Red ginseng fermentation liquor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to red ginseng fermentation broth and a preparation method and application thereof.The preparation method of the red ginseng fermentation broth comprises the following steps that red ginseng powder is dissolved in water, and a red ginseng fermentation medium is obtained by breaking cell walls in red ginseng; mixing the seed solution of the plant lactobacillus and the seed solution of the fermentation mucus lactobacillus to obtain a compound strain seed solution; inoculating the compound strain seed solution into a red ginseng fermentation culture medium for fermentation culture to obtain a fermentation solution; centrifuging the fermentation liquor, collecting supernate, extruding and filtering the supernate, and collecting filtrate, so as to obtain the red ginseng fermentation liquor. According to the invention, compound strain fermentation is adopted, and a mechanical extrusion mode is combined to increase the content of total saponins of ginseng. According to the method disclosed by the invention, the concentration of the total saponins of ginseng is increased from 2.19 mg / mL to 7.53 mg / mL.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a red ginseng fermentation broth, a preparation method thereof and an application thereof. Background Art

[0002] Ginseng belongs to the plant of the genus Panax in the Araliaceae family and has extensive and good medicinal and health care functions in aspects such as cardiovascular system diseases, mental system diseases, digestive system diseases, blood hematopoietic and immune system diseases, endocrine system diseases, anti-tumor, anti-aging, supplementing qi and blood, promoting hair growth and preventing hair loss. Red ginseng is a cooked product made by steaming and drying ginseng, and its function is more inclined to warm nourishment. It not only retains various active ingredients of ginseng, has better effects of invigorating qi and cultivating yuan, benefiting blood and promoting fluid production, strengthening the heart and stomach, etc., but also has a milder property compared with ginseng. The total ginsenoside content in ginseng accounts for about 4% of its active ingredients, but it plays a significant effect in medicine. Among them, the contents of natural prototype saponins such as Rb1, Rb2, Rc, Re and Rg1 account for 70% - 80% of the total ginsenosides in ginseng; while the contents of rare saponins such as Rg3, Rh1, Rh2, F2 and CK account for a very low proportion, but have high pharmacological activities. For example, the rare saponin Rg3 is considered to be a comprehensive regulator of human functions and has effects such as anti-inflammatory, anti-cancer, protecting the cardiovascular system, improving cognition, protecting nerves and immunomodulation. The rare saponin F2 is one of the core efficacy components of red ginseng and has multiple effects such as antioxidant, anti-photoaging, reducing wrinkles, promoting skin hydration and repairing the skin barrier. A large number of studies have shown that natural prototype saponins can be transformed into rare saponins through the metabolism of intestinal microbiota in vivo, improving their pharmacological activities. At the same time, rare saponins, with a smaller molecular weight and stronger liposolubility, can easily cross the blood-brain barrier and reach the brain directly, further improving their absorption efficiency and bioavailability in vivo, opening up new possibilities for the treatment and health care fields.

[0003] As a key active ingredient in ginseng, total ginsenosides have broad application prospects in the fields of medicine, health products and cosmetics. However, at present, a key problem faced in the process of preparing total ginsenosides is the low yield, which seriously restricts the large-scale production and application of total ginsenosides.

[0004] In the existing microbial fermentation technology, the utilization efficiency of ginseng raw materials by microorganisms is generally low. On the one hand, complex chemical components in ginseng, such as polysaccharides and proteins, may inhibit the growth and metabolic activities of microorganisms, making it impossible for microorganisms to fully exert their conversion ability for total ginsenosides. On the other hand, the growth characteristics and metabolic regulation mechanisms of microorganisms themselves also limit the yield of total ginsenosides. For example, during the fermentation process of some microorganisms, the conversion enzyme activity for total ginsenosides is not high, resulting in low substrate conversion rate; or in the later stage of microbial growth, due to the consumption of nutrients and the accumulation of metabolites, the growth is inhibited, thereby affecting the continuous synthesis of total ginsenosides.

[0005] Due to the low yield of total ginsenosides prepared by microbial fermentation, the production cost remains high, resulting in high prices for total ginsenoside-related products and difficulty in meeting market demand. This not only hinders the wide application of total ginsenosides in the medical field but also limits their large-scale promotion in the health care product and cosmetics industries. Therefore, there is an urgent need to develop a method that can significantly improve the yield of total ginsenosides prepared by microbial fermentation. Summary of the Invention

[0006] To solve the problem of low yield of total ginsenosides prepared by microbial fermentation, the present invention provides a method for preparing a red ginseng fermentation broth.

[0007] The technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a method for preparing a red ginseng fermentation broth is provided, including the following steps: Dissolve red ginseng powder in water, add cellulase and pectinase to break the cell wall in red ginseng, then inactivate the enzymes and sterilize to obtain a red ginseng fermentation medium; prepare a seed solution of Lactiplantibacillus plantarum; prepare a seed solution of Limosilactobacillus fermentum; mix the seed solution of Lactiplantibacillus plantarum and the seed solution of Limosilactobacillus fermentum to obtain a composite strain seed solution; inoculate the composite strain seed solution into the red ginseng fermentation medium for fermentation culture to obtain a fermentation broth; centrifuge the fermentation broth, collect the supernatant, and subject the supernatant to extrusion filtration to collect the filtrate, and the obtained filtrate is the red ginseng fermentation broth.

[0008] Preferably, the pore size of the filter membrane used for extrusion filtration is 0.8 μm.

[0009] Preferably, the volume ratio of the seed solution of Lactiplantibacillus plantarum to the seed solution of Limosilactobacillus fermentum is 1:1, and the inoculation amount of the composite strain seed solution is 4% by volume fraction.

[0010] Preferably, the viable count of Lactiplantibacillus plantarum in the seed solution of Lactiplantibacillus plantarum is 1×10 8 CFU / mL; the viable count of Limosilactobacillus fermentum in the seed solution of Limosilactobacillus fermentum is 1×10 8 CFU / mL.

[0011] Preferably, the addition amounts of the cellulase and the pectinase are: 0.05 mL of cellulase and 0.05 mL of pectinase are added to every 1 g of red ginseng powder.

[0012] Preferably, the conditions for fermentation culture are static culture at 37°C for 48 h.

[0013] In the second aspect of the present invention, a red ginseng fermentation broth obtained according to the preparation method is provided.

[0014] The third aspect of the present invention provides an application of the red ginseng fermentation broth, and the red ginseng fermentation broth is used for preparing a product for enhancing immunity.

[0015] Preferably, the product further comprises a pharmaceutically acceptable excipient, and the excipient is a sweetening agent.

[0016] Preferably, the sweetening agent is xylitol.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a preparation method of a red ginseng fermentation broth, comprising the following steps: Dissolve red ginseng powder in water, add cellulase and pectinase to break the cell wall in red ginseng, then inactivate the enzymes and sterilize to obtain a red ginseng fermentation medium; prepare a seed solution of Lactiplantibacillus plantarum; prepare a seed solution of Limosilactobacillus fermentum; mix the seed solution of Lactiplantibacillus plantarum and the seed solution of Limosilactobacillus fermentum to obtain a composite strain seed solution; inoculate the composite strain seed solution into the red ginseng fermentation medium for fermentation culture to obtain a fermentation broth; centrifuge the fermentation broth, collect the supernatant, and squeeze and filter the supernatant to collect the filtrate, and the obtained filtrate is the red ginseng fermentation broth. The present invention adopts composite strain fermentation and combines mechanical extrusion to increase the content of total ginsenosides. Through the method of the present invention, the concentration of total ginsenosides is increased from 2.19 mg / mL to 7.53 mg / mL.

[0018] As probiotics, Lactiplantibacillus plantarum and Limosilactobacillus fermentum can spontaneously produce bacterial outer vesicles during the fermentation process. After collecting the supernatant of the fermentation broth fermented by the composite strain in the present invention, a red ginseng fermentation broth containing bacterial outer vesicles is prepared by mechanical extrusion. On the one hand, the bacterial outer vesicles can improve the health status of the human intestinal flora, and can also effectively encapsulate total ginsenosides and improve their storage stability. In addition, the red ginseng fermentation broth described in the present invention has anti-inflammatory and antioxidant effects.

[0019] The present invention uses red ginseng as a substrate for cultivation. On the one hand, probiotics can produce bacterial outer vesicles during growth, and at the same time, the growth and metabolism of probiotics can transform natural prototype saponins in red ginseng to produce secondary metabolites with high physiological activities such as rare saponins. During the mechanical extrusion process, the bacterial outer vesicles can enrich and encapsulate rare saponins, and it can be applied in the fields of food and beverage and cosmetic efficacy raw materials. This method simplifies the process of engineering bacterial outer vesicles to encapsulate rare saponins, and can effectively improve the problems of poor water solubility and poor stability of rare saponins.

[0020] The present invention combines probiotic fermentation technology with bacterial outer membrane vesicle preparation technology, effectively solving the problem that probiotics only function as fermentation agents. At the same time, the present invention also overcomes the problem of poor stability of rare saponins in aqueous solutions. In the present invention, a system in which bacterial outer membrane vesicles encapsulate rare saponins to form nanoemulsions can significantly improve the stability of rare saponins and their resistance to gastric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the characterization result of bacterial outer membrane vesicles. A is the bacterial outer membrane vesicles in the filtrate passing through a 0.8 μm filter membrane, and B is the bacterial outer membrane vesicles in the filtrate passing through a 0.1 μm filter membrane.

[0022] Figure 2 It is the particle size distribution diagram of bacterial outer membrane vesicles. A is the bacterial outer membrane vesicles in the filtrate passing through a 0.8 μm filter membrane, and B is the bacterial outer membrane vesicles in the filtrate passing through a 0.1 μm filter membrane.

[0023] Figure 3 It is the concentration of total ginsenosides in the red ginseng fermentation broth prepared in Example 1 and Comparative Example 1.

[0024] Figure 4 It is the characterization result of the structure of bacterial outer membrane vesicles after simulated in vitro digestion. A is the transmission electron microscope photograph of the blank control group; B is the transmission electron microscope photograph after simulated digestion with artificial saliva, artificial gastric juice and artificial intestinal juice.

[0025] Figure 5 It is the characterization of the structure of bacterial outer membrane vesicles under different storage conditions. A is 5°C for 30 days; B is 5°C for 60 days; C is 25°C for 30 days; D is 25°C for 60 days. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further described below through specific examples, but the scope of the present invention is not limited. Without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0027] The inventive concept of the present invention is as follows: In the existing microbial fermentation technology, the utilization efficiency of microbial raw ginseng materials is generally low. The main reasons are as follows: The complex chemical components in ginseng will inhibit the growth and metabolic activities of microorganisms, making it impossible for microorganisms to fully exert their conversion ability of total ginsenosides, and the growth characteristics and metabolic regulation mechanisms of organisms themselves also limit the yield of total ginsenosides.

[0028] Based on this, the present invention provides a method for preparing a red ginseng fermentation broth, comprising the following steps: Dissolve red ginseng powder in water, add cellulase and pectinase to break the cell wall in red ginseng, then inactivate the enzymes and sterilize to obtain the red ginseng fermentation medium; prepare the seed liquid of Lactiplantibacillus plantarum; prepare the seed liquid of Limosilactobacillus fermentum; mix the seed liquid of Lactiplantibacillus plantarum and the seed liquid of Limosilactobacillus fermentum to obtain the composite strain seed liquid; inoculate the composite strain seed liquid into the red ginseng fermentation medium for fermentation culture to obtain the fermentation broth; centrifuge the fermentation broth, collect the supernatant, and squeeze and filter the supernatant to collect the filtrate, and the obtained filtrate is the red ginseng fermentation broth.

[0029] As probiotics, Lactiplantibacillus plantarum and Limosilactobacillus fermentum, on the one hand, cellulase, hemicellulase, etc. in the probiotics can degrade the cell wall of red ginseng and release the natural prototype saponins in red ginseng cells; on the other hand, glycosidase, especially β-glucosidase in the probiotics can hydrolyze the glycosidic bond in the natural prototype saponins and convert them into rare saponins, thus increasing the content of rare saponins.

[0030] Probiotics can spontaneously produce BEVs. BEVs are spherical structures with a lipid bilayer, and their diameters are generally 20nm - 400nm. As small molecule active substances derived from bacteria, BEVs have the advantages of stable structure and long effective circulation time. They can encapsulate a variety of bioactive molecules, including proteins, lipids, nucleic acids, and metabolites. A large number of studies have shown the potential mechanisms of BEVs in immune responses, antioxidant, etc., and there is application potential as a new type of postbiotic in functional foods, disease treatment alternatives, immune regulation adjuvants, etc. In addition, BEVs are also ideal carriers for bioactive compound nanodelivery systems because they have good immunological properties and pharmacokinetic properties, as well as the ability to penetrate physiological barriers. Therefore, high-value functional ingredients can be loaded by technical means to prepare a delivery system. After humans take related products, it can achieve an accurate targeted nutritional intervention effect and can be an important carrier for constructing a food nutrient factor delivery system.

[0031] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0032] The abbreviation list of the present invention is shown in Table 1.

[0033] Table 1 Abbreviation list The strains and manufacturer information used in the present invention are shown in Table 2.

[0034] Table 2 Strains and manufacturer information The cellulase and pectinase used in the present invention were purchased from Novozymes. The enzyme activity of the cellulase was 700 U / mL, and the enzyme activity of the pectinase was 10,000 U / mL. The red ginseng powder was purchased from Jilin Hongjiu Biotechnology Co., Ltd. The artificial saliva, artificial gastric juice, and artificial intestinal juice were purchased from Shanghai Xinfan Biotechnology Co., Ltd.

[0035] In the present invention, the detection method for the total ginsenoside content: Add 20 μL of the red ginseng fermentation broth to 0.2 mL of a 5 g / L vanillin solution and mix well. Then add 0.8 mL of analytical grade perchloric acid and mix well. Incubate in a constant temperature water bath at 60 °C for 10 min. After cooling on ice, add glacial acetic acid to make up the volume to 5 mL and shake well. Measure the absorbance value at a wavelength of 560 nm.

[0036] Example 1 A preparation method of a red ginseng fermentation broth is as follows: S1. Dissolve the red ginseng powder in water, add cellulase and pectinase to break the cell wall in the red ginseng, and then inactivate the enzyme and sterilize to obtain a red ginseng fermentation medium.

[0037] Mix the red ginseng powder and distilled water at a mass ratio of 1:10. After fully dissolving the red ginseng powder, add cellulase and pectinase. At 60 °C, shake and enzymatically hydrolyze at a rotation speed of 100 rpm / min for 3 h. Inactivate the enzyme at 85 °C for 10 min, and then perform sterilization treatment at 121 °C for 20 min. After cooling to room temperature, obtain the red ginseng fermentation medium. Among them, the addition amounts of cellulase and pectinase are: add 0.05 mL of cellulase and 0.05 mL of pectinase to every 1 g of red ginseng powder.

[0038] S2. Prepare the seed liquid of Lactiplantibacillus plantarum and the seed liquid of Lactobacillus mucosae fermentum respectively, and mix the seed liquid of Lactiplantibacillus plantarum and the seed liquid of Lactobacillus mucosae fermentum to obtain a composite strain seed liquid.

[0039] Dissolve YZ-Lactiplantibacillus plantarum LP28 in sterile water to obtain a YZ-Lactiplantibacillus plantarum LP28 bacterial solution. Gently streak the YZ-Lactiplantibacillus plantarum LP28 bacterial solution on an MRS solid medium using an inoculation loop, and culture at 37 °C until single colonies of YZ-Lactiplantibacillus plantarum LP28 appear. Pick the single colonies of YZ-Lactiplantibacillus plantarum LP28 with an inoculation loop and transfer them to a sterilized test tube containing 5 mL of MRS liquid medium for strain activation, and culture statically at 37 °C for 18 h to obtain the seed liquid of YZ-Lactiplantibacillus plantarum LP28. The viable count of YZ-Lactiplantibacillus plantarum LP28 in the seed liquid of YZ-Lactiplantibacillus plantarum LP28 is 1×10 8 CFU / mL.

[0040] The fermented Lactobacillus mucosae JYLF-71 was dissolved in sterile water to obtain a bacterial solution of Lactobacillus mucosae JYLF-71. The bacterial solution of Lactobacillus mucosae JYLF-71 was gently streaked on MRS solid medium using an inoculation loop and cultured at 37 °C until single colonies of Lactobacillus mucosae JYLF-71 appeared. The single colonies of Lactobacillus mucosae JYLF-71 were picked up with an inoculation loop and transferred into a sterilized test tube containing 5 mL of MRS liquid medium for strain activation. It was statically cultured at 37 °C for 18 h to obtain the seed liquid of Lactobacillus mucosae JYLF-71. The viable count of Lactobacillus mucosae JYLF-71 in the seed liquid of Lactobacillus mucosae JYLF-71 was 1×10 8 CFU / mL.

[0041] The seed liquid of Lactobacillus plantarum LP28 and the seed liquid of Lactobacillus mucosae JYLF-71 were mixed in a volume ratio of 1:1 to obtain a composite strain seed liquid.

[0042] S3. The composite strain seed liquid was inoculated into the red ginseng fermentation medium for fermentation culture to obtain a fermentation broth.

[0043] The composite strain seed liquid was inoculated into the red ginseng fermentation medium at an inoculation amount of 4% (v / v) and statically cultured in an incubator at 37 °C for 48 h. After fermentation, the fermentation broth was collected.

[0044] S4. The fermentation broth was centrifuged, and the supernatant was collected. Then, the supernatant was subjected to extrusion filtration, and the filtrate was collected. The obtained filtrate was the red ginseng fermentation broth.

[0045] The fermentation broth was centrifuged at 4000 rpm / min for 5 min at room temperature to remove the powder of red ginseng powder in the fermentation broth, and the supernatant in the fermentation broth was retained. Since Lactobacillus plantarum LP28 and Lactobacillus mucosae JYLF-71 can spontaneously produce bacterial outer vesicles during fermentation, the following operations were performed on the supernatant:

[0046] The supernatant was successively passed through filter membranes with pore sizes of 8 μm, 5 μm, 1 μm, 0.8 μm, and 0.1 μm. At the same time, when the supernatant passed through each stage of the filter membrane, a hand-pushed liposome extruder was used for extrusion. The supernatant passing through each stage of the filter membrane was unidirectionally extruded 6 times to obtain the filtrate under the corresponding pore size. 50 mL of the filtrate under each pore size was collected for subsequent detection, and the remaining filtrate was subjected to repeated extrusion operations after changing to the next-stage filter membrane until the supernatant completely passed through the 0.1-μm filter membrane. The filtrate passing through the 0.8-μm filter membrane was the red ginseng fermentation broth.

[0047] Comparative Example 1 A preparation method of red ginseng fermentation broth is as follows: S1 to S3 are exactly the same as those in Example 1. In S4, after the fermentation is completed, the fermentation broth is centrifuged at 4000 rpm / min for 5 min at room temperature to remove the powder of red ginseng powder in the fermentation broth, and the supernatant in the fermentation broth is the red ginseng fermentation broth.

[0048] The filtrates under various pore size filter membranes in Example 1 were characterized as follows: Whether the Tyndall effect exists was observed for the filtrates passing through 8 μm, 5 μm, 1 μm, 0.8 μm, and 0.1 μm filter membranes respectively. Subsequently, the filtrates passing through 0.8 μm and 0.1 μm filter membranes were observed by transmission electron microscopy, and the laser particle size distribution and protein concentration were measured.

[0049] The results of transmission electron microscopy observation are as Figure 1 shown, and the particle size analysis results are as Figure 2 shown. It can be seen from Figure 1 that the bacterial outer vesicles in the filtrate are in a teacup-like structure. It can be seen from Figure 2 that the average particle size of the bacterial outer vesicles in the filtrate passing through the 0.8 μm filter membrane is 147 nm, and the average particle size of the bacterial outer vesicles in the filtrate passing through the 0.1 μm filter membrane is 132.4 nm.

[0050] In addition, the vesicle concentration and protein concentration of the bacterial outer vesicles in the filtrate were also measured. The detection results are as follows: the concentration of the bacterial outer vesicles in the filtrate passing through the 0.8 μm filter membrane is 1.8×10 10 Particles / mL, and the protein concentration is 7.30 mg / mL; the concentration of the bacterial outer vesicles in the filtrate passing through the 0.1 μm filter membrane is 1.4×10 10 Particles / mL, and the protein concentration is 6.24 mg / mL.

[0051] The total ginsenosides in the red ginseng fermentation broth prepared in Example 1 and Comparative Example 1 were measured as follows: It can be seen from Figure 3 that the total ginsenoside concentration in the red ginseng fermentation broth prepared in Comparative Example 1 is 2.19 mg / mL; while the total ginsenoside concentration in the red ginseng fermentation broth prepared in Example 1 is increased to 7.53 mg / mL. Thus, it can be seen that after extrusion, the total ginsenoside concentration is significantly increased.

[0052] In addition, SDS with a mass fraction of 20% and an equal volume was added to the red ginseng fermentation broth prepared in Example 1 for demulsification, and the total ginsenoside concentration reached 9.55 mg / mL after demulsification, indicating that the bacterial outer vesicles can effectively encapsulate the total ginsenosides.

[0053] Example 2 An application of the red ginseng fermentation broth is as follows: 1. The immunity effect of the red ginseng fermentation broth.

[0054] Using macrophage Raw264.7 as the research object, the red ginseng fermentation broth prepared in Example 1 was used to treat the cells, and the changes in cell viability were observed and statistically analyzed to evaluate the immunity efficacy. The grouping and treatment methods of the cell proliferation test are shown in Table 3.

[0055] Table 3 Grouping and treatment methods of cell proliferation test Using macrophage Raw264.7 as the research object, the red ginseng fermentation broth prepared in Example 1 was used to treat the cells, and the changes in cell phagocytosis ability were observed and statistically analyzed to evaluate the immunity efficacy. The grouping and treatment methods of the cell phagocytosis ability test are shown in Table 4.

[0056] Table 4 Grouping and treatment methods of cell phagocytosis ability test Note: In Table 4, " / " indicates that there is no such item.

[0057] The detection results of cell proliferation rate are shown in Table 5. Compared with the blank control group, the cell proliferation rate of the positive control group increased significantly, with an increase rate of 22.27%, indicating that the model was successfully constructed. Compared with the blank control group, the cell viability in Sample Group 1 increased by 36.98%; compared with the blank control group, the cell viability in Sample Group 2 increased by 36.18%, indicating that the red ginseng fermentation broth in the present invention has the effect of enhancing immunity. In Table 5, compared with the blank control group, the significance in the positive control group is indicated by #, and p < 0.01 is indicated by ##; compared with the blank control group, the significance in the sample group is indicated by *, and p < 0.01 is indicated by **.

[0058] The detection results of cell phagocytosis index are shown in Table 6. Compared with the blank control group, the phagocytosis index in Sample Group 3 increased significantly, with an increase of 55.20%; compared with the blank control group, the phagocytosis index in Sample Group 4 increased significantly, with an increase of 32.58%. In Table 6, compared with the blank control group, the significance in the sample group is indicated by *, and p < 0.01 is indicated by **.

[0059] The above results indicate that the red ginseng fermentation broth of the present invention has the effect of enhancing immunity. The red ginseng fermentation broth of the present invention is prepared into a product for enhancing immunity after adding the sweetener xylitol.

[0060] Table 5 Detection results of cell proliferation viability Table 6 Detection results of cell phagocytosis index 2. Simulate in vitro digestion.

[0061] The red ginseng fermentation broth prepared in Example 1 was successively reacted in artificial saliva, artificial gastric juice, and artificial intestinal juice to simulate the digestion process. The method is as follows: a. Take 500 μL of the red ginseng fermentation broth and add 500 μL of artificial saliva kept at 37°C in a water bath. Mix well and place in a 37°C water bath for 5 min.

[0062] b. Add 1000 μL of artificial gastric juice kept at 37°C in a water bath to the red ginseng fermentation broth digested by artificial saliva. Mix well and place in a 37°C water bath for 2 h.

[0063] c. Continue to add 2000 μL of artificial intestinal juice kept at 37°C in a water bath. Mix well and place in a 37°C water bath for 1 h.

[0064] d. After the previous step is completed, place in a 90°C water bath for 5 min.

[0065] e. Centrifuge at 8000 rpm for 10 min, collect the supernatant, and conduct detection.

[0066] It can be seen from Figure 4 that after the bacterial outer vesicles in the red ginseng fermentation broth are successively digested by artificial saliva, artificial gastric juice, and artificial intestinal juice, they still retain a complete vesicle structure, indicating that they can resist gastrointestinal juice digestion. The bacterial outer vesicles in the red ginseng fermentation broth have a stable structure and have a good ability to retain their contents.

[0067] 3. Storage stability.

[0068] After adding 0.1% potassium sorbate preservative by mass fraction to the red ginseng fermentation broth prepared in Example 1, it was stored at 5°C and 25°C for a certain period of time, and the pH stability, soluble solid content, color and appearance morphology, and structural indexes of bacterial outer vesicles were observed and detected respectively. The detailed results are shown in Table 7 and Figure 5 .

[0069] The results show that the red ginseng fermentation broth prepared in Example 1 remains clear after being stored at 5°C and 25°C for 2 months.

[0070] Table 7 Results of storage stability In Table 7, "-" indicates that the structure of bacterial outer vesicles was not detected. Due to the short detection time interval and little change in the structure of bacterial outer vesicles in the short term, it was not detected.

[0071] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0072] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing red ginseng fermented liquid, characterized in that: The following steps are involved: Dissolving red ginseng powder in water, adding cellulase and pectinase to destroy the cell wall of the red ginseng, then inactivating the enzymes and sterilizing to obtain a red ginseng fermentation medium; preparing a seed solution of Lactobacillus plantarum; preparing a seed solution of fermented Lactobacillus mucilaginosus; Mixing the seed liquid of Lactobacillus plantarum and the seed liquid of Lactobacillus mucilaginosus fermentation to obtain a composite strain seed liquid; inoculating the composite strain seed liquid into the red ginseng fermentation medium for fermentation and culturing to obtain a fermentation liquid; The fermentation liquid is centrifuged, the supernatant is collected, and the supernatant is squeezed and filtered to collect the filtrate, and the obtained filtrate is the red ginseng fermentation liquid.

2. The preparation method according to claim 1, characterized in that The pore size of the filter membrane used in the extrusion filtration is 0.8 μm.

3. The preparation method according to claim 1, characterized in that: The volume ratio of the seed liquid of the plant lactobacillus to the seed liquid of the fermented mucus lactobacillus is 1:1, and the inoculation amount of the composite strain seed liquid is 4% by volume.

4. The preparation method according to claim 1, characterized in that: The number of viable Lactobacillus plantarum in the seed solution of the Lactobacillus plantarum is 1×10 8 CFU / mL; The number of viable bacteria of fermented Lactobacillus mucilaginosus in the seed liquid of fermented Lactobacillus mucilaginosus is 1×10 8 CFU / mL.

5. The preparation method according to claim 1, characterized in that: The addition amount of the cellulase and pectinase is: Add 0.05 mL of cellulase and 0.05 mL of pectinase to every 1 g of red ginseng powder.

6. The preparation method according to claim 1, characterized in that: The fermentation culture conditions are static culture at 37° C. for 48 hours.

7. A red ginseng fermented liquid obtained according to the preparation method according to claim 1.

8. The use of red ginseng fermented liquid as claimed in claim 7, characterized in that: The red ginseng fermented liquid is used for preparing products for improving immunity.

9. The use according to claim 8, characterized in that The product further comprises a pharmaceutically acceptable auxiliary material, which is a sweetener.

10. The use according to claim 9, characterized in that The sweetener is xylitol.

Images