Dendrobium officinale fermentation liquor for promoting stem cell proliferation as well as preparation method and application of dendrobium officinale fermentation liquor

Abstract

The invention provides dendrobium officinale fermentation liquor for promoting stem cell proliferation as well as a preparation method and application of the dendrobium officinale fermentation liquor, and belongs to the technical field of microbial fermentation. The preparation method comprises the following steps: drying fresh stems of dendrobium officinale, crushing and sieving to obtain fresh stem powder of dendrobium officinale; mixing the fresh dendrobium officinale stem powder with water to obtain a mixture, and sterilizing the mixture; inoculating zymophyte powder into the sterilized mixture for fermentation, performing solid-liquid separation after fermentation is finished, and taking liquid, thereby obtaining the dendrobium officinale fermentation liquor for promoting stem cell proliferation. According to the invention, the compound bacterium powder containing bifidobacterium lactis, lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus and lactobacillus plantarum is adopted to ferment the dendrobium officinale, and all strains play a role synergistically, so that the content and the extraction rate of water-soluble mannose in the dendrobium officinale fermentation liquor are increased, and meanwhile, the dendrobium officinale fermentation liquor also contains a small amount of polysaccharide; under the combined action of mannose and polysaccharide, the fermentation liquor has antioxidant activity and can promote stem cell proliferation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a Dendrobium officinale fermentation liquid for promoting stem cell proliferation, and a preparation method and application thereof. Background Art

[0002] Dendrobii Officinalis Caulis is the first of the "Nine Immortal Herbs" in my country and a rare and precious Chinese medicinal material in the orchid family. Dendrobii Officinalis Caulis is rich in polysaccharides that have the effects of protecting the liver, lowering blood lipids, and promoting the proliferation of embryonic stem cells. The main active ingredient of Dendrobii Officinalis polysaccharides is mannose D(+)-Mannose. In the field of medicine, mannose is currently the only glyconutrient used in clinical practice. It can be directly used by the body for the synthesis of glycoproteins, and has anti-inflammatory, immune system regulation, and increased wound healing effects.

[0003] In recent years, the research on polysaccharides and mannose in Dendrobium officinale has mainly focused on its content. For example, in 2024, Shao Yijuan et al. used fully automatic Soxhlet extraction-high performance liquid chromatography to determine the mannose content of 92 samples of Dendrobium officinale collected in Guangdong Province, and found that the mannose content in Dendrobium officinale was 1.05% to 34.80% (Shao Yijuan, Long Chaoyang, Wu Ximei, et al. Determination of mannose content in commercially available Dendrobium officinale in Guangdong Province by fully automatic Soxhlet extraction-high performance liquid chromatography [J]. South China Preventive Medicine, 2024, 50(06): 583-586); in 2022, Tan Shancai et al. reported that by comparing the same planting The polysaccharide and mannose contents in the stems, leaves and flowers of Dendrobium officinale were measured by culture. It was found that the polysaccharide and mannose contents in the stems were the highest, ranging from 29.32% to 33.78% and 18.17% to 21.05%, respectively (Tan Shancai, Tan Changjin, Wu Xiaolan, et al. Optimization of hydrolysis process of Dendrobium officinale polysaccharides and determination of mannose content [J]. Food Industry Science and Technology, 2022, 43(19): 220-227. DOI: 10.13386 / j.issn1002-0306.2021120032.).

[0004] Embryonic stem cells are the original source of human and various tissue cells, with high potential for self-replication, high proliferation and multidirectional differentiation. Some studies have reported that Dendrobium officinale polysaccharides have this effect, but there are no reports on the effect of Dendrobium officinale fermentation liquid on promoting the proliferation of mouse embryonic stem cells. At the same time, the content of mannose is also affected by the extraction factors. Therefore, it is particularly important to develop a method for preparing Dendrobium officinale fermentation liquid that promotes stem cell proliferation. This not only helps to further explore the medicinal value of Dendrobium officinale, but also provides new ideas and methods for the treatment of related diseases. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a Dendrobium officinale fermentation liquid for promoting stem cell proliferation, and a preparation method and application thereof, so as to provide a technical reference for the fermentation liquid to have antioxidant effects while promoting stem cell proliferation.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a Dendrobium officinale fermentation liquid for promoting stem cell proliferation, comprising the following steps:

[0008] 1) drying the fresh stem of Dendrobium officinale, crushing and sieving to obtain the fresh stem powder of Dendrobium officinale;

[0009] 2) mixing the fresh stem powder of Dendrobium officinale and water to obtain a mixture, and sterilizing the mixture;

[0010] 3) inoculating fermentation bacteria powder into the sterilized mixture for fermentation, performing solid-liquid separation after the fermentation is completed, and taking the liquid to obtain the Dendrobium officinale fermentation liquid that promotes the proliferation of stem cells.

[0011] Preferably, the inoculation amount of the fermentation bacteria powder is 0.025-0.2% of the volume of the mixture.

[0012] Preferably, in step 2), the mass volume ratio of the fresh stem powder of Dendrobium officinale and water is 1 g: 45-65 mL.

[0013] Preferably, in step 3), the fermentation temperature is 32-48° C., and the fermentation time is 12-60 h.

[0014] Preferably, the sterilization temperature in step 2) is 80-120° C., and the sterilization time is 20-40 min.

[0015] Preferably, the drying in step 1) is vacuum freeze drying, the temperature of the vacuum freeze drying is -40 to -60°C, and the time of the vacuum freeze drying is 18 to 30 hours.

[0016] Preferably, the mesh size of the crushing and screening in step 1) is 20 to 50 meshes.

[0017] The present invention also provides a Dendrobium officinale fermentation liquid for promoting stem cell proliferation prepared by the preparation method.

[0018] The present invention also provides the Dendrobium officinale fermentation liquid for promoting stem cell proliferation prepared by the preparation method or the use of the Dendrobium officinale fermentation liquid for promoting stem cell proliferation in the preparation of a drug for promoting stem cell proliferation.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The preparation method of the Dendrobium officinale fermented liquid provided by the present invention adopts the composite bacterial powder containing Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus plantarum to ferment the Dendrobium officinale, and each bacterial species works synergistically, which can not only increase the content and extraction rate of mannose, but also contain a small amount of polysaccharides, and the combined effect of mannose and polysaccharides makes the fermented liquid have antioxidant properties and can also promote stem cell proliferation. The synergistic fermentation effect of the composite lactic acid bacteria of Chinese herbal medicine can significantly reduce the time of fermentation extraction, reduce energy consumption, and be beneficial to industrial production. There is no research report on the Dendrobium officinale fermented liquid with the effect of promoting stem cell proliferation. Our discovery not only helps to deeply explore the medicinal value of Dendrobium officinale, but also provides new ideas and methods for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of different solid-liquid ratios on the mannose content and mannose extraction rate in the fermentation broth of Dendrobium officinale;

[0022] Figure 2 The effect of different fermentation bacteria powder addition amounts on the mannose content and mannose extraction rate in the fermentation liquid of Dendrobium officinale;

[0023] Figure 3 The effect of different fermentation temperatures on the mannose content and mannose extraction rate in the fermentation liquid of Dendrobium officinale;

[0024] Figure 4 The effect of different fermentation times on the mannose content and mannose extraction rate in the fermentation liquid of Dendrobium officinale. DETAILED DESCRIPTION

[0025] The present invention provides a method for preparing a Dendrobium officinale fermentation liquid for promoting stem cell proliferation, comprising the following steps:

[0026] 1) drying the fresh stem of Dendrobium officinale, crushing and sieving to obtain the fresh stem powder of Dendrobium officinale;

[0027] 2) mixing the fresh stem powder of Dendrobium officinale and water to obtain a mixture, and sterilizing the mixture;

[0028] 3) inoculating fermentation bacteria powder into the sterilized mixture for fermentation, performing solid-liquid separation after the fermentation is completed, and taking the liquid to obtain the Dendrobium officinale fermentation liquid that promotes the proliferation of stem cells.

[0029] In the present invention, the fresh stem of Dendrobium officinale is dried and then crushed and sieved to obtain the fresh stem powder of Dendrobium officinale. The drying is preferably vacuum freeze drying, the temperature of the vacuum freeze drying is preferably -40 to -60°C, more preferably -45 to -55°C; the time of the vacuum freeze drying is preferably 18 to 30 hours, more preferably 20 to 28 hours; the mesh number of the crushing and sieving is preferably 20 to 50 meshes, more preferably 25 to 40 meshes.

[0030] In the present invention, the fresh stem powder of Dendrobium officinale and water are mixed to obtain a mixture, and the mixture is sterilized. The mass volume ratio of the fresh stem powder of Dendrobium officinale and water is preferably 1g:45-65mL, and more preferably 1g:50-60mL; the sterilization method is preferably steam sterilization, and the temperature is preferably 80-120°C, and more preferably 90-110°C; the sterilization time is preferably 20-40min, and more preferably 25-35min; and the sterilization is cooled to room temperature after completion.

[0031] In the present invention, fermentation powder is inoculated into the sterilized mixture for fermentation, and solid-liquid separation is performed after the fermentation is completed, and the liquid is taken to obtain the Dendrobium officinale fermentation liquid that promotes the proliferation of stem cells. The fermentation powder includes Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus plantarum; the inoculation amount of the fermentation powder is preferably 0.025-0.2% of the volume of the mixture, and more preferably 0.05-0.15% of the volume of the mixture; the fermentation temperature is preferably 32-48°C, and more preferably 36-44°C; the fermentation time is preferably 12-60h, and more preferably 24-48h; the solid-liquid separation method is preferably gauze filtration, and the mesh number of the gauze is preferably 180-220 mesh, and more preferably 190-210 mesh.

[0032] The present invention also provides a Dendrobium officinale fermentation liquid for promoting stem cell proliferation prepared by the preparation method.

[0033] The present invention also provides the Dendrobium officinale fermentation liquid for promoting stem cell proliferation prepared by the preparation method or the use of the Dendrobium officinale fermentation liquid for promoting stem cell proliferation in the preparation of a drug for promoting stem cell proliferation.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Fermented bacteria powder: probiotic freeze-dried powder-five bacteria compound purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.; live bacteria concentration: live bacteria count ≥1.0×10 10 CFU / g;

[0036] The mannose content determination kit was purchased from Hepeng (Shanghai) Biotechnology Co., Ltd.

[0037] Example 1

[0038] (1) The fresh stems of Dendrobium officinale were freeze-dried at -50°C for 24 h using a vacuum freeze dryer to remove moisture, and then ground into powder with a particle size of 40 mesh using a pulverizer to obtain the fresh stem powder of Dendrobium officinale, which was then placed in a dry sub-bottle and stored in a desiccator for later use.

[0039] (2) Weigh 1 g of fresh stem powder of Dendrobium officinale, add 60 mL of purified water, and stir evenly to obtain a mixture.

[0040] (3) The mixture is subjected to steam sterilization at 100° C. for 30 min, cooled to room temperature after sterilization, and then inoculated with 0.1% fermentation bacteria powder and sealed and fermented at 36° C. for 48 h at a fermentation speed of 50 r / min. After fermentation, it is filtered using 200-mesh gauze to separate the solid and liquid, and the liquid is obtained to obtain the Dendrobium officinale fermentation liquid.

[0041] Example 2

[0042] (1) The fresh stems of Dendrobium officinale were freeze-dried at -40°C for 18 h using a vacuum freeze dryer to remove moisture, and then ground into powder with a particle size of 20 mesh using a pulverizer to obtain the fresh stem powder of Dendrobium officinale, which was then placed in a dry sub-bottle and stored in a desiccator for later use.

[0043] (2) Weigh 1 g of fresh stem powder of Dendrobium officinale, add 45 mL of purified water, and stir evenly to obtain a mixture.

[0044] (3) The mixture was steam sterilized at 80° C. for 40 min, cooled to room temperature after sterilization, and then inoculated with 0.025% fermentation bacteria powder and sealed and fermented at 32° C. for 12 h at a fermentation speed of 50 r / min. After fermentation, it was filtered using 180-mesh gauze to separate the solid and liquid, and the liquid was obtained to obtain the Dendrobium officinale fermentation liquid.

[0045] Example 3

[0046] (1) The fresh stems of Dendrobium officinale were freeze-dried at -60°C for 30 h using a vacuum freeze dryer to remove moisture, and then ground into powder with a particle size of 50 mesh using a pulverizer to obtain the fresh stem powder of Dendrobium officinale, which was then placed in a dry sub-bottle and stored in a desiccator for later use.

[0047] (2) Weigh 1 g of fresh stem powder of Dendrobium officinale, add 65 mL of purified water, and stir evenly to obtain a mixture.

[0048] (3) The mixture is subjected to steam sterilization at 120° C. for 20 min, cooled to room temperature after sterilization, and then inoculated with 0.2% fermentation bacteria powder and sealed and fermented at 48° C. for 60 h at a fermentation speed of 50 r / min. After fermentation, it is filtered through 220-mesh gauze to separate the solid and liquid, and the liquid is obtained to obtain the Dendrobium officinale fermentation liquid.

[0049] Comparative Example 1

[0050] The difference from Example 1 is that fermentation is not carried out by inoculating fermentation bacteria powder.

[0051] Experimental Example 1

[0052] 1. Effects of different preparation methods on the contents of water-soluble Dendrobium officinale polysaccharides and water-soluble mannose in Dendrobium officinale fermentation broth

[0053] The Dendrobium officinale fermentation broths prepared in Example 1, Example 2, Example 3 and Comparative Example 1 were selected to determine the contents of water-soluble Dendrobium officinale polysaccharides and water-soluble mannose in the Dendrobium officinale fermentation broths.

[0054] Polysaccharide content determination method: The polysaccharide content was determined using the phenol-sulfuric acid method. Preparation of standard solution:

[0055] Dextran standard solutions were prepared with concentrations ranging from 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.

[0056] Determination process:

[0057] Take 1.0mL of standard solution and add it to the test tube. Add 1.0mL of phenol solution (5%) and mix well. Add 5.0mL of concentrated sulfuric acid and mix quickly. Let stand at room temperature for 30 minutes away from light. Use a UV spectrophotometer to measure the absorbance value at a wavelength of 490nm. Measure the absorbance value of each set of standard solutions and draw a concentration-absorbance standard curve. Linear fitting equation: Absorbance (A 490 )=k×concentration+b.

[0058] Mannose content determination method: Mannose content determination kit was used for determination.

[0059] (1) Reagent preparation: Prepare the reagents according to the instructions.

[0060] (2) This sample is acidic and needs to be adjusted to pH 7.4 with NaOH (2M) first. Then, let it stand at room temperature for 30 minutes and take the clear liquid for testing. Preheat the UV spectrophotometer for 30 minutes, set the temperature at 25°C, set the wavelength to 340nm, and adjust the zero value with distilled water. Incubate in a 25°C water bath for 15 minutes; add the reagents in a 1mL quartz cuvette (optical path 1cm) in sequence according to the instructions.

[0061] (3) Calculated by volume: Mannose content (mg / mL) = [ΔA ÷ (ε × d) × V2 × Mr × 10 3 ]÷V1×D=0.343×ΔA×D

[0062] ε: molar extinction coefficient of NADPH, 6.3×10 3 L / mol / cm;

[0063] d: light path, 1 cm;

[0064] V1: added sample volume, 0.06 mL;

[0065] V2: total reaction volume, 7.2×10 -4 L;

[0066] Mr: molecular weight of mannose, 180.16;

[0067] D: dilution factor, undiluted is 1;

[0068] ΔA = (A2-A1) measurement - (A2-A1) blank.

[0069] Extraction rate (%) = mannose content of fermentation broth × volume of fermentation broth / total mass of Dendrobium officinale before fermentation × 100

[0070] The absorbance value of the polysaccharide solution was substituted into the linear fitting equation to calculate the content of soluble polysaccharide in the solution.

[0071] The linear equation of the polysaccharide concentration-absorbance standard curve is A 490 =0.084×concentration (mg / mL)+0.093, R 2 =0.993. The detailed data are shown in Table 1.

[0072] Table 1 Polysaccharide solution concentration-absorbance standard curve

[0073] Standard solution concentration (mg / mL) <![CDATA[Absorbance (A 490 )]]> 0.1 0.102 0.2 0.110 0.4 0.127 0.6 0.144 0.8 0.161 1.0 0.178

[0074] Experimental results: as shown in Table 2.

[0075] Table 2 Soluble Dendrobium Officinale Polysaccharide and Soluble Mannose Content in Different Examples

[0076]

[0077] Experimental results: As shown in Table 2, the mannose content of Examples 1 to 3 is much higher than that of Comparative Example 1, and the soluble mannose content in the Dendrobium officinale fermentation liquid prepared in Example 1 is the highest. Next, the fermentation process of this example is optimized.

[0078] 2. Effect of solid-liquid ratio on mannose content and extraction rate in Dendrobium officinale fermentation broth

[0079] The other conditions of Example 1 remained unchanged, and the solid-liquid ratio of Dendrobium officinale fresh stem powder and purified water was set to 1 g:45 mL, 1 g:50 mL, 1 g:55 mL, 1 g:60 mL, and 1 g:65 mL, respectively, and the mannose content and extraction rate in the Dendrobium officinale fermentation broth were determined.

[0080] Experimental results: Figure 1 As shown in the figure. In the liquid-to-solid ratio range of 1g:45mL to 1g:65mL, the mannose content and extraction rate stabilized with the increase of the liquid-to-solid ratio. When the liquid-to-solid ratio was 1g:60mL, the mannose content and extraction rate were the highest. This shows that the liquid-to-solid ratio has a significant effect on the mannose content and extraction rate of the fermented liquid of Dendrobium officinale.

[0081] 3. Effect of fermentation bacterial powder inoculation amount on mannose content and extraction rate in Dendrobium officinale fermentation broth

[0082] The other conditions of Example 1 remain unchanged, and the inoculation amount of the fermentation bacteria powder is set to 0.025%, 0.05%, 0.1%, 0.15%, and 0.20% of the volume of the mixture, respectively, to determine the mannose content and extraction rate in the fermentation liquid of Dendrobium officinale. The detection method is the same as above.

[0083] Experimental results: Figure 2 As shown. In the range of 0.025% to 0.1%, the content and extraction rate of mannose increased with the increase of the fermentation powder inoculation amount. When the fermentation powder inoculation amount was 0.1%, the mannose content and extraction rate were the highest, with the content reaching 2g / L and the extraction rate being 11%. In the range of 0.1% to 0.2%, the content and extraction rate of mannose decreased with the increase of the fermentation powder inoculation amount, which indicates that the fermentation powder inoculation amount has a significant effect on the change trend of the mannose content and extraction rate of Dendrobium officinale fermentation liquid.

[0084] 4. Effect of fermentation temperature on mannose content and extraction rate in Dendrobium officinale fermentation broth

[0085] The other conditions of Example 1 remain unchanged, and the fermentation temperature is set to 32°C, 36°C, 40°C, 44°C, and 48°C, respectively, to determine the mannose content and extraction rate in the fermentation liquid of Dendrobium officinale. The detection method is the same as above.

[0086] Experimental results: Figure 3 As shown in the figure, within the range of 32-48°C, the content and extraction rate of mannose increased first and then decreased with the increase of fermentation temperature. When the fermentation temperature was 36°C, the content and extraction rate of mannose were the highest, reaching 2g / L and the extraction rate was 11%. This shows that the appropriate fermentation temperature is conducive to increasing the content and extraction rate of mannose in the fermentation liquid of Dendrobium officinale.

[0087] 5. Effect of fermentation time on mannose content and extraction rate in Dendrobium officinale fermentation broth

[0088] The other conditions of Example 1 remain unchanged, and the fermentation time is set to 12h, 24h, 36h, 48h, and 60h, respectively, to determine the mannose content and extraction rate in the fermentation liquid of Dendrobium officinale. The detection method is the same as above.

[0089] Experimental results: Figure 4 As shown. In the range of 12h to 48h, the content and extraction rate of mannose increased with the increase of fermentation time. When the fermentation time was 48h, the mannose content and extraction rate were the highest, with the content reaching 2.0g / L and the extraction rate being 11%. In the range of 48h to 60h, the content and extraction rate of mannose decreased with the increase of fermentation time, which indicates that the extension of fermentation time will reduce the content and extraction rate of mannose in the fermentation liquid of Dendrobium officinale. The above results show that the optimal process conditions for the fermentation of Dendrobium officinale are: liquid-to-solid ratio of 1g:60mL, fermentation powder inoculation amount of 0.1%, fermentation temperature of 36℃, and fermentation time of 48h.

[0090] Experimental Example 2

[0091] Free radical scavenging activity assay

[0092] 100 μL of Example 1, Example 2, Example 3 and the control group (deionized water) and 100 μL of DPPH (100 μM, dissolved in methanol) were added to a 96-well plate, mixed thoroughly, and incubated at room temperature in the dark for 30 minutes. The absorbance was measured at 517 nm. Three parallel determinations were performed for each group. Deionized water was used as a negative control. DPPH free radical scavenging activity (%) = [1-(A S -A S0 ) / (A C -A C0 )]×100%, where A S : absorbance of samples of different concentrations; A S0 : Background absorbance of the sample without DPPH; A C : Absorbance of the control without sample; A C0 : Background absorbance without sample and DPPH.

[0093] Experimental results: as shown in Table 3.

[0094] Table 3 DPPH free radical scavenging rate in different embodiments

[0095] sample <![CDATA[A S ]]> <![CDATA[A S0 ]]> <![CDATA[A C ]]> <![CDATA[A C0 ]]> DPPH scavenging activity Example 1 0.270 0.050 0.450 0.060 43.59％ Example 2 0.350 0.050 0.450 0.060 23.08％ Example 3 0.320 0.050 0.450 0.060 30.77％ Control group 0.440 0.050 0.450 0.060 0.00％

[0096] As shown in Table 3, Example 1 has the best free radical scavenging effect, followed by Example 3, and Example 2 has the worst free radical scavenging effect.

[0097] Experimental Example 3

[0098] Experiment on promoting proliferation of mouse embryonic stem cells by fermented liquid of Dendrobium officinale

[0099] D3 mouse embryonic stem cells in the logarithmic growth phase were counted and seeded into 96-well plates coated with 0.5% gelatin at a rate of 4.0 × 10 cells per well. 3 cells. Divide into five groups, add 100 μL of Example 1, Example 2, Example 3, 200 μg / mL of Dendrobium officinale polysaccharide solution (purchased from Shanxi Fushengda Biotechnology Co., Ltd., specification model is 98%) and complete culture medium to each group. Set up 4 replicate wells for each group. After culturing for 24 hours, 48 ​​hours, and 72 hours, use MTT staining to determine cell proliferation, and read the absorbance (A) at 490 nm. 490 ).

[0100] Experimental results: as shown in Table 4.

[0101] Table 4 Absorbance of different groups after 0h, 24h, 48h, 72h of culture

[0102]

[0103]

[0104] As shown in Table 4, Example 1 has the best effect on promoting the proliferation of mouse embryonic stem cells, and the effect of Dendrobium officinale polysaccharide is second. The effects of Example 2 and Example 3 are relatively poor, which may be because the excessively high concentration of Dendrobium officinale polysaccharide inhibits the proliferation of mouse embryonic stem cells. In addition, the mannose contained in Example 1 can reduce the effect of free radicals on the proliferation of mouse embryonic stem cells. Therefore, Example 1 has the best effect on the proliferation of mouse embryonic stem cells.

[0105] It can be seen from the above embodiments that the method for preparing the Dendrobium officinale fermentation liquid provided by the present invention adopts specific Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus plantarum to ferment the Dendrobium officinale with synergistic effect, which can not only increase the content and extraction rate of mannose, but also contain some polysaccharides. The combined effect of mannose and polysaccharides makes the fermentation liquid have antioxidant activity and can also promote stem cell proliferation.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a Dendrobium officinale fermentation broth for promoting stem cell proliferation, characterized in that: The following steps are involved: 1) drying the fresh stem of Dendrobium officinale, crushing and sieving to obtain the fresh stem powder of Dendrobium officinale; 2) mixing the fresh stem powder of Dendrobium officinale and water to obtain a mixture, and sterilizing the mixture; 3) inoculating fermentation bacteria powder into the sterilized mixture for fermentation, performing solid-liquid separation after the fermentation is completed, and taking the liquid to obtain the Dendrobium officinale fermentation liquid that promotes the proliferation of stem cells.

2. The preparation method according to claim 1, characterized in that: The inoculation amount of the fermentation bacteria powder is 0.025-0.2% of the volume of the mixture.

3. The preparation method according to claim 1, characterized in that: Step 2) The mass volume ratio of the fresh stem powder of Dendrobium officinale and water is 1 g: 45-65 mL.

4. The preparation method according to claim 1, characterized in that: Step 3) The fermentation temperature is 32-48° C. and the fermentation time is 12-60 hours.

5. The preparation method according to claim 1, characterized in that: Step 2) The sterilization temperature is 80-120° C. and the sterilization time is 20-40 min.

6. The preparation method according to claim 1, characterized in that: The drying in step 1) is vacuum freeze drying, the temperature of the vacuum freeze drying is -40 to -60°C, and the time of the vacuum freeze drying is 18 to 30 hours.

7. The preparation method according to claim 1, characterized in that: The mesh size of the crushed and sieved product in step 1) is 20 to 50 meshes.

8. The Dendrobium officinale fermentation liquid for promoting stem cell proliferation prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the Dendrobium officinale fermentation liquid for promoting stem cell proliferation prepared by the preparation method according to any one of claims 1 to 7 or the Dendrobium officinale fermentation liquid for promoting stem cell proliferation according to claim 8 in the preparation of a drug for promoting stem cell proliferation.

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