcellulose E50, the disintegrated tablets are more easily passed through the stainless steel screen of the disintegration tester. Considering the weight loss ratio of sticking wall and the disintegration time, the amount of hydroxypropyl methylcellulose E50 is selected as 3 mg / tablet

[0125] 5. Freeze-drying process parameter screening

[0126] In this experiment, appearance and moisture were used as the main evaluation indexes to screen the optimal freeze-drying curve.

[0127] Table 13 Note: The total duration does not include the time for entering the box.

[0128] Experimental conclusion:

[0129] From the results of the freeze-drying curve evaluation in Table 13, it can be seen that during the prescription screening, the curve of 240522-2-1 batch (curve 1) was used, with a total duration of 305 min. The appearance of the freeze-dried samples was good, and the moisture also met the requirements. Compared with curve 1 (240522-2-1 batch), curve 2 (240620-1 batch) deleted the temperature segments of -25℃ and 10℃, and the overall time was shortened by 70 min. The samples were fully loaded and freeze-dried, and the moisture of the finished product was qualified, but the tablets showed slight bottom melting. Curve 3 (240624-1 batch) increased the time at 0℃ by 30 min based on curve 2, and the total freeze-drying time was 265 min. The moisture and properties of the fully loaded freeze-dried samples were good, and therefore curve 3 was determined as the final freeze-drying curve.

[0130] 6: Flavoring additive screening

[0131] Objective: To evaluate the taste and mouthfeel of different additives of milk exosomes, and to screen the best additive and formulation type for export.

[0132] Test method:

[0133] 1) Different additive milk exosome taste experience test: 5% (w / v) sucrose, 5% (w / v) xylitol, physiological saline, PBS, 3% (w / v) casein were added to milk exosomes respectively. Five volunteers were recruited for oral test, each volunteer orally took one kind of milk exosome sucking sugar every 1 h, and 5 h completed the oral test of 5 kinds of formula, and feedback was recorded after each oral completion. The order of distribution was random, and each volunteer did not know the distribution order of various oral formulas.

[0134] 2) Different formulation types of taste experience test: milk exosomes were made into sucking sugar, oral liquid, and conventional freeze-dried powder (with 5% w / v mannitol as freeze-drying protectant). Five volunteers were recruited for oral test, and the oral dose of different formulations was kept consistent, each volunteer orally took one kind of formulation every 1 h, and feedback was recorded after each oral completion.

[0135] Results:

[0136] From Table 14. Different additive milk exosome taste evaluation results, Table 15 different formulation types of taste experience test, it can be known that:

[0137] 1) The taste of milk exosomes with added sucrose and xylitol is more suitable, with milk aroma and sweetness.

[0138] 2) Compared with conventional formulations, the taste of sucking sugar is the best, retains milk aroma, and is easy to disintegrate in the oral cavity.

[0139] Table 14

[0140] Table 15

[0141] Example 3: Stability of mEV sucking sugar and conventional freeze-dried powder, frozen oral liquid

[0142] Objective: To evaluate the dose stability of exosomes of different formulations

[0143] Test method: Milk exosomes were made into sucking sugar, oral liquid, and conventional freeze-dried powder, sucking sugar and freeze-dried powder were stored at -20℃, and oral liquid was stored at -80℃ after quick freezing in liquid nitrogen. After the same storage time, the remaining particle number of exosomes was detected, and the theoretical particle concentration of freeze-dried powder was kept consistent with that of liquid formulation before detection, and the concentration before freeze-drying was 4.00E+12 particles / ml.

[0144] Results: As shown in Table 16(a), 16(b) and Figure 5, the stability of the lozenge particles is the best, and the recovery rate of the particles is still 85% after 3 months of storage after freeze-drying; the stability of the liquid particles is the worst, and the number of particles has decreased significantly after 7 days of storage, and only about 25% of the particles remain after 90 days of storage.

[0145] Table 16(a)

[0146] Table 16(b)

[0147] Further, as shown in Figure 6, the present disclosure also provides a packaging box for the above-mentioned product, specifically, a special aluminum foil developed by the applicant, which solves the problems of curling, light blocking, and air permeability during the stamping process of the aluminum foil. The new packaging material serves as a liquid storage freeze-drying carrier, solving the problems of freeze-drying process and product stability. To facilitate the transportation and carrying of the product, the applicant has developed a special money clip type packaging box.

[0148] In summary, the present disclosure first discloses the application of chymosin precipitation method to the preparation of milk exosomes, which can produce high-purity, high-yield, and high-activity milk exosome products. At the same time, it promotes the application of milk exosomes in oral drug delivery systems and other biomedical fields. The above-mentioned lozenge preparation method of milk-derived exosomes can maximize the activity of mEVs in lozenges (freeze-dried oral disintegrating tablets); not only reduces the amount of freeze-drying protectants, but also helps to maintain the biological activity of mEVs, ensures the rapid release of mEVs in the oral cavity, significantly improves the taste and onset time. Through technical innovation, formula optimization and improvement, the application potential of milk-derived exosomes in the food field will be further explored, and more diversified solutions will be provided for human health.

[0149] Based on the above, those skilled in the art can understand that the technical solutions claimed by the present disclosure and their equivalent technical solutions will be obvious. In addition, those skilled in the art can also make appropriate modifications and changes to the disclosed technical solutions according to needs, and these modified and improved technical solutions are also within the protection scope of the claims of the present disclosure.

Claims

1. A method for preparing a milk-derived exosome buccal sugar, characterized in that, comprising the following steps: taking milk-derived exosomes and mixing them with a skeleton agent, a binder, and an auxiliary material to prepare a dispersion solution; the skeleton agent is selected from mannitol, glycine, or dextran 70; the binder includes pullulan and / or hypromellose; the solution is placed in a -30℃ freezer and vacuumized; freeze-drying is performed according to the following freeze-drying program settings, thereby obtaining a milk-derived exosome buccal sugar: phase 1, the temperature is raised from -30℃ to -10℃ for 20 min, and maintained at -10℃ for 60 min; phase 2, the temperature is raised from -10℃ to 0℃ for 10 min, and maintained at 0℃ for 60-90 min; phase 3, the temperature is raised from 0℃ to 25℃ for 25 min, and maintained at 25℃ for 60 min; wherein the active ingredient of the milk-derived exosome buccal sugar is milk-derived exosomes, and does not contain other drug ingredients.

2. The method for preparing according to claim 1, characterized in that, the phase 1 comprises: phase 1-1, the temperature is raised from -30℃ to -25℃ for 5 min, and maintained at -25℃ for 30 min; phase 1-2, the temperature is raised from -25℃ to -10℃ for 15 min, and maintained at -10℃ for 60 min.

3. The method for preparing according to claim 1, characterized in that, the phase 3 comprises: phase 3-1, the temperature is raised from 0℃ to 10℃ for 10 min, and maintained at 10℃ for 40 min; phase 3-2, the temperature is raised from 10℃ to 25℃ for 15 min, and maintained at 25℃ for 60 min. taking milk-derived exosomes and mixing them with a skeleton agent, a binder, and an auxiliary material, adding purified water to 400 mg to prepare the dispersion solution. The amount of milk-derived exosomes is 50 mg. The skeleton agent is mannitol, which is used as a freeze-drying protective agent in the freeze-drying program, and the amount is 8-12 mg. The binder is pullulan and hypromellose; the amount of pullulan is 4 mg, and the amount of hypromellose is 3 mg. The auxiliary material includes polysorbate 80, xanthan gum, sucralose, sweet orange powder essence, and purified water. The auxiliary material includes a flavoring additive accounting for 5% (w / v) of the mass percentage of the dispersion solution, and the flavoring additive is sucrose or xylitol. The preparation of the milk-derived exosomes comprises the following steps: 1) taking milk and adjusting the pH to 5.5 with citric acid, adding 0.8 mg / ml rennet, heating to 42-43℃, removing the precipitate, and obtaining supernatant A; 2) taking the supernatant A and washing it with a 300-750 KD pore size tangential flow hollow fiber ultrafiltration for 5-8 times to obtain supernatant B; 3) taking the supernatant B and purifying it to obtain milk-derived exosomes. The rennet includes rennet gold drops and bovine-derived rennet. The milk includes skimmed fresh milk. ​ ​ ​ ​ 4. The production method according to claim 1, wherein ​ 5. The production method according to claim 4, wherein ​ 6. The production method according to claim 4, wherein ​ 7. The production method according to claim 4, wherein ​ 8. The production method according to claim 1, wherein ​ 9. The production method according to claim 4, wherein ​ 10. The production method according to claim 1, wherein ​ ​ ​ ​ 11. The production method according to claim 10, wherein ​ 12. The production method according to claim 10, wherein ​ 13. A milk-derived exosome buccal sweet, characterized in that, The quality raw material comprises: milk-derived exosomes 50 mg, a skeleton agent 8-12 mg, and a binder 8 mg, wherein the active ingredient of the milk-derived exosomes for oral sucking is the milk-derived exosomes, and no other pharmaceutical ingredient is contained.

14. The chewing gum of claim 13, wherein the gum base comprises from about 0.5 to about 2.0 wt% of the chewing gum. The skeleton agent is mannitol, and the mass of the mannitol is 8 mg.

15. The chewing gum of claim 13, wherein the gum base comprises about 20% to about 40% of the chewing gum. The binder is pullulan and hypromellose, the mass of the pullulan is 4 mg, and the mass of the hypromellose is 3 mg.

16. The chewing gum of claim 13, wherein the gum base comprises from about 0.5 to about 2.0 wt% of the chewing gum. The raw material further comprises sucrose with a mass percentage of 5% (w / v) or xylitol with a mass percentage of 5% (w / v).

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