Streptomyces homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses a Streptomyces rochei D74 homogeneous polysaccharide and a preparation method and application thereof.The Streptomyces rochei D74 homogeneous polysaccharide is obtained by smashing Streptomyces rochei D74 mycelia obtained through liquid fermentation, then conducting water extraction, concentration, alcohol precipitation and deproteinization to obtain crude polysaccharide, and further conducting ion-exchange chromatography, dialysis and gel column chromatography purification to obtain the Streptomyces rochei D74 homogeneous polysaccharide. The method is simple and efficient, and the obtained streptomyces homogeneous polysaccharide remarkably inhibits proliferation of human gastric cancer cells SGC-7901 by inducing tumor cell apoptosis; meanwhile, immune cells RAW264.7 can be activated, the phagocytic ability and the nitric oxide (NO) release ability of the immune cells RAW264.7 can be improved, and the polysaccharide can become a potential polysaccharide drug for treating human liver cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide extraction, in particular to a streptomyces uniform polysaccharide and a preparation method and application thereof. BACKGROUND

[0002] As the second largest cause of death in the world, in addition to surgical treatment and radiotherapy, chemotherapy is one of the most commonly used treatment methods. However, severe side effects and drug resistance of tumor cells to chemotherapeutic drugs are considered to be the main reasons for the failure of tumor chemotherapy. According to statistics, about 50% of chemotherapy failure cases are related to multi-drug resistance. Therefore, it is essential to develop new anticancer drugs with low toxicity and high efficiency.

[0003] As a biological macromolecule, polysaccharides exhibit a wide range of biological activities such as anti-tumor, antioxidant, immunomodulatory, anti-inflammatory and anti-viral activities, and have potential medical application value, and have become a new research hotspot. Natural polysaccharides are listed as one of the top ten anticancer candidate molecules in 2025 due to their unique "multi-target-low toxicity" characteristics. Many microbial polysaccharides exhibit significant anti-tumor activity; at the same time, these polysaccharides can also improve the immune function of the body; in addition, compared with traditional chemotherapeutic drugs, microbial-derived polysaccharides (such as shiitake and lentinan) have been clinically verified and have significant safety. Therefore, developing natural microbial polysaccharides is a promising strategy for treating tumors. SUMMARY

[0004] The present application aims to provide a streptomyces uniform polysaccharide and a preparation method and application thereof, which can not only induce gastric cancer cell apoptosis and inhibit the proliferation of gastric cancer cells, but also activate macrophages and enhance the immune response capacity of the body, and is expected to become a potential polysaccharide drug for treating human liver cancer.

[0005] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0006] The present application provides a preparation method of a streptomyces uniform polysaccharide, which comprises the following steps:

[0007] (a) fermenting and culturing Streptomyces rouxii D74 in a liquid medium, then separating the mycelium and freeze-drying and crushing to obtain mycelium powder;

[0008] (b) adding the mycelium powder to deionized water for extraction to obtain an extraction liquid;

[0009] (c) adding Sevag reagent to the extraction liquid and mixing, standing, centrifuging, collecting the supernatant, repeating the above operation until there is no protein precipitate in the supernatant, then adding an ethanol solution to the supernatant, standing, centrifuging, collecting the precipitate, and freeze-drying to obtain the mycelium crude polysaccharide;

[0010] (d) dissolving the mycelium crude polysaccharide in deionized water and filtering with a microporous filter membrane, collecting the filtrate, and then adding the filtrate to a DEAE-52 cellulose column for chromatography, eluting with deionized water, 0.2 mol / L NaCl solution and 0.5 mol / L NaCl solution in sequence, collecting the eluate of the 0.2 mol / L NaCl solution, and then dialyzing with a dialysis bag with a molecular weight cut-off of 3000 Da, and freeze-drying to obtain a uniform polysaccharide crude product;

[0011] (e) dissolving the uniform polysaccharide crude product in deionized water, and then adding to a Sephadex G-100 gel chromatography column and eluting with deionized water, collecting one tube every 10-20 ml and detecting the absorbance at 490 nm by the phenol-sulfuric acid method, and drawing an elution curve according to the absorbance, collecting the eluate at the first elution peak, and then freeze-drying to obtain the uniform polysaccharide of Streptomyces.

[0012] Preferably, in the step (a), the liquid medium is Gao's No. 1 liquid medium; the fermentation culture temperature is 27-29℃, and the time is 6-10 days.

[0013] Preferably, in the step (b), adding the mycelium powder to deionized water for extraction includes:

[0014] The mycelium powder is added to deionized water for water bath extraction at a mass-volume ratio of 1:(10-30) for 1-3 h, the supernatant is collected by centrifugation, the extraction is repeated 2-4 times, the supernatants are combined, and concentrated to 0.05-0.2 times the original volume to obtain the extract.

[0015] Preferably, in the step (c), the volume ratio of Sevag reagent to extract is 1:(3-5); and the standing time after adding the Sevag reagent is 3-5 h.

[0016] Preferably, in the step (c), the concentration of the ethanol solution is not less than 90%; the addition amount of the ethanol solution to the supernatant is 3-5 times the volume of the supernatant; and the standing time after adding the ethanol solution is 10-14 h.

[0017] Preferably, in the step (d), the concentration of the mycelium crude polysaccharide in the filtrate is 0.05-0.1 g / mL.

[0018] Preferably, in the step (d), the inner diameter of the DEAE-52 cellulose column is 2-3 cm, and the length is 70-90 cm; the elution flow rate is 1.5-2.5 mL / min, and one tube is collected every 50 mL during the elution process; the number of tubes eluted with deionized water is 20-23 tubes; and the number of tubes eluted with 0.2 mol / L NaCl solution is 27-34 tubes.

[0019] Preferably, in step (e), the Sephadex G-100 gel chromatography column has a diameter of 1.6-2.0 cm, a length of 140-160 cm, and an elution flow rate of 0.8-1.2 mL / min.

[0020] The second aspect of the present application provides the streptomyces homogeneous polysaccharide prepared by the above preparation method.

[0021] The third aspect of the present application provides the use of the streptomyces homogeneous polysaccharide prepared by the above preparation method in the preparation of an anti-gastric cancer drug or a drug for enhancing the immunity of the body.

[0022] Compared with the prior art, the present application has at least the following beneficial effects:

[0023] The streptomyces homogeneous polysaccharide provided by the present application can not only induce the apoptosis of gastric cancer cells and inhibit the proliferation of gastric cancer cells, but also activate macrophages and enhance the immune response ability of the body, and is expected to become a potential polysaccharide drug for treating human liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the specific embodiments or prior art description will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0025] Figure 1 Elution curve of DEAE-52 cellulose column chromatography in the embodiment of the present application;

[0026] Figure 2 Elution curve of Sephadex G-100 gel chromatography column in the embodiment of the present application;

[0027] Figure 3 High-performance gel chromatography analysis result of SRP-1-1 in experimental example 3 of the present application;

[0028] Figure 4 Infrared spectroscopy analysis result of SRP-1-1 in experimental example 3 of the present application;

[0029] Figure 5 Monosaccharide composition analysis result of SRP-1-1 in experimental example 3 of the present application;

[0030] Figure 6 Nuclear magnetic resonance analysis result of SRP-1-1 in experimental example 3 of the present application;

[0031] Figure 7 Influence result of different concentrations of SRP-1-1 on the viability of human gastric cancer cells SCG-7901 in experimental example 4 of the present application;

[0032] Figure 8 Results of the influence of different concentrations of SRP-1-1 on the apoptosis of human gastric cancer cells SCG-7901 in the experimental example 4 of the present application;

[0033] Figure 9 Results of the influence of SRP-1-1 on the proliferation of RAW 264.7 cells in the experimental example 4 of the present application;

[0034] Figure 10 Results of the influence of SRP-1-1 on the NO release amount of RAW 264.7 cells in the experimental example 4 of the present application;

[0035] Figure 11 Results of the influence of SRP-1-1 on the phagocytic ability of RAW 264.7 cells in the experimental example 4 of the present application. DETAILED DESCRIPTION

[0036] The embodiments of the technical solutions of the present application will be described in detail below in combination with the examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0037] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by the skilled person in the field of the present application.

[0038] The present application provides a preparation method of streptomyces homopolysaccharide, which comprises the following steps:

[0039] (a) fermenting and culturing Streptomyces rouxii D74 in a liquid culture medium, then separating mycelium, freeze-drying and crushing to obtain mycelium powder;

[0040] (b) adding the mycelium powder to deionized water for extraction to obtain an extraction liquid;

[0041] (c) adding Sevag reagent to the extraction liquid and mixing, standing, centrifuging, collecting the supernatant, repeating the above operation until there is no protein precipitate in the supernatant, then adding ethanol solution to the supernatant, standing, centrifuging, collecting the precipitate, freeze-drying to obtain mycelium crude polysaccharide;

[0042] (d) dissolving the mycelium crude polysaccharide in deionized water and filtering by using a microporous filter, collecting the filtrate, and then adding the filtrate into a DEAE-52 cellulose column for chromatography, eluting in sequence by using distilled water, 0.2 mol / L NaCl solution and 0.5 mol / L NaCl solution, collecting the eluate of the 0.2 mol / L NaCl solution, and then dialyzing by using a dialysis bag with a molecular weight cut-off of 3000 Da, and freeze-drying to obtain a uniform polysaccharide crude product;

[0043] (e) dissolving the uniform polysaccharide crude product in deionized water, and then adding into a Sephadex G-100 gel chromatography column and eluting by using deionized water, collecting one tube every 10-20 ml and detecting the absorbance by using a phenol-sulfuric acid method at 490 nm, and then drawing an elution curve according to the absorbance, collecting the eluate at the first elution peak, and then freeze-drying to obtain the uniform polysaccharide of Streptomyces.

[0044] The uniform polysaccharide of Streptomyces provided by the application can not only induce apoptosis of gastric cancer cells and inhibit proliferation of gastric cancer cells, but also activate macrophages and enhance the immune response ability of the body, and is expected to become a potential polysaccharide drug for treating human liver cancer.

[0045] In some embodiments, in step (a), the liquid medium is Gao's No. 1 liquid medium; the fermentation culture temperature is 27-29℃, and the time is 6-10 days.

[0046] In some embodiments, in step (b), adding the mycelium powder into deionized water for extraction includes:

[0047] According to a mass-volume ratio of 1: (10-30), the mycelium powder is added into deionized water for water bath extraction for 1-3 h, the supernatant is collected by centrifugation, the extraction is repeated for 2-4 times, the supernatants are combined, and the combined supernatant is concentrated to 0.05-0.2 times of the original volume to obtain the extract.

[0048] In some embodiments, in step (c), the volume ratio of the Sevag reagent to the extract is 1: (3-5); and the standing time after adding the Sevag reagent is 3-5 h.

[0049] In some embodiments, in step (c), the concentration of the ethanol solution is not less than 90%; the addition amount of the ethanol solution to the supernatant is 3-5 times of the volume of the supernatant; and the standing time after adding the ethanol solution is 10-14 h.

[0050] In some embodiments, in step (d), the concentration of the mycelium crude polysaccharide in the filtrate is 0.05-0.1 g / mL.

[0051] In some embodiments, in step (d), the DEAE-52 cellulose column has an inner diameter of 2-3 cm and a length of 70-90 cm; the elution flow rate is 1.5-2.5 mL / min, and during the elution, one tube is collected every 50 mL; the number of tubes eluted with distilled water is 20-23; the number of tubes eluted with 0.2 mol / L NaCl solution is 27-34; and the number of tubes eluted with 0.5 mol / L NaCl solution is not less than 10.

[0052] In some embodiments, in step (e), the Sephadex G-100 gel chromatography column has an inner diameter of 1.6-2.0 cm and a length of 140-160 cm, and the elution flow rate is 0.8-1.2 mL / min.

[0053] Another embodiment of the present application provides a streptomyces uniform polysaccharide prepared by the above preparation method.

[0054] Still another embodiment of the present application provides a use of the streptomyces uniform polysaccharide prepared by the above preparation method in the preparation of an anti-gastric cancer drug or a drug for enhancing the immunity of a body.

[0055] The technical solutions of the present application are further described in detail below through specific embodiments.

[0056] Embodiment

[0057] The present embodiment is a preparation method of a streptomyces uniform polysaccharide, which comprises the following steps:

[0058] (a) Streptomyces rochei strain D74 is inoculated into 10 L of Gause No. 1 liquid medium, and cultured at 28 ℃ for 8 days; after the mycelium is separated using gauze, it is freeze-dried and crushed to obtain 325 g of mycelium powder;

[0059] (b) 200 g of the mycelium dry powder is added to 2 L of deionized water, and extracted in a 90 ℃ water bath for 2 h; the supernatant is collected by centrifugation; the extraction is repeated for 3 times, and the supernatants are combined; the supernatants are concentrated to 500 mL to obtain an extract;

[0060] (c) 100 mL of Sevag reagent is added to the extract, and after being shaken thoroughly, it is placed for 4 h; the supernatant is collected by centrifugation; the above step is repeated until there is no protein precipitation in the liquid; then 2 L of anhydrous ethanol is added, and placed at 4 ℃ for 12 h; the precipitate is collected by centrifugation at 4500 rpm for 15 min, and freeze-dried to obtain 18.7 g of mycelium crude polysaccharide;

[0061] (d) Dissolve 15 g of the obtained crude polysaccharide in 200 mL of deionized water, filter through a 0.45 µm microporous membrane, and pass the filtrate through a DEAE-52 cellulose column chromatography. Elute sequentially with distilled water and 0.2 and 0.5 mol / L NaCl solutions, collecting one tube for every 50 mL. Detect the absorbance at 490 nm using the phenol-sulfuric acid method and plot the elution curve (e.g., ...). Figure 1 As shown in the figure, there were 23 tubes for distilled water elution; 34 tubes for 0.2 mol / L NaCl solution elution; and 23 tubes for 0.5 mol / L NaCl solution elution. The distilled water elution phases were combined and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3000 Da. After freeze-drying, 2.12 g of crude first polysaccharide (denoted as SRP-W) was obtained. The 0.2 mol / L NaCl elution phases were combined and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3000 Da. After freeze-drying, 2.78 g of crude second polysaccharide (denoted as SRP-1) was obtained. The 0.5 mol / L NaCl elution phases were combined and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3000 Da. Dialysis with the dialysis bag for 24 hours, followed by freeze-drying, yielded 1.37 g of crude third polysaccharide (denoted as SRP-2); the DEAE-52 cellulose column had an inner diameter of 2.6 cm and a length of 80 cm; the elution flow rate was 2 mL / min.

[0062] (e) Dissolve 2 g of SRP-1 in 30 mL of deionized water and add it to a Sephadex G-100 gel chromatography column. Use deionized water as the eluent for further purification. Collect one tube every 20 mL and detect the absorbance at 490 nm using the phenol-sulfuric acid method. Plot an elution curve based on the absorbance (e.g., ...). Figure 2 As shown in the figure, the eluent from the first elution peak was collected and then freeze-dried to obtain 1.32 g of the first Streptomyces homogeneous polysaccharide (denoted as SRP-1-1). The eluent from the second elution peak was collected and then freeze-dried to obtain 0.25 g of the second Streptomyces homogeneous polysaccharide (denoted as SRP-1-2). The Sephadex G-100 gel chromatography column had an inner diameter of 1.8 cm, a length of 150 cm, and an elution flow rate of 1 mL / min.

[0063] Experimental Example 1

[0064] This experimental case studies the effects of SRP-W, SRP-1, and SRP-2 on the proliferation of human gastric cancer cells SCG-7901.

[0065] The effects of SRP-W, SRP-1, and SRP-2 on the proliferation of human gastric cancer cells SCG-7901 were detected using the MTT assay: SCG-7901 cells were seeded in 96-well plates and incubated at 37°C and 5% CO2 for 12 h. Then, each well was treated with 500 μg / mL SRP-W, SRP-1, and SRP-2 for 24 h. After incubation, 20 μL of MTT solution was added to each well and incubated for 4 h, discarding the supernatant. Then, 150 μL of DMSO was added and incubated for 10 min. Finally, the absorbance (A) was measured at 570 nm using a microplate reader to calculate cell viability.

[0066] The study found that SRP-W, SRP-1, and SRP-2 inhibited the proliferation of SCG-7901 cells by 2.3%, 49.8%, and 11.6%, respectively. Based on the results, SRP-1 was determined to have anti-proliferation activity against human gastric cancer cells SCG-7901.

[0067] Experiment Example 2

[0068] This experimental example studies the effects of SRP-1-1 and SRP-1-2 on the proliferation of human gastric cancer cells SCG-7901:

[0069] The effects of SRP-1-1 and SRP-1-2 on the proliferation of human gastric cancer cells SCG-7901 were detected using the MTT assay: SCG-7901 cells were seeded in 96-well plates and incubated at 37°C and 5% CO2 for 12 h. Then, the cells were treated with 500 μg / mL SRP-1-1 and SRP-1-2 for 24 h, respectively. After incubation, 20 μL of MTT solution was added to each well and incubated for 4 h, after which the supernatant was discarded. Then, 150 μL of DMSO was added and incubated for 10 min. Finally, the absorbance (A) was measured at 570 nm using a microplate reader to calculate cell viability.

[0070] The study found that SRP-1-1 and SRP-1-2 inhibited the proliferation of SCG-7901 by 62.4% and 18.2%, respectively. Based on the results, SRP-1-1 was determined to be the most effective inhibitor.

[0071] Experimental Example 3

[0072] This experimental example demonstrates the analysis and study of SRP-1-1's molecular weight, purity, infrared spectroscopy, and monosaccharide composition.

[0073] The molecular weight and purity of SRP-1-1 were analyzed, and the results are as follows: Figure 3 As shown;

[0074] Infrared spectroscopy analysis of SRP-1-1 was performed, and the results are as follows: Figure 4 As shown;

[0075] The monosaccharide composition of SRP-1-1 was analyzed, and the analysis results are shown in Figure 5 . Figure 5 , wherein A is a monosaccharide standard chart, and 1-10 represent mannose, glucosamine, rhamnose, glucuronic acid, galactosamine, glucose, galactose, arabinose, and xylose, respectively; and B is the monosaccharide analysis results of SRP-1-1.

[0076] The nuclear magnetic resonance analysis of SRP-1-1 was performed, and the analysis results are shown in Figure 6 . Figure 6 , wherein A is a hydrogen spectrum chart; and B is a carbon spectrum chart.

[0077] It can be seen from Figures 3-6 that:

[0078] The total sugar content of SRP-1-1 is 94.53%, the relative molecular weight is 14.8 kDa, the monosaccharide composition and molar percentage are mannose, rhamnose, glucose, galactose, and xylose (molar ratio is 1:0.38:1.75:0.68:0.21), and the main glycoside type is β-pyranoside.

[0079] Experimental Example 4

[0080] This experimental example is a study on the effects of different concentrations of SRP-1-1 on human gastric cancer cells SCG-7901 and immune cells RAW 264.7:

[0081] The MTT method was used to detect the inhibitory activity of SRP-1-1 on human gastric cancer cells SCG-7901: SCG-7901 cells were inoculated in a 96-well plate and incubated at 37°C, 5% CO2 for 12 hours, then treated with different concentrations of SRP-1-1 (0, 100, 300, 500, 700, and 900 μg / mL) for 24 hours, and after incubation, MTT solution (20 μL / well) was added to each well and incubated for 4 hours, and the supernatant was discarded. Then 150 μL of DMSO was added and incubated for 10 minutes. Finally, the absorbance (A) was measured at 570 nm wavelength by an enzyme-labeled instrument, and the cell viability was calculated, and the results are shown in Figure 7 .

[0082] SCG-7901 cells (1×10 6 cells) were inoculated in a 6-well plate. After being cultured with different concentrations of SRP-1-1 (0, 250, and 500 μg / mL) for 48 hours, the cells were washed twice with cold PBS, stained with FITC-labeled annexin V, and incubated in the dark for 15 minutes. After 15 minutes, PI (50 μg / mL) was added to the sample and stained for 15 minutes, and then analyzed using a flow cytometer. The results are shown in the accompanying Figure 8 .

[0083] It can be seen that: Figures 7-8

[0084] SRP-1-1 can inhibit the proliferation of SCG-7901 by inducing apoptosis of human gastric cancer cells SCG-7901, and the half inhibitory concentration (IC50) is 329 μg / mL, which shows that SRP-1-1 has the potential to be developed as an anti-tumor drug.

[0085] The effect of SRP-1-1 on the proliferation of RAW 264.7 cells was detected by MTT method: RAW 264.7 was inoculated in 96-well plates (10 5 cells / well), 100 μL per well, and 5 duplicate wells were set at the same time, and incubated at 37℃, 5% CO2 for 12 hours. After the cells adhered, high glucose medium containing different concentrations of SRP-1-1 (0, 50, 100, 200, 400, 800 μg / mL) was added, the final volume of the medium was 200 μL, and the incubation was continued for 24 hours. Discard the supernatant, add 20 μL of MTT (5 mg / mL) to each well, continue to incubate for 4h. Discard the MTT solution, add 150 μL of DMSO, incubate for 15 min, and measure the absorbance (A) at 570 nm wavelength with a microplate reader. Calculate the cell viability.

[0086] Lipopolysaccharide (LPS) was used as a positive control, and the phagocytic ability of RAW 264.7 cells was determined by neutral red colorimetry: RAW 264.7 was inoculated in 96-well plates (10 5 cells / well), 100 μL per well, and 5 duplicate wells were set at the same time, and incubated at 37℃, 5% CO2 for 12 hours. After the cells adhered, high glucose medium containing different concentrations of SRP-1-1 (0, 50, 100, 200, 400, 800 μg / mL) and LPS (10 μg / mL) was added, and the incubation was continued for 24 hours. Discard the supernatant, add 100 μL of neutral red solution (0.075%) to each well, continue to incubate for 1 hour. Discard the supernatant, wash with PBS for 3 times, add 100 μL of cell lysis solution (ethanol- glacial acetic acid 1:1), and after cell lysis, measure the absorbance at 540 nm wavelength with a microplate reader, and calculate the phagocytic rate.

[0087] Lipopolysaccharide (LPS) was used as a positive control, and the NO content of RAW 264.7 cells was determined by Griess method: RAW 264.7 was inoculated in 24-well plates (10 6 ​Cells were cultured in 5 replicates at 37°C and 5% CO2 for 12 h. After cell attachment, different concentrations of SRP-1-1 (0, 50, 100, 200, 400, 800 μg / mL) and LPS (2 μg / mL) were added, and incubation continued for 24 h. 100 μL of cell culture supernatant was taken from each well and transferred to a 96-well plate. Then, 100 μL of Griess reagent was added, mixed, and incubated at 25°C and 5% CO2 for 10 min. The absorbance (A) was measured at 540 nm using a microplate reader, and the NO content of RAW 264.7 cells was calculated based on the standard curve.

[0088] The results of the effects of SRP-1-1 on the proliferation, phagocytic capacity, and NO release of RAW 264.7 cells are shown in the attached figure. Figures 9-11 As shown.

[0089] Depend on Figures 9-11 It can be seen that, compared with the blank control group, co-culturing RAW264.7 cells with different concentrations of SRP-1-1 for 24 h did not reduce the cell survival rate. Moreover, when the concentration was 100~800 μg / mL, it could significantly promote the proliferation of RAW264.7 cells, enhance the phagocytic ability of cells, increase the release of NO from cells, and show a clear dose-dependent relationship.

[0090] The above experimental results are sufficient to prove that SRP-1-1 can induce apoptosis in gastric cancer cells, inhibit the proliferation of gastric cancer cells, activate macrophages, and enhance the body's immune response, thus becoming a potential polysaccharide drug for the treatment of human liver cancer.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A process for the preparation of a homopolysaccharide of Streptomyces, characterized in that, The preparation method comprises the following steps: (a) fermenting Streptomyces rouxii D74 in a liquid medium, then separating mycelium and freeze-drying and crushing to obtain mycelium powder; (b) adding the mycelium powder into deionized water for extraction to obtain an extract; (c) adding Sevag reagent into the extract and mixing, standing, centrifuging, collecting supernatant, repeating the above operation until there is no protein precipitate in the supernatant, then adding an ethanol solution into the supernatant, standing, centrifuging, collecting the precipitate, and freeze-drying to obtain mycelium crude polysaccharide; (d) dissolving the mycelium crude polysaccharide in deionized water and filtering by using a microporous filter, collecting the filtrate, then adding the filtrate into a DEAE-52 cellulose column for chromatography, eluting in sequence by using distilled water, 0.2 mol / L NaCl solution and 0.5 mol / L NaCl solution, collecting the eluate of the 0.2 mol / L NaCl solution, and dialyzing by using a dialysis bag with a molecular weight cut-off of 3000 Da, and freeze-drying to obtain uniform polysaccharide crude product; (e) dissolving the uniform polysaccharide crude product in deionized water, then adding into a Sephadex G-100 gel chromatography column and eluting by using deionized water, collecting one tube every 10-20 ml and detecting the absorbance at 490 nm by using the phenol-sulfuric acid method, drawing an elution curve according to the absorbance, collecting the eluate at the first elution peak, and freeze-drying to obtain the uniform Streptomyces polysaccharide.

2. The production method according to claim 1, characterized by, In the step (a), the liquid medium is Gao's No. 1 liquid medium; the fermentation culture temperature is 27-29 ℃, and the time is 6-10 days.

3. The preparation method according to claim 1, characterized in that, In the step (b), the adding of the mycelium powder into deionized water for extraction comprises: adding the mycelium powder into deionized water for water bath extraction at a mass-volume ratio of 1:(10-30) for 1-3 h, centrifuging to collect the supernatant, repeating the extraction for 2-4 times, combining the supernatants, and concentrating to 0.05-0.2 times of the original volume to obtain the extract.

4. The production method according to claim 1, characterized by, In the step (c), the volume ratio of the Sevag reagent to the extract is 1:(3-5); the standing time after adding the Sevag reagent is 3-5 h.

5. The method of claim 1, wherein, In the step (c), the concentration of the ethanol solution is not less than 90%; the adding amount of the ethanol solution to the supernatant is 3-5 times of the volume of the supernatant; and the standing time after adding the ethanol solution is 10-14 h.

6. The method of claim 1, wherein, In the step (d), the concentration of the mycelium crude polysaccharide in the filtrate is 0.05-0.1 g / mL.

7. The preparation method according to claim 1, characterized in that, In the step (d), the inner diameter of the DEAE-52 cellulose column is 2-3 cm, and the length is 70-90 cm; the elution flow rate is 1.5-2.5 mL / min, and one tube is collected every 50 mL during the elution process; the number of the tubes for elution by using distilled water is 20-23; and the number of the tubes for elution by using 0.2 mol / L NaCl solution is 27-34.

8. The method of claim 1, wherein, In the step (e), the inner diameter of the Sephadex G-100 gel chromatography column is 1.6-2.0 cm, and the length is 140-160 cm; and the elution flow rate is 0.8-1.2 mL / min.

9. The streptomyces homogeneous polysaccharide prepared by the method of any one of claims 1 to 8.

10. The use of the streptomyces homogeneous polysaccharide prepared by the method of any one of claims 1 to 8 in the preparation of a drug for resisting stomach cancer or a drug for enhancing immunity.

Images