Milk-derived exosome buccal sugar and preparation method thereof

Abstract

The invention relates to the field of health food, in particular to a preparation method of milk-derived exosome buccal sugar, which comprises the following steps: mixing milk-derived exosome with a skeleton agent and auxiliary materials to prepare a dispersion solution; and placing the dispersion solution in an environment of-30 DEG C for vacuumizing, and then performing freeze drying according to freeze-drying program setting to obtain the milk-derived exosome buccal sugar. Through the preparation method, the activity of mEV in the product can be maintained to the maximum extent; the use amount of the freeze-drying protective agent is reduced, the mEV biological activity can be maintained, the mEV can be quickly released in the oral cavity, the taste is obviously improved, and the onset time is obviously prolonged.

Description

Technical Field

[0001] This disclosure relates to the field of health food, and in particular to a milk exosome freeze-dried orally disintegrating tablet and its preparation method. Background Technology

[0002] Milk-derived exosomes (mEVs), as unique bionanocarriers, exhibit properties drastically different from cell-derived exosomes, and their potential as oral drug delivery systems has attracted widespread research interest in recent years. Milk exosomes, 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 maintain their stability under the highly acidic environment of the stomach and the complex degradation conditions of the intestine, and their unique ability to cross the gastrointestinal barrier offers broad application prospects as an oral drug delivery tool.

[0003] However, the extraction process of milk exosomes faces significant technical challenges. Due to the complexity of milk composition, proteins such as casein and whey protein, which are abundant in milk, do not directly participate in the formation of exosomes. These proteins are not present on the surface of exosomes or inside vesicles, so these impurities need to be effectively removed during the extraction process.

[0004] Currently, although methods for removing impurities using salt precipitation and tangential flow ultrafiltration have been disclosed in invention patent CN114790439B, practical applications using high-concentration salt precipitation result in excessive salt consumption and lack specificity for protein precipitation. Most importantly, the addition of high-concentration salts (such as ammonium sulfate, sodium citrate, or disodium EDTA) conflicts with the intended use of milk exosomes as food ingredients, requiring additional desalting and purification steps to remove the previously added salt, leading to increased costs and limited scale. Furthermore, the method of preparing exosomes by precipitating casein with hydrochloric acid significantly damages exosome activity. Therefore, the preparation of milk exosomes currently faces problems such as low impurity removal efficiency, high reagent consumption, and insufficient yield and purity.

[0005] Furthermore, milk exosomes not only possess strong immunomodulatory potential but are also rich in key nutrients such as phospholipids, nucleic acids, and whey protein. These characteristics open up new avenues for their application in the food industry, providing not only important supplements of the aforementioned nutrients but also demonstrating significant advantages in promoting gastrointestinal health (such as relieving symptoms of gastric ulcers, enteritis, colitis, and constipation). Oral administration of milk exosomes has significant gastrointestinal health benefits. However, although research and development of milk exosomes in the pharmaceutical field (such as drug delivery systems and pharmaceutical excipients) is relatively advanced, their application in the food industry is still in its early stages. Existing mEV oral products are usually prepared by reconstitution of frozen solutions to obtain oral liquids or by lyophilizing powders using conventional freeze-drying technology, and still face a series of technical bottlenecks.

[0006] On the one hand, the liquid dosage form of mEV faces the problems of poor stability and difficulty in preservation. It is extremely unstable at room temperature, and even when stored under low temperature freezing (below -40 degrees Celsius), its shelf life is still limited. The active ingredients usually decay after one month of storage, and it has strict requirements for storage and transportation conditions, resulting in high costs.

[0007] On the other hand, the solid dosage form of mEVs is mainly freeze-dried powder. During the processing, in order to overcome the physical stress damage during freeze-drying, a large amount of freeze-drying protectants or excipients are often added. These additives may change the natural composition of exosomes and affect their biological activity. Freeze-dried powders often have a bitter taste and poor solubility when dissolved orally, and the original "fatty taste" may not be acceptable to all consumers, limiting the market penetration of the product.

[0008] In conclusion, there is an urgent need to develop a novel milk exosome extraction technology that can significantly improve the yield and purity of milk exosomes while reducing reagent consumption 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 milk-derived exosome food products that are low-cost to store and transport, easy to consume, have a long shelf life, and a pleasant taste. Summary of the Invention

[0009] In view of this, the purpose of this disclosure is to solve at least one of the problems existing in the prior art. More specifically, this invention aims to further explore the application potential of milk exosomes in the food field through technological innovation, formula optimization, and other research and development, providing more diversified solutions for human health. This disclosure aims to provide a method for preparing milk exosome oral sugar (lyophilized orally disintegrating tablets) and its application, to obtain milk exosome oral sugar with high unit yield, high vesicle purity, high bioactivity, and a taste, flavor, and effective ingredient release rate far superior to lyophilized powder, while also having significantly better storage stability and lower storage and transportation costs than frozen oral liquids.

[0010] To achieve the above-mentioned objectives, this disclosure provides the following technical solution:

[0011] Specifically, the first aspect of this disclosure provides a method for preparing oral saccharides from milk-derived exosomes, comprising the following steps:

[0012] Milk-derived exosomes were mixed with matrix agents, binders, and excipients to prepare a dispersion solution;

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

[0014] The adhesive comprises pullulan and / or hydroxypropyl methylcellulose;

[0015] The solution was placed in a -30°C freeze-drying chamber and evacuated, then freeze-dried according to the following procedure to obtain milk-derived exosome oral sugars:

[0016] Phase 1: The temperature is raised from -30℃ to -10℃ over a period of 20 minutes, and then maintained at -10℃ for 60 minutes.

[0017] Phase 2: Raise the temperature from -10℃ to 0℃ over a period of 10 minutes, and maintain the temperature at 0℃ for 60 to 90 minutes.

[0018] Phase 3 involves raising the temperature from 0°C to 25°C over a period of 25 minutes, and then maintaining the temperature at 25°C for 60 minutes.

[0019] In the aforementioned stages, stage 1 is a drying stage, which mainly controls the temperature and pressure below the product's eutectic point to remove water in the form of ice crystals. During this stage, a high vacuum is maintained, and there is a water vapor pressure difference so that water vapor has enough power to escape from the product. This stage can remove most of the moisture.

[0020] Phase 2 is the desorption drying phase, which is to remove some of the adsorbed water and bound water. This water is not frozen and cannot be removed in one drying cycle. In order to avoid the growth of microorganisms and to stabilize product quality, desorption drying, i.e., Phase 2, is adopted.

[0021] Phase 3 is the final finishing touch, adjusting the product temperature to near room temperature to achieve normal conditions for product preservation and stabilize the product's condition.

[0022] Optionally, Phase 1 specifically includes:

[0023] Phase 1-1: Raise the temperature from -30℃ to -25℃ over a period of 5 minutes, and then maintain the temperature at -25℃ for 30 minutes.

[0024] In stages 1-2, the temperature is raised from -25℃ to -10℃ over a period of 15 minutes, and then maintained at -10℃ for 60 minutes.

[0025] Optionally, Phase 3 specifically includes:

[0026] Stage 3-1: Raise the temperature from 0℃ to 10℃ over a period of 10 minutes, and maintain the temperature at 10℃ for 40 minutes.

[0027] Phase 3-2: Raise the temperature from 10℃ to 25℃ over a period of 15 minutes, and maintain the temperature at 25℃ for 60 minutes.

[0028] Preferably, in stage 2, the temperature is set to rise from -10℃ to 0℃, the heating time is 10 minutes, and the holding time is 60 minutes.

[0029] More preferably, in stage 2, the temperature is set to rise from -10°C to 0°C, the heating time is 10 min, and the holding time is 90 min.

[0030] Freeze-drying is a core process in the production of oral saccharides from milk-derived exosomes. By rationally setting the freeze-drying curve, the activity of mEV (mEV) is maximized, product quality is improved, and production costs are reduced. On one hand, the freeze-drying curve removes most of the water from the product during the sublimation stage. The technical solution disclosed here directly affects the sublimation time through the rational setting of the freeze-drying curve, avoiding product melting and collapse. On the other hand, by influencing the desorption drying temperature through the freeze-drying curve, potential impacts on related substances in the product are avoided, such as product collapse due to excessively high drying temperature or prolonged drying time due to excessively low drying temperature, ultimately avoiding energy waste. Furthermore, the freeze-drying curve also directly affects the product's moisture content, improving the water content and the stability of the product and its active ingredients. The technical solution disclosed here not only improves product quality but also significantly shortens the freeze-drying time. Further, milk-derived exosomes are mixed with a matrix agent, binder, and excipients, and purified water is added to 400 mg to prepare the dispersion solution. Preferably, the amount of milk-derived exosomes (mEV) used is 50 mg. In this disclosure, the active ingredient (active pharmaceutical ingredient) of the oral sugar-containing milk exosome is only the milk exosome and does not contain any other drug components. The function of the milk 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 more preferably, the matrix agent is mannitol; preferably, the dosage is 8-12 mg, and more preferably, the dosage is 8 mg.

[0032] Preferably, mannitol is used as the matrix agent, and simultaneously functions as a lyophilization protectant. Compared to existing technologies, where a typical 10mg lyophilized powder injection generally requires more than 50mg of lyophilization protectant or matrix agent (e.g., ginkgo biloba lyophilized powder injection), the amount of lyophilization protectant in this disclosure can be reduced from more than 50mg to 8mg. Clearly, the technical solution of this disclosure significantly reduces the amount of lyophilization protectant required. This not only solves the problem of poor solubility but also further improves the significant impact on the taste and flavor of the product.

[0033] In oral lyophilized tablet formulations, matrix agents serve as carriers to form a rigid, uniform matrix, thereby improving the appearance of the oral lyophilized tablets and providing a certain degree of hardness. In this disclosure, the amount of mannitol used has no significant effect on tablet appearance, wall adhesion, and disintegration time. The samples exhibited good appearance, wall adhesion, and disintegration time, and disintegrated rapidly within 1 second after being placed in the disintegration apparatus.

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

[0035] In oral lyophilized tablet formulations, binders play a role in shaping, providing tablet strength and toughness. The amount of binder affects the tablet shape, adhesion to the wall, disintegration, and dissolution of the lyophilized tablet. The binder and its amount disclosed herein prevent edge cracking and tablet breakage, and increase the disintegration rate, thereby affecting the dissolution effect; in addition, the use of binder improves the wall adhesion effect.

[0036] Pullulan replaces the traditional gelatin excipient in Zydis technology, solving the safety issue of excipient sourcing. Compared to gelatin, pullulan is a natural polysaccharide amylopectin with excellent adhesiveness and adhesion, better aqueous solution stability, and avoids the safety issues associated with animal-derived gelatin, such as mad cow disease and foot-and-mouth disease. Furthermore, animal-derived gelatin is difficult to gain acceptance among some religious groups; therefore, the milk-derived exosome oral sugar obtained through this disclosed technology has a wider target audience.

[0037] Preferably, the excipients include polysorbate 80, xanthan gum, sucralose, sweet orange powder flavoring, and purified water. Preferably, the dosage is: 0.4 mg polysorbate 80, 0.2 mg xanthan gum, 0.5 mg sucralose, 0.5 mg sweet orange powder flavoring, with the remainder being purified water to fill to 400 mg.

[0038] Among them, polysorbate 80 can also be used as a solubilizer, cosolvent, and dispersant; xanthan gum is mainly used as a suspending agent and stabilizer; sucralose is used as a flavoring agent, powdered flavoring is used as a flavor masking agent; and water is used as a solvent.

[0039] Furthermore, the excipients also include flavoring additives (sweeteners). Preferably, the flavoring additives account for 5% (w / v) of the dispersion solution by mass. More preferably, the flavoring additives are sucrose or xylitol. The above technical solution can prepare oral sugars from milk exosomes. Milk exosomes with added sucrose and xylitol have a more suitable taste, with a milky aroma and sweetness. Compared with conventional dosage forms, oral sugars have the best taste, retain the milky aroma, and disintegrate easily in the oral cavity.

[0040] The oral candy granules prepared using the flavoring formula, oral candy excipient formula, freeze-drying process technology and parameters provided in this disclosure exhibit the best stability. After freeze-drying, they can be stored at -20℃, and after 3 months, the granule count recovery rate remains at 85%. Natural flavoring and modification: Natural ingredients are used to flavor the milk-derived exosome product, masking unpleasant flavors while ensuring the product's health attributes are not affected. Compound formula design: Compound formulas are designed by combining other functional ingredients (such as probiotics and dietary fiber) to further enhance the product's benefits to gastrointestinal health and improve consumer acceptance.

[0041] This disclosed technical solution involves encapsulating the drug raw material in a water-soluble matrix material, injecting the drug suspension into a blister pack, flash-freezing with liquid nitrogen, sublimating to remove moisture, and then using freeze-drying technology to obtain a loose and porous formulation. This formulation disintegrates rapidly, dissolves instantly in the mouth, has no gritty feel, and is convenient to take. The resulting freeze-dried orally disintegrating tablets do not require water to take; saliva is sufficient for rapid disintegration or dissolution in the mouth. The proportion of inactive ingredients and excipients used in the preparation of the orally disintegrating tablets is low, having no impact on taste or flavor.

[0042] Furthermore, a second aspect of this disclosure provides a method for preparing milk-derived exosomes, comprising the following steps:

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

[0044] 2) Take the supernatant A and wash it with tangential flow hollow fiber ultrafiltration with a pore size of 300-750KD to obtain supernatant B;

[0045] 3) The supernatant was purified by column B to obtain milk exosomes.

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

[0047] In some embodiments, the pH of the milk is adjusted with citric acid. Preferably, after adjusting the pH, the temperature is adjusted. Preferably, the temperature is 35°C.

[0048] In some embodiments, the rennet includes rennet gold drops or bovine rennet.

[0049] Although rennet is widely recognized in the industrial application of casein coagulation, its direct application in the preparation of milk exosomes lacks sufficient theoretical basis and experimental verification. This disclosure innovatively explores the application of rennet, a commonly used additive in cheese production, in the preparation of milk exosomes without affecting the surface proteins of the exosomes. However, preliminary practice revealed that rennet can only coagulate proteins under suitable pH conditions, and the protease activity of rennet may potentially affect the integrity of the surface proteins of milk exosomes, thereby potentially affecting their biological activity. To address this issue, this disclosure, through systematic research screening and experimental verification, deeply explores the applicability of rennet in the production of milk exosomes. Experimental results show that under specific addition conditions and processing methods, rennet can effectively precipitate casein while maintaining the high purity, high yield, and high biological activity of milk exosomes. This discovery provides new ideas and methods for the preparation of milk exosomes, possessing significant academic value and application prospects.

[0050] This disclosure describes a rennet that requires a small amount of rennet and can specifically precipitate casein, and that the food-grade rennet complies with regulatory requirements for food ingredient production. The rennet specifically hydrolyzes casein without affecting exosome surface functional proteins, thus maximizing the preservation of the biological activity of exosomes in milk.

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

[0052] In some embodiments, supernatant A is microfiltered using a 0.45μm Capillary filter to further remove large particles, precipitates, and impurities. After microfiltration, the supernatant is subjected to 300KD-750kD tangential flow hollow fiber ultrafiltration and washing to obtain supernatant B.

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

[0054] This disclosure describes the preparation of milk exosomes via rennet and specific tangential flow hollow fiber ultrafiltration. Compared to traditional filtration methods, this avoids clogging caused by excessively large protein volumes and achieves high-efficiency filtration even at high flow rates. Furthermore, the preparation method described above does not require chromatographic chromatography, making the preparation simpler and less costly. Moreover, when used in food raw material production, it eliminates concerns about residual chromatographic packing resin components and contamination, thus making it safer.

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

[0056] In some implementations, the above purification is performed using a Capto Core 700 column.

[0057] A third aspect of this 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 the supernatant A and wash it with tangential flow hollow fiber ultrafiltration with a pore size of 300-750KD to obtain supernatant B;

[0060] 3) Take the supernatant B and purify it using a Capto Core 700 column to obtain milk exosomes.

[0061] This disclosure provides milk exosomes for the preparation of drugs for treating gastric ulcers.

[0062] In some embodiments, the aforementioned drug includes milk exosomes and pharmaceutically acceptable excipients or adjuvants.

[0063] The fourth aspect of this disclosure provides milk exosomes for use in oral drug delivery systems.

[0064] The fifth aspect of this disclosure provides milk exosomes for use in food preparation.

[0065] In some embodiments, the aforementioned food includes milk exosomes and food-acceptable additives.

[0066] The aforementioned technical solution, by combining the process of removing casein through rennet precipitation with the process of removing whey protein through tangential flow filtration, does not affect the surface proteins of milk exosomes, thus significantly improving exosome purity. Using the technology of this invention, the number of exosome particles can reach 1×10⁻⁶ per 50L of milk. 16 ~2×10 16 particles.

[0067] The milk exosomes with specific surface proteins obtained by this disclosure significantly improve the bioactivity of milk exosomes and are more conducive to improving the prevention and treatment of gastric ulcers. At the same time, this disclosure applies rennet to the production and preparation of milk exosomes, which can efficiently remove precipitates and impurities. This disclosure can prepare high-yield, high-bioactivity milk exosomes with fewer preparation steps and has developed a set of large-scale, low-cost, high-quality production processes centered around rennet.

[0068] Furthermore, in a sixth aspect, this disclosure provides a milk-derived exosome oral saccharide comprising the following raw materials by weight: 50 mg of milk-derived exosomes, 8-12 mg of matrix agent, and 8 mg of binder.

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

[0070] Preferably, the binder is pullulan polysaccharide 4 mg and hydroxypropyl methylcellulose 3 mg.

[0071] Preferably, the excipients also include 5% (w / v) sucrose or 5% (w / v) xylitol by weight. Attached Figure Description

[0072] The above and other features, advantages, and aspects of the embodiments of this disclosure will become more apparent from the accompanying drawings and the following detailed description. In the drawings, the same or similar reference numerals denote the same or similar elements, wherein:

[0073] Figure 1 This is a flowchart of the milk exosome preparation process involved in Embodiment 1 of this disclosure;

[0074] Figure 2 This is a schematic diagram of the particle size distribution of milk exosomes prepared in Embodiment 1 of this disclosure;

[0075] Figure 3 This is a schematic diagram of an electron microscope (negative staining) of milk exosomes in Embodiment 1 of this disclosure;

[0076] Figure 4 This is a SEC-HPLC chromatogram of milk exosomes involved in Example 1 of this disclosure.

[0077] Figure 5 This is a bar chart comparing the particle stability of different dosage forms in the embodiments of this disclosure;

[0078] Figure 6 This is a schematic diagram of the packaging box for the milk exosome oral sugar provided in this disclosure. Detailed Implementation

[0079] This disclosure will be described more fully below with reference to the accompanying drawings, in which various embodiments are illustrated. However, this disclosure may be implemented in many different ways and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be exhaustive and complete, and will fully convey the scope of this disclosure to those skilled in the art.

[0080] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0081] It should be understood that the order of steps or the sequence of actions is not important as long as this disclosure remains operational. Furthermore, two or more steps or actions may be performed simultaneously.

[0082] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0083] Unless otherwise specified, all terms used herein, including technical and scientific terms, shall have the same meaning as commonly understood by one of ordinary skill in the art. Terms defined in commonly used dictionaries shall be interpreted as having the same meaning as in the relevant technical context, and shall not be construed as having a formal meaning in an idealized or overly formal sense unless expressly defined in the specification.

[0084] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0085] All reagents, raw materials, instruments or equipment mentioned below 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] Rennet precipitation of casein: Take 50L of skim fresh milk out of the 4℃ refrigerator, heat it to 13℃, add citric acid to the milk to adjust the pH to 5.5, then heat the milk to 35℃, add bovine rennet to a final concentration of 0.8mg / ml, stir well, let it stand at room temperature for 40min, heat it to 42℃, gently break up the coagulated solids, and let it stand at room temperature for 1h.

[0090] Exosome preparation: Milk precipitated by rennet pretreatment was filtered through a 400-mesh screen to remove the precipitate. The supernatant was further microfiltered through a 0.45 μm Capone filter to remove large particles and impurities. The supernatant after microfiltration was then subjected to 750 kD tangential flow hollow fiber ultrafiltration with washing (50 L of purified water each time, for a total of 8 washes). Finally, the exosomes were purified by a Capto Core 700 column to obtain the final product.

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

[0092] Results: Comparing three batches of exosome products produced using the same process, the exosome yield, vesicle purity, particle size distribution, and size purity were all relatively stable and consistent with the electron microscopy results; the top 50 most abundant proteins in the proteomics results showed little difference.

[0093] Table 1 shows a comparison of the yield and purity of exosomes from different batches:

[0094] Table 1

[0095]

[0096] Based on the results in Table 1 of Example 1 and Figure 2-4 It can be seen that the milk exosome preparation technology in this invention yields exosomes with higher unit yield and higher purity.

[0097] The proteomic results of three batches of milk exosomes (the top 50 most abundant proteins) are shown in Table 2-4:

[0098] Table 2. Proteomics results of milk exosomes (batch 1)

[0099]

[0100]

[0101]

[0102] Table 3. Proteomics results of milk exosomes (batch 2)

[0103]

[0104]

[0105]

[0106] Table 4. Proteomics results of milk exosomes (batch 3)

[0107]

[0108]

[0109]

[0110] Example 2: Preparation of oral sugars from milk exosomes

[0111] 1. Screening of matrix agents

[0112] The matrix agents were mannitol, glycine, and dextran 70. Following the established preparation process, mEV oral sugars were prepared, and the matrix agent types were screened using appearance, wall adhesion, and disintegration time as evaluation indicators. The specific experimental design is shown in Table 5, which provides formulation information for matrix agent screening.

[0113] Table 5

[0114]

[0115]

[0116] Note: Purified water is used and removed during the preparation process, as in the following prescription.

[0117] Table 6

[0118]

[0119] Experimental conclusion:

[0120] Table 6 shows the screening and evaluation results of the matrix agents: batch 240129-3-1 (matrix agent: dextran 70) has an irregular surface, uneven color, raised areas, and poor tablet shape; batches 240129-1-1 (matrix agent: mannitol) and 240129-2-1 (matrix agent: glycine) have smooth and clean surfaces, uniform color, and good tablet shape, but batch 240129-2-1 (matrix agent: glycine) exhibits severe wall adhesion, resulting in a weight loss of 3.84%. Therefore, mannitol was selected as the matrix agent for this product.

[0121] 2. Investigation on the dosage of the matrix agent

[0122] The formulation used mannitol as the matrix agent. Roxadustat oral lyophilized tablets were prepared according to the established preparation process. The dosage of the matrix agent was investigated using appearance, wall adhesion, and disintegration time as evaluation indicators. The specific experimental design is shown in Table 7, which provides information on the formulation for screening the dosage of the matrix agent.

[0123] Table 7

[0124]

[0125] Table 8

[0126]

[0127]

[0128] Experimental conclusion:

[0129] Table 8 shows the results of the screening and evaluation of the matrix agent dosage. It can be seen that some tablets in batch 240218-1-1 (mannitol: 6mg) have uneven color and poor tablet shape; while batches 240129-1-1 (mannitol: 8mg), 240218-2-1 (mannitol: 10mg), and 240218-3-1 (mannitol: 12mg) have uniform color, smooth and clean surface, and good tablet shape. Moreover, there is no significant difference in the weight loss ratio of adhering to the wall and the disintegration time. This indicates that the dosage of the matrix agent mannitol in the range of 8 to 12mg / tablet has no significant effect on appearance, adhering to the wall, and disintegration time. Therefore, the dosage of mannitol is 8mg / tablet.

[0130] 3. Investigation of adhesive manufacturers and types

[0131] The formulation used pullulan and hydroxypropyl methylcellulose as binders. Following a pre-defined preparation process, mEV oral sugars were prepared, and the manufacturers and types of binders were investigated. Specific experimental design information for the binder manufacturer and type screening is shown in Table 9.

[0132] Table 9

[0133]

[0134] Table 10

[0135]

[0136]

[0137] Experimental conclusion:

[0138] As shown in Table 10, the screening and evaluation results of adhesive manufacturers and types indicate that batches 240220-1-1 (adhesive: pullulan), 240220-2-1 (adhesive: hydroxypropyl methylcellulose E50), and 240220-3-1 (adhesive: pullulan + hydroxypropyl methylcellulose E50) all have uniform color, smooth and clean surfaces, and no significant differences in sheet shape; compared with batches 240220-2-1 and 240220-3-1, batch 240220-1-1 exhibits more severe wall adhesion. By observing disintegration phenomena, batch 240220-1-1 showed a better disintegration time than batches 240220-2-1 and 240220-3-1. Compared to batch 240220-3-1, batch 240220-3-1 had fewer residual tablets after 60 seconds. This indicates that hydroxypropyl methylcellulose E50 has a higher viscosity than pullulan. Using hydroxypropyl methylcellulose E50 alone as a binder can improve wall adhesion but prolongs the disintegration time; using pullulan alone as a binder results in severe wall adhesion but faster disintegration. In conclusion, the tentative binder combination of pullulan and hydroxypropyl methylcellulose E50 will be used. Further adjustments to the hydroxypropyl methylcellulose E50 dosage will optimize tablet form, wall adhesion, and disintegration time.

[0139] 4. Adhesive dosage assessment

[0140] According to the established preparation process, the amount of pullulan was fixed at 4 mg / tablet, and the amount of hydroxypropyl methylcellulose E50 was reduced to prepare mEV oral sugar. The dosage was first evaluated based on appearance, wall adhesion, and disintegration time. The specific experimental design is shown in Table 11 for the formulation information on the amount of binder.

[0141] Table 11

[0142]

[0143]

[0144] Table 12

[0145]

[0146] Experimental conclusion:

[0147] Table 12 shows the results of the adhesive dosage screening test. The results indicate that batches 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) all exhibited uniform color, smooth and glossy surfaces, and no significant differences in tablet shape. With decreasing hydroxypropyl methylcellulose E50 dosage, the weight loss due to wall adhesion slightly increased, and the wall adhesion phenomenon slightly worsened, but the difference was minor. There was no significant difference in disintegration time, but with decreasing hydroxypropyl methylcellulose E50 dosage, the disintegrated tablets passed through the stainless steel tube sieve of the disintegrator more easily. Considering both the weight loss due to wall adhesion and the disintegration time, a hydroxypropyl methylcellulose E50 dosage of 3 mg / tablet was selected.

[0148] 5. Screening of freeze-drying process parameters

[0149] This experiment uses appearance and moisture content as the main evaluation indicators to screen the optimal freeze-drying curve.

[0150] Table 13

[0151]

[0152]

[0153] Note: The total duration does not include the time spent maintaining the container inside.

[0154] Experimental conclusion:

[0155] Table 13 shows the results of the freeze-drying curves. Curve 1 (batch 240522-2-1) was used during formulation screening, with a total time of 305 min. The freeze-dried samples showed good appearance and met moisture requirements. Curve 2 (batch 240620-1) eliminated the -25℃ and 10℃ temperature ranges compared to Curve 1 (batch 240522-2-1), shortening the overall time by 70 min. The samples were fully freeze-dried, and the finished product moisture content was acceptable, but slight bottom melting occurred in the tablets. Curve 3 (batch 240624-1) increased the freeze-drying time by 30 min at 0℃ compared to Curve 2, with a total freeze-drying time of 265 min. The fully freeze-dried samples showed good moisture content and properties; therefore, Curve 3 was determined to be the final freeze-drying curve.

[0156] 6: Screening of flavoring additives

[0157] Objective: To evaluate the taste and mouthfeel of milk exosomes with different additives, and to screen out the additives and formulations with the best taste.

[0158] Test method:

[0159] 1) Taste Experience Test of Milk Exosomes with Different Additives: Milk exosomes were supplemented with 5% (w / v) sucrose, 5% (w / v) xylitol, physiological saline, PBS, and 3% (w / v) casein, respectively. Five volunteers were recruited to participate in the oral administration test. Each volunteer orally administered one type of milk exosome oral supplement every 1 hour, completing the oral administration test of the five formulations over 5 hours. After each oral administration, volunteers provided feedback and recorded the taste and flavor. The order of administration was randomized, and each volunteer was unaware of the order in which the various formulations were administered.

[0160] 2) Taste experience test of different formulation types: Milk exosomes were formulated into three dosage forms: oral sugar, oral liquid, and conventional lyophilized powder (with 5% w / v mannitol as a lyophilization protectant). Five volunteers were recruited to participate in the oral test. The oral dosage of each formulation was kept consistent. Each volunteer took one formulation orally every 1 hour, and the taste and flavor were recorded after each oral administration.

[0161] result:

[0162] From Table 14, the evaluation results of milk exosome flavor with different additives, and Table 15, the taste experience test results of different formulation types, we can see that:

[0163] 1) Milk exosomes with added sucrose and xylitol have a more suitable taste, with a milky and sweet flavor.

[0164] 2) Compared with conventional dosage forms, oral sugar has the best taste, retains the milky flavor, and is easy to disintegrate in the mouth.

[0165] Table 14

[0166]

[0167] Table 15

[0168]

[0169] Example 3: Stability of mEV oral sugars compared to conventional lyophilized powders and frozen oral liquids

[0170] Objective: To evaluate the dose stability of exosomes in different formulations.

[0171] Experimental Methods: Milk exosomes were prepared into three dosage forms: oral lozenges, oral liquid, and conventional lyophilized powder. The oral lozenges and lyophilized powder were stored at -20°C, while the oral liquid was flash-frozen in liquid nitrogen and then stored at -80°C. After storage for the same period, the number of remaining exosome particles was measured. Before testing, the lyophilized powder was dissolved to maintain the same theoretical particle concentration as the liquid formulation, with a pre-lyophilized concentration of 4.00E+12 particles / ml.

[0172] Results: Tables 16(a) and 16(b) show the particle stability data for different dosage forms and Figure 5A comparison of particle stability in different dosage forms showed that oral sugar granules had the best stability, with a particle count recovery rate of 85% after 3 months of freeze-drying; liquid formulation granules had the worst stability, with the particle count decreasing significantly after 7 days and only about 25% remaining after 90 days.

[0173] Table 16(a)

[0174]

[0175] Table 16(b)

[0176]

[0177] Furthermore, such as Figure 6 As shown, this disclosure also provides packaging boxes for the aforementioned products, specifically: a special aluminum foil developed by the applicant, which solves problems such as curling, light blocking, and air permeability during the aluminum foil stamping process. This novel packaging material, as a liquid storage freeze-drying carrier, solves the problems of freeze-drying process and finished product stability. To facilitate product transportation and carrying, the applicant has developed a special wallet-style packaging box.

[0178] In summary, this disclosure presents for the first time the application of rennet precipitation to the preparation of milk exosomes, resulting in high-purity, high-yield, and highly active milk exosome products. This also promotes the application of milk exosomes in oral drug delivery systems and other biomedical fields. The above-described method for preparing oral sugars from milk exosomes maximizes the preservation of mEV activity in oral sugars (lyophilized orally disintegrating tablets); it not only reduces the amount of lyophilization protectant used but also helps maintain the biological activity of mEVs, ensuring rapid release of mEVs in the oral cavity and significantly improving taste and onset time. Furthermore, through technological innovation, formulation optimization, and other research and development efforts, the application potential of milk exosomes in the food industry will be further explored, providing more diversified solutions for human health.

[0179] Based on the foregoing, those skilled in the art will understand that the technical solutions claimed in this disclosure and their equivalents will be readily apparent. Furthermore, those skilled in the art can make appropriate modifications and alterations to the disclosed technical solutions as needed, and these modifications and improvements are also within the scope of protection of the claims in this disclosure.

Claims

1. A method for preparing oral saccharides from milk-derived exosomes, characterized in that, Includes the following steps: Milk-derived exosomes were mixed with matrix agents, binders, and excipients to prepare a dispersion solution; The matrix agent is selected from: mannitol, glycine, or dextran 70; The adhesive comprises pullulan and / or hydroxypropyl methylcellulose; The solution was placed in a -30°C chamber and evacuated. Freeze-drying was performed according to the following freeze-drying procedure to obtain oral sugars from milk-derived exosomes: Phase 1: The temperature is raised from -30℃ to -10℃ over a period of 20 minutes, and then maintained at -10℃ for 60 minutes. Phase 2: Raise the temperature from -10℃ to 0℃ over a period of 10 minutes, and maintain the temperature at 0℃ for 60 to 90 minutes. Phase 3: Raise the temperature from 0°C to 25°C over a period of 25 minutes, and maintain the temperature at 25°C for 60 minutes. The active ingredient in the milk exosomes, which contains sugar, is milk exosomes and does not contain other drug components.

2. The preparation method according to claim 1, characterized in that, Phase 1 includes: Phase 1-1: Raise the temperature from -30℃ to -25℃ over a period of 5 minutes, and then maintain the temperature at -25℃ for 30 minutes. In stages 1-2, the temperature is raised from -25℃ to -10℃ over a period of 15 minutes, and then maintained at -10℃ for 60 minutes.

3. The preparation method according to claim 1, characterized in that, Phase 3 includes: Stage 3-1: Raise the temperature from 0℃ to 10℃ over a period of 10 minutes, and maintain the temperature at 10℃ for 40 minutes. Phase 3-2: Raise the temperature from 10℃ to 25℃ over a period of 15 minutes, and maintain the temperature at 25℃ for 60 minutes.

4. The preparation method according to claim 1, characterized in that, Milk-derived exosomes were mixed with a matrix agent, binder, and excipients, and purified water was added to a final volume of 400 mg to prepare the dispersion solution.

5. The preparation method according to claim 4, characterized in that, The dosage of the milk-derived exosomes is 50 mg.

6. The preparation method according to claim 4, characterized in that, The skeleton agent is mannitol, which is used as a freeze-drying protectant in the freeze-drying process at a dosage of 8-12 mg.

7. The preparation method according to claim 4, characterized in that, The binder is pullulan and hydroxypropyl methylcellulose; the amount of pullulan is 4 mg and the amount of hydroxypropyl methylcellulose is 3 mg.

8. The preparation method according to claim 1, characterized in that, The excipients include polysorbate 80, xanthan gum, sucralose, sweet orange powder flavoring, and purified water.

9. The preparation method according to claim 4, characterized in that, The excipients include a flavoring additive comprising 5% (w / v) of the dispersion solution by mass, wherein the flavoring additive is sucrose or xylitol.

10. The preparation method according to claim 1, characterized in that, The preparation of the milk-derived exosomes includes the following steps: 1) Take milk, adjust the pH to 5.5 with citric acid, add 0.8 mg / ml rennet, heat to 42-43℃, remove the precipitate, and obtain supernatant A; 2) Take the supernatant A and use tangential flow hollow fiber ultrafiltration with a pore size of 300-750KD for washing 5-8 times to obtain supernatant B; 3) The supernatant B was purified to obtain milk-derived exosomes.

11. The preparation method according to claim 10, characterized in that, The rennet includes rennet gold drops and bovine rennet.

12. The preparation method according to claim 10, characterized in that, The milk includes skim milk.

13. A type of oral sugar derived from milk exosomes, characterized in that, The product contains the following raw materials: 50 mg of milk exosomes, 8-12 mg of matrix agent, and 8 mg of binder. The active ingredient of the milk exosomes containing sugar is milk exosomes, and it does not contain other drug components.

14. The oral candy as described in claim 13, characterized in that, The matrix agent is mannitol, and the mass of the mannitol is 8 mg.

15. The oral candy as described in claim 13, characterized in that, The binder is pullulan and hydroxypropyl methylcellulose, wherein the pullulan has a mass of 4 mg and the hydroxypropyl methylcellulose has a mass of 3 mg.

16. The oral candy as described in claim 13, characterized in that, The raw materials also include 5% (w / v) sucrose or 5% (w / v) xylitol by weight.

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