Mesenchymal stem cell exosome and enzyme compounded health-care beverage and preparation method thereof

Abstract

The invention discloses a mesenchymal stem cell exosome and enzyme compounded health-care beverage and a preparation method thereof, and belongs to the technical field of health-care food preparation. The health-care beverage is formed by compounding human umbilical cord mesenchymal stem cell exosomes, an enzyme stock solution, normal saline and a sweetening agent, and the preparation method comprises the following steps: preparing the human umbilical cord mesenchymal stem cell exosomes, preparing the enzyme stock solution, then mixing the exosomes and the enzyme stock solution with the normal saline, and adding the sweetening agent. The health-care beverage has the effects of resisting aging, regulating immunity, resisting inflammation and resisting oxidation. The preparation method is simple and convenient in process and controllable in operation, and the prepared product has excellent health-preserving and health-care functions.

Description

Technical Field

[0001] This invention relates to the field of health food preparation technology, specifically to a health beverage composed of mesenchymal stem cell exosomes and enzymes, and its preparation method. Background Technology

[0002] Stem cells are the origin cells of the body, possessing the ability to self-renew and differentiate into multiple lineages. Mesenchymal stem cells (MSCs) are a branch of stem cells, also possessing self-renewal and multi-lineage differentiation capabilities. Umbilical cord MSCs, isolated from the umbilical cord of newborns, have advantages such as stronger proliferative and differentiation capabilities, low immunogenic activity, ease of isolation, high purity, absence of tumor cell contamination, ease of quality control, cryopreservation capability, low risk of infection, harmless collection, and fewer ethical controversies. MSCs also possess multi-lineage differentiation potential, support hematopoiesis, promote hematopoietic stem cell engraftment, regulate immunity, and are easy to isolate and culture.

[0003] Exosomes are extracellular vesicles with a diameter of approximately 30-150 nm, released into the extracellular matrix after the fusion of intracellular multivesicular bodies with the cell membrane. Almost all cells can release exosomes. Exosomes are classified into three types: exosomes (30-150 nm), ectosomes (100-1000 nm), and apoptotic bodies (50-5000 nm). Exosomes are released via exocytosis, carrying molecules such as proteins and RNA. Through endocytosis, they form primary endosomes, which bud inward to form multivesicular bodies, fuse with the plasma membrane, and are released extracellularly, or transported to lysosomes for degradation. Exosomes serve as a pathway for intercellular communication, secreting exosomes carrying different components, which are then absorbed by recipient cells, facilitating the exchange of substances and signals. Exosome production involves proteins such as Rab GTPases, ESCRT proteins, CD9, CD81, CD63, flotilin, TSG101, ceramides, and Alix. Exosomes from different sources exhibit heterogeneity, containing mRNA, miRNA, gDNA fragments, and proteins within their cavities, reflecting the characteristics of the cells from which they originate. They can be taken up at a systemic level, altering the state of target cells. Umbilical cord mesenchymal stem cell exosomes, reflecting the characteristics of their source cells, possess application potential. Formulating umbilical cord mesenchymal stem cell exosomes may help to exert health-improving effects.

[0004] Traditional Chinese medicine (TCM) is a creation of the Chinese nation. Approximately 90% of Chinese medicinal herbs are plants, with their active ingredients encased within cell walls composed of cellulose, hemicellulose, pectin, lignin, and other components. Traditional processing methods, such as decoction, soaking, and percolation, cannot break down these cell walls, resulting in significant losses of active ingredients, low yields, inadequate resource utilization, long processing cycles, numerous steps, high temperatures, high energy consumption, and high costs. Modern artificially cultivated Chinese medicinal herbs have shorter growth cycles, lower levels of active ingredients, and require larger quantities according to pharmacopoeias. Traditional methods cannot achieve the desired therapeutic effects, thus limiting their application.

[0005] Overcoming the drawbacks of traditional methods and maximizing the value and efficiency of traditional Chinese medicine (TCM) through modern technology has become crucial. Enzymatic hydrolysis technology, which utilizes enzymes to degrade target materials, is applied to TCM to improve the extraction and separation of effective components, increase their content, promote the transformation of active ingredients, and drive industrial leapfrog development. Enzymatic hydrolysis of TCM herbs fully utilizes and enhances their efficacy, produces low-molecular-weight cellulose, and complexes and removes harmful components, reducing toxic side effects.

[0006] Enzymes are produced from animal, plant, fungal, and medicinal materials through microbial fermentation. They contain active substances, biological enzymes, and probiotics, and are rich in nutrients and have high economic value. Enzyme fermentation involves inoculating microorganisms and conducting multi-stage, directional fermentation to produce functional beverages rich in organic acids, oligosaccharides, enzymes, amino acids, and vitamins. The enzymatic hydrolysis products of medicinal and edible herbs are fermented to extract and enhance active ingredients such as polyphenols, ketones, alkenes, terpenes, polysaccharides, and saponins, thus creating health benefits.

[0007] Traditional enzyme fermentation involves natural fermentation by a mixed microbial community, taking three months or longer. Without proper regulation, it is susceptible to contamination by harmful bacteria, molds, and pathogens, failing to meet hygiene standards. Combining enzymatic hydrolysis and fermentation in the development of traditional Chinese medicinal materials effectively solves these problems. Summary of the Invention

[0008] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0009] The purpose of this invention is to provide a health beverage composed of mesenchymal stem cell exosomes and enzymes, and its preparation method, for improving health.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A health beverage composed of mesenchymal stem cell exosomes and enzymes is made from human umbilical cord mesenchymal stem cell exosomes, enzyme stock solution, physiological saline and sweeteners. The human umbilical cord mesenchymal stem cell exosomes are obtained by culturing human umbilical cord mesenchymal stem cells in vitro, secreting exosomes, and then extracting and purifying them. The enzyme stock solution is obtained by mixing the first component with water, adding a compound enzyme for enzymatic hydrolysis to obtain the first enzymatic hydrolysis extract, and then adding a compound engineered bacteria to the first enzymatic hydrolysis extract for fermentation. The saline solution is an aqueous solution containing 0.9% sodium chloride; The sweetener is selected from one or more of D-mannitol, xylitol, isomaltitol, sorbitol, high maltose powder, and white sugar.

[0011] The human umbilical cord mesenchymal stem cell exosomes are exosomes secreted by human umbilical cord mesenchymal stem cells, containing some of the bioactive substances of human umbilical cord mesenchymal stem cells, and have anti-aging and immune-regulating functions.

[0012] The first component is selected from at least one of Polygonatum sibiricum, Pueraria lobata and Dioscorea opposita.

[0013] By mass fractions, the first component comprises 80-150 parts of Polygonatum sibiricum, 100-170 parts of Pueraria lobata, and 80-150 parts of Dioscorea opposita.

[0014] The concentration of human umbilical cord mesenchymal stem cell exosomes in health drinks is 1-10 billion exosomes / ml.

[0015] The optimal value is 2 billion exosomes / mL.

[0016] The preparation method of health beverages includes the following steps: S1. Culture of human umbilical cord mesenchymal stem cells: The umbilical cord specimen was removed from a laminar flow hood and placed in a disposable sterile culture dish. It was thoroughly washed with PBS, and the umbilical cord was cut into segments approximately 1 cm long using tissue scissors. Arteries and veins were removed, and the Warton gel was cut to 1 mm. 3 Size, inoculated at T-25 cm 2 Add 6 mL of complete culture medium to the culture flask and culture in a 37 ℃, 5% CO2 cell culture incubator. When the cells reach 70%-80% confluence, passage them and identify them at the 4th generation.

[0017] When the fourth-generation umbilical cord mesenchymal stem cells reached 90% fusion and were in good growth condition, they were digested with trypsin. After the cells became rounded, fetal bovine serum was added to stop the digestion. The cells were repeatedly pipetted to form a single-cell suspension. The suspension was centrifuged at 2000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in 10 mL of PBS. 20 μL of CD29-PE, CD34-PE, CD44-PE, CD45-PC5, and CD105-PE antibody fluorescent dyes were added sequentially to the assay tubes. An equal amount of IgG-PC5 and IgG-PE fluorescent dyes were added to the isotype control tubes. The cells were incubated at 4 ℃ in the dark for 20 min. The cells were washed twice with PBS and resuspended to a final volume of 500 μL. The cells were then analyzed by flow cytometry.

[0018] S2. Exosome preparation: Exosomes were extracted from the culture supernatant collected by starvation culture method.

[0019] To prepare a 32% (w / v) PEG6000 solution: Weigh 320 g of PEG6000 powder and 117.5 g of sodium chloride, dissolve them in ultrapure water, heat and stir until completely dissolved, then bring the volume to 1000 ml and autoclave. The resulting PEG6000 solution is a 4X stock solution.

[0020] The collected cell supernatant was transferred to 50 ml centrifuge tubes and centrifuged at 3000 rpm for 20 min at room temperature to remove cells and cell debris. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove large particles and vesicles.

[0021] Add the PEG6000 stock solution to make the final concentration of PEG6000 8% (W / V), mix well, and let stand overnight at 4 ℃.

[0022] The solution obtained in the previous step was centrifuged for the first time at 4 ℃ and 5000 rpm for 30 min, and the supernatant was discarded.

[0023] After discarding the supernatant, perform a second short centrifugation to remove the liquid from the tube wall, then discard the supernatant.

[0024] If necessary, add an appropriate amount of physiological saline to resuspend the exosome solution.

[0025] S3. Quality Control: Particle size analysis 30-250 nm (nanoparticle tracking analysis), concentration ≥1×10⁻⁶ 10 Particles / mL, total protein BCA detection <100 μg / mL, positive rate of markers CD63 / CD81 / CD9 ≥95%, calnexin negative, and clear exosome structure visible under transmission electron microscopy.

[0026] Finally, the sample was resuspended in particle-free saline by shaking at room temperature for up to 30 minutes.

[0027] S4. Preparation of enzyme stock solution: Mix the first component with water and add a compound enzyme for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 35℃-45℃ and the enzymatic hydrolysis time is 2.5 h-4 h to obtain the first enzymatic hydrolysis extract. Add compound engineered bacteria to the first enzymatic hydrolysis extract for fermentation. The fermentation temperature is 25℃-35℃ and the fermentation time is ≤5 d. Stop fermentation when the residual sugar content is ≤1.2 g / L to obtain the enzyme stock solution.

[0028] The complex enzyme is selected from at least one of α-amylase, cellulase and pectinase; the ratio of α-amylase, cellulase and pectinase in the complex enzyme is (1.2-1.6):1:1.

[0029] Based on the first enzymatic hydrolysis extract, the material-to-liquid ratio of the compound enzyme to the first enzymatic hydrolysis extract is (17-21):1.

[0030] Before adding the compound enzyme, the mixture of the first component and water is boiled for ≥2 hours; the compound enzyme is added after the mixture is cooled to 35℃-45℃.

[0031] The preparation method also includes inactivating the enzyme in the first enzymatic hydrolysis extract. The enzyme inactivation method is selected from any one of high-temperature enzyme inactivation, enzyme inhibitor enzyme inactivation, antibody-mediated enzyme inactivation, and protein-bound enzyme inactivation.

[0032] After adjusting the pH of the first enzymatic hydrolysis extract to 5.0-6.0, a carbon source and a compound engineered bacteria are added for fermentation. The pH adjustment reagent is sodium bicarbonate solution. Based on the first enzymatic hydrolysis extract, the amount of carbon source added is 11%-16%. By mass fraction, the carbon source includes 3%-4% amino acids and 8%-12% sugars. The amino acids are selected from at least one of monosodium glutamate, lysine, and leucine. The sugars are selected from at least one of brown sugar, fructose, and glucose.

[0033] Based on the first enzymatic hydrolysis extract, the amount of compound engineered bacteria added is 0.3%-0.7%; the compound engineered bacteria are selected from at least one of yeast, acetic acid bacteria and lactic acid bacteria; by mass fraction, the compound engineered bacteria include 0.28%-0.33% yeast, 0.10%-0.15% acetic acid bacteria and 0.04%-0.08% lactic acid bacteria.

[0034] S5. Compounding: Mix the exosome solution with the enzyme stock solution, dilute with physiological saline to an exosome concentration of 1-10 billion exosomes / mL, add sweetener at a concentration of 1%-2% of the total amount, and obtain a health beverage.

[0035] The health beverage also contains nano-bubble hydrogen gas at a concentration of 1 ppm to 1.5 ppm, preferably 1.2 ppm.

[0036] The beneficial effects of this invention are as follows: By organically integrating the enzyme stock solution with the fermentation products and exosomes, the exosomes are stably dispersed in the beverage system, avoiding the loss of bioactivity caused by the easy agglomeration of exosomes. At the same time, the bioactive components of the enzyme help enhance the intercellular communication efficiency of exosomes, forming a non-obvious synergistic mechanism. The preparation method is simple and controllable, effectively solving the stability problem of existing exosome preparations in oral health care applications, and facilitating industrial promotion.

[0037] The health beverage prepared by this invention uses exosomes as the main component, which exerts its core biological functions of anti-aging and immune regulation. With the assistance of enzymes, it further enhances the overall anti-inflammatory and antioxidant efficacy, reduces inflammation in the body, and improves the body's disease resistance. It has comprehensive health benefits that exceed the sum of simple components. Detailed Implementation

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0039] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0040] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single embodiment or an embodiment selectively excluded from other embodiments.

[0041] Example 1 This embodiment provides a method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes, including the following steps: S1. Culture of human umbilical cord mesenchymal stem cells: The umbilical cord specimen was removed from a clean bench and placed in a disposable sterile culture dish. It was thoroughly washed with PBS, and the umbilical cord was cut into segments approximately 1 cm long using tissue scissors. Arteries and veins were removed, and the Warton gel was cut to 1 mm. 3 Size, inoculated at T-25 cm 2 Add 6 mL of complete culture medium to the culture flask and culture in a 37 ℃, 5% CO2 cell culture incubator. When the cells reach 70%-80% confluence, passage them and identify them at the 4th generation.

[0042] When the fourth-generation umbilical cord mesenchymal stem cells reached 90% fusion and were in good growth condition, they were digested with trypsin. After the cells became rounded, fetal bovine serum was added to stop the digestion. The cells were repeatedly pipetted to prepare a single-cell suspension, centrifuged at 2000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in 10 mL PBS. 20 μL of CD29-PE, CD34-PE, CD44-PE, CD45-PC5, and CD105-PE antibody fluorescent dyes were added sequentially to the assay tubes. An equal amount of IgG-PC5 and IgG-PE fluorescent dyes were added to the isotype control tubes. The cells were incubated at 4 ℃ in the dark for 20 min, washed twice with PBS, and resuspended to a final volume of 500 μL. The cells were then analyzed by flow cytometry. The results showed that the cells expressed CD29, CD44, and CD105 positively, and CD34 and CD45 negatively, confirming them as human umbilical cord mesenchymal stem cells.

[0043] S2. Exosome preparation: Exosomes were extracted from the supernatant of fourth-generation cell culture using a starvation culture method.

[0044] To prepare a 32% (W / V) PEG6000 solution: Weigh 320 g of PEG6000 powder and 117.5 g of sodium chloride, dissolve them in ultrapure water, heat and stir until completely dissolved, then bring the volume to 1000 ml and autoclave to obtain the 4X stock solution.

[0045] The collected cell supernatant was transferred to 50 ml centrifuge tubes and centrifuged at 3000 rpm for 20 min at room temperature to remove cells and cell debris. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove large particles and vesicles.

[0046] Add the PEG6000 stock solution to make the final concentration of PEG6000 8% (W / V), mix well, and let stand overnight at 4 ℃.

[0047] The resulting solution was centrifuged for the first time at 4 ℃ and 5000 rpm for 30 min, and the supernatant was discarded.

[0048] After discarding the supernatant, perform a second short centrifugation to remove the liquid from the tube wall, then discard the supernatant.

[0049] Resuspend the exosomes in an appropriate amount of physiological saline to obtain an exosome solution.

[0050] S3. Quality Control: A nanoparticle tracking analyzer was used to detect particle sizes of 30-250 nm and a concentration of 1.5 × 10⁻⁶. 10 The total protein concentration was 80 μg / mL as determined by BCA. Western blot analysis showed a 98% positivity rate for CD63, CD81, and CD9, and a negative result for calnexin. Clearly defined bimembrane exosomes with a diameter of approximately 100 nm were observed under transmission electron microscopy.

[0051] The sample was resuspended in particulate-free saline by shaking at room temperature for 20 minutes.

[0052] S4. Preparation of enzyme stock solution: By mass, the first component includes 150 parts of Polygonatum sibiricum, 100 parts of Pueraria lobata, and 150 parts of Dioscorea opposita. The first component is mixed with water at a ratio of 10:1, pulped, and then steamed for 2 hours before cooling to room temperature to obtain the slurry.

[0053] 2.0% of a compound enzyme was added to the slurry for enzymatic hydrolysis. The ratio of α-amylase, cellulase, and pectinase in the compound enzyme was 1.5:1:1. The hydrolysis temperature was 40 ℃, and the time was 3 h. Enzymatic hydrolysis was performed with ultrasonic assistance at a power of 200 W. After hydrolysis, the mixture was filtered, and the filtrate was inactivated by enzymes in a 100 ℃ water bath for 1 h. The filtrate was then cooled to room temperature to obtain the first enzymatic extract. The material-to-liquid ratio was 19:1.

[0054] The pH of the first enzymatic hydrolysis extract was adjusted to 5.55 using 1 mol / L sodium bicarbonate solution, and 3.05% monosodium glutamate and 10% brown sugar were added as carbon sources.

[0055] The activated composite engineered bacteria were inoculated into the mixed system at an inoculation amount of 0.5%, including 0.13% acetic acid bacteria, 0.06% lactic acid bacteria, and 0.31% yeast.

[0056] Seal the opening with sealing film and apply ultrasonic intensity of 0.16 W / cm. 3 After treatment with constant-frequency ultrasound for 88 min, the mixture was allowed to ferment statically. The fermentation temperature was 30 ℃, and the fermentation lasted for 4 days. The residual sugar content was 1.0 g / L. Fermentation was then terminated, and the enzyme stock solution was obtained by filtration.

[0057] S5. Compounding: Mix the exosome solution with the enzyme stock solution, dilute with physiological saline to an exosome concentration of 2 billion exosomes / mL, add 1.5% xylitol for flavoring, then add nano-bubble hydrogen to 1.2 ppm, fill into hydrogen-barrier containers, sterilize by irradiation, and store.

[0058] The resulting health beverage is light purple in color, with a sweet and sour, refreshing taste, and the aroma of Polygonatum sibiricum and purple yam, along with a fermented fragrance, and has no off-odors. Tests showed that the exosome concentration was stable, the active enzyme components were fully preserved, and it possesses anti-aging, immune-regulating, anti-inflammatory, and antioxidant functions.

[0059] Example 2 This embodiment provides a method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes, which differs from Embodiment 1 in that: S1. The cultivation and identification processes are the same, and the identification results are consistent.

[0060] S2, the preparation process of exosomes is the same.

[0061] S3. Quality control: Particle size 30-250 nm, concentration 1.2×10⁻⁶ 10 The protein content was 90 μg / mL, with a positive rate of 96% for CD63 / CD81 / CD9 and a negative result for calnexin. The structure was clearly visible under transmission electron microscopy.

[0062] S4. Preparation of Enzyme Stock Solution: The first component consists of 100 parts of Polygonatum sibiricum, 100 parts of Pueraria lobata, and 120 parts of Dioscorea opposita. Mix, pulp, and cook for 2 hours, then cool. Add a compound enzyme at a ratio of 1.2:1:1, hydrolyze at 35 ℃ for 4 hours, and use ultrasonic power of 180 W. After enzyme inactivation, adjust the pH to 5.0, and add 3% monosodium glutamate and 8% brown sugar. The inoculum size is 0.3% (0.28% yeast, 0.10% acetic acid bacteria, and 0.04% lactic acid bacteria). Ferment at 25 ℃ for 5 days, yielding a residual sugar of 1.2 g / L. Filter to obtain the enzyme stock solution.

[0063] S5, Compound: Dilute to an exosome concentration of 100 million exosomes / mL, add 1% D-mannitol, and 1 ppm of nanobubble hydrogen.

[0064] The resulting beverage has a uniform purple color, a glossy appearance, a slightly mild aroma, a pure and mellow taste, and health benefits.

[0065] Example 3 This embodiment provides a method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes, which differs from Embodiment 1 in that: S1. The cultivation and identification processes are the same.

[0066] S2, the preparation process of exosomes is the same.

[0067] S3. Quality control: Particle size 30-250 nm, concentration 1.8×10⁻⁶ 10 Particles / mL, total protein 70 μg / mL, positive rate 97%, negative, clear structure.

[0068] S4. Preparation of Enzyme Stock Solution: The first component consists of 120 parts of Polygonatum sibiricum, 100 parts of Pueraria lobata, and 120 parts of Dioscorea opposita. The mixture is steamed for 2.5 hours and then cooled. The ratio of the compound enzyme is 1.6:1:1, the enzymatic hydrolysis temperature is 45 ℃, the time is 2.5 hours, and the power is 220 W. After enzyme inactivation, the pH is 6.0, and 4% lysine and 12% fructose are added.

[0069] The inoculum was 0.7%, with yeast accounting for 0.33%, acetic acid bacteria 0.15%, and lactic acid bacteria 0.08%. The fermentation temperature was 35 ℃, and the fermentation time was 3 days, resulting in a residual sugar content of 0.8 g / L.

[0070] S5, Compound: Dilute to 10 billion exosomes / mL, add 2% sorbitol, and 1.5 ppm hydrogen.

[0071] The beverage has a light purple color, a glossy sheen, a harmonious aroma, and a refreshing taste.

[0072] To verify the synergistic effect of the beverage of this invention, a comparative experiment was conducted on the compound beverage, the exosome group alone, and the enzyme group alone: Exosome stability test: The particle size distribution of exosomes in the three groups of samples was tested after storage at 4℃ for 30 days. The particle size change rate was 4.8% in the compound beverage group, 22.5% in the exosome group, and not applicable to the enzyme group.

[0073] Free radical scavenging experiment: The antioxidant capacity was determined by the DPPH method. The free radical scavenging rate was 82% in the compound beverage group, 45% in the exosome group, and 60% in the enzyme group.

[0074] Mouse inflammation model: In LPS-induced mouse models, IL-6 decreased by 35% in the compound beverage group, TNF-α decreased by 40%, exosome group decreased by 15%-20%, and enzyme group decreased by 20%-25%.

[0075] Experimental results show that the compound beverage has significant synergistic anti-inflammatory and antioxidant effects, and the stability of exosomes is greatly improved.

[0076] Example 4 This embodiment provides a method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes, which differs from Embodiment 1 in that: S4. Preparation of Enzyme Stock Solution: The first component consisted of only 80 parts of Polygonatum sibiricum. It was boiled for 2 hours, with the complex enzymes being only α-amylase and cellulase in a ratio of 1.4:1. Enzymatic hydrolysis was performed at 40°C for 3 hours without ultrasound. Enzyme inactivation was achieved using an enzyme inhibitor. The pH was 5.5, and the carbon source consisted of 3.5% leucine and 10% glucose. The composite engineered bacteria consisted of only 0.30% yeast. Fermentation was carried out at 30°C for 4 days, resulting in a residual sugar content of 1.1 g / L.

[0077] S5. Dilute to 5 billion exosomes / mL, add 1.2% isomaltitol, without hydrogen gas.

[0078] The beverage is light purple in color, has a moderate aroma, a mild taste, and basic health benefits.

[0079] Example 5 This embodiment provides a method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes, which differs from Embodiment 1 in that: S4, the first component consists of only 170 parts of kudzu root and 80 parts of purple yam. Steaming for 3 hours. Enzymatic hydrolysis with a 1:1 ratio of pectinase and cellulase at 38°C for 3.5 hours. Enzyme inactivation at high temperature. pH 5.2, carbon source: 3% monosodium glutamate and 9% brown sugar. Engineered bacteria: 0.12% acetic acid bacteria and 0.05% lactic acid bacteria. Fermentation at 28°C for 4.5 days, residual sugar 1.0 g / L.

[0080] S5. Dilute to 1 billion exosomes / mL, add 1.8% high maltose powder, and 1.3 ppm hydrogen.

[0081] The beverage has a uniform color, a strong fermented aroma, a sweet and sour taste, and good functionality.

[0082] Comparative Example 1 The enzyme was prepared using traditional natural fermentation, without enzymatic hydrolysis or engineered bacteria. The first component (150 parts Polygonatum sibiricum, 100 parts Pueraria lobata, and 150 parts Dioscorea opposita) was mixed with water and naturally fermented for 90 days to obtain an enzyme solution. This solution was then mixed with an exosome solution, diluted with physiological saline to 2 billion exosomes / mL, and 1.5% xylitol was added, without hydrogen gas.

[0083] The resulting beverage has an uneven color, is light brown, and lacks luster; it has a pungent aroma and an off-putting odor; it tastes bitter and has a poor mouthfeel. The long fermentation time makes it susceptible to contamination, and its health benefits are weak.

[0084] Comparative Example 2 The enzyme-free stock solution was diluted with only exosomes and physiological saline to 2 billion exosomes / mL, and 1.5% xylitol was added.

[0085] The beverage has no color change, no aroma, a monotonous taste, and no anti-inflammatory or antioxidant enhancements.

[0086] Comparative Example 3 No steaming or boiling was used in the preparation of the enzyme; it was directly hydrolyzed. The first component consisted of 150 parts of Polygonatum sibiricum, and the hydrolysis was carried out at 40 °C for 3 h. Other steps were the same as in Example 1.

[0087] Incomplete enzymatic hydrolysis results in low extraction of active ingredients, leading to a bitter taste and reduced functionality in the beverage.

[0088] Comparative Example 4 The proportions of the engineered bacteria were changed: yeast 0.20%, acetic acid bacteria 0.20%, lactic acid bacteria 0.10%, and the rest were the same as in Example 1.

[0089] The fermentation time was extended to 7 days, the residual sugar was 1.5 g / L, the aroma of the beverage was not harmonious, and the taste was too sour.

[0090] Comparative Example 5 The exosome concentration was 0.5 billion / mL, and other parameters were the same as in Example 1.

[0091] It has weak anti-aging and immune regulation functions, resulting in poor overall health benefits.

[0092] In summary, sensory evaluation (color 20 points, aroma 30 points, taste 30 points, overall satisfaction 20 points) and functional testing (in vitro antioxidant activity DPPH scavenging rate %, immunomodulatory IL-6 reduction rate %) were conducted on Examples 1-5 and Comparative Examples 1-5.

[0093] The results are as follows: Example 1: Color 18, Aroma 21, Taste 28, Satisfaction 18; DPPH 85%, IL-6 70%.

[0094] Example 2: Color 19, Aroma 20, Taste 28, Satisfaction 17; DPPH 82%, IL-6 68%.

[0095] Example 3: Color 18, Aroma 19, Taste 26, Satisfaction 16; DPPH 84%, IL-6 69%.

[0096] Example 4: Color 17, Aroma 18, Taste 25, Satisfaction 15; DPPH 80%, IL-6 65%.

[0097] Example 5: Color 18, Aroma 20, Taste 27, Satisfaction 17; DPPH 83%, IL-6 67%.

[0098] Comparative Example 1: Color 12, Aroma 15, Taste 13, Satisfaction 8; DPPH 60%, IL-6 40%.

[0099] Comparative Example 2: Color 10, Aroma 10, Taste 15, Satisfaction 10; DPPH 50%, IL-6 55%.

[0100] Comparative Example 3: Color 15, Aroma 16, Taste 20, Satisfaction 12; DPPH 70%, IL-6 50%.

[0101] Comparative Example 4: Color 16, Aroma 17, Taste 22, Satisfaction 13; DPPH 75%, IL-6 52%.

[0102] Comparative Example 5: Color 17, Aroma 18, Taste 24, Satisfaction 14; DPPH 78%, IL-6 60%.

[0103] The results showed that the sensory and functional aspects of the embodiment were superior to those of the comparative embodiment, and the combination enhanced the health benefits.

[0104] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A health beverage composed of mesenchymal stem cell exosomes and enzymes, characterized in that: This health drink is composed of human umbilical cord mesenchymal stem cell exosomes, enzyme stock solution, physiological saline and sweetener; The human umbilical cord mesenchymal stem cell exosomes are obtained by culturing human umbilical cord mesenchymal stem cells, secreting exosomes, and extracting and purifying them. The enzyme stock solution is obtained by mixing the first component with water, adding a compound enzyme for enzymatic hydrolysis to obtain the first enzymatic hydrolysis extract, and then adding a compound engineered bacteria to the first enzymatic hydrolysis extract for fermentation. The saline solution is an aqueous solution containing 0.9% sodium chloride; The sweetener is selected from one or more of D-mannitol, xylitol, isomaltitol, sorbitol, high maltose powder, and white sugar.

2. The health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 1, characterized in that: The human umbilical cord mesenchymal stem cell exosomes are exosomes secreted by human umbilical cord mesenchymal stem cells. These exosomes contain some of the bioactive substances of human umbilical cord mesenchymal stem cells and have anti-aging and immune-regulating functions. The first component is selected from at least one of Polygonatum sibiricum, Pueraria lobata and Dioscorea opposita.

3. The health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 1, characterized in that: The first component comprises, by mass parts, 80-150 parts of Polygonatum sibiricum, 100-170 parts of Pueraria lobata, and 80-150 parts of Dioscorea opposita.

4. A health beverage comprising mesenchymal stem cell exosomes and enzymes as described in claim 1, characterized in that: The concentration of human umbilical cord mesenchymal stem cell exosomes in the health beverage is 1-10 billion exosomes / ml.

5. A health beverage comprising mesenchymal stem cell exosomes and enzymes as described in claim 1, characterized in that: The concentration of human umbilical cord mesenchymal stem cell exosomes in the health beverage is 2 billion exosomes / ml.

6. The method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 1, characterized in that: Includes the following steps: S1. Culture of human umbilical cord mesenchymal stem cells: Wash the umbilical cord specimen, cut it into pieces, remove blood vessels, cut it into small pieces, seed it in a culture flask, add complete culture medium, culture it until 70%-80% confluence, passage it, and identify it at the 4th generation. S2. Exosome preparation: Collect cell culture supernatant, add PEG6000 solution to a final concentration of 8%, let stand overnight, centrifuge and discard the supernatant, resuspend in physiological saline to obtain exosome solution; S3. Preparation of enzyme stock solution: Mix the first component with water and add a compound enzyme for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 35℃-45℃ and the enzymatic hydrolysis time is 2.5 h-4 h to obtain the first enzymatic hydrolysis extract. Add compound engineered bacteria to the first enzymatic hydrolysis extract for fermentation. The fermentation temperature is 25℃-35℃ and the fermentation time is ≤5 d. Stop fermentation when the residual sugar content is ≤1.2 g / L to obtain the enzyme stock solution. S4. Compounding: Mix the exosome solution with the enzyme stock solution, dilute with physiological saline to an exosome concentration of 1-10 billion exosomes / mL, add sweetener, and obtain a health beverage.

7. The method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 6, characterized in that: The complex enzyme is selected from at least one of α-amylase, cellulase and pectinase; the ratio of α-amylase, cellulase and pectinase in the complex enzyme is (1.2-1.6):1:

1.

8. The method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 6, characterized in that: Before adding the compound enzyme, the mixture of the first component and water is boiled for ≥2 hours; after cooling to 35℃-45℃, the compound enzyme is added.

9. The method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 6, characterized in that: The composite engineered bacteria are selected from at least one of yeast, acetic acid bacteria and lactic acid bacteria; by mass fraction, the composite engineered bacteria include 0.28%-0.33% yeast, 0.10%-0.15% acetic acid bacteria and 0.04%-0.08% lactic acid bacteria.

10. The method for preparing a health beverage composed of mesenchymal stem cell exosomes and enzymes as described in claim 6, characterized in that: The health beverage also contains nano-bubble hydrogen gas at a concentration of 1 ppm to 1.5 ppm.