Preparation method of ganoderma lucidum mutant strain active polysaccharide and application of ganoderma lucidum mutant strain active polysaccharide in oxidation resistance
The preparation of active polysaccharides from Ganoderma lucidum mutant strains by water extraction and alcohol precipitation solved the problems of large differences in antioxidant activity and low yield of ordinary Ganoderma lucidum strains, provided a high-efficiency raw material for antioxidant products, and improved the antioxidant performance of Ganoderma lucidum polysaccharides.
Patent Information
- Application Number
- CN202511814891.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-02-06
AI Technical Summary
In existing technologies, common Ganoderma lucidum strains exhibit significant differences in antioxidant activity, have long cultivation cycles, and low yields, making it difficult to meet the needs of large-scale industrial production.
The mycelium of Ganoderma lucidum mutant strain CGMCC No.23283 was used to prepare active polysaccharides of Ganoderma lucidum mutant strain by water extraction and alcohol precipitation method, including water bath extraction, filtration, concentration, precipitation by adding anhydrous ethanol and dialysis treatment.
It provides Ganoderma lucidum polysaccharides with high antioxidant activity, which significantly improves total antioxidant capacity and free radical scavenging rate, filling the gap in the study of nutritional components of Ganoderma lucidum mycelium induced by spacecraft mutation.
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Figure CN121471390A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing active polysaccharides from Ganoderma lucidum mutant strains and their application in antioxidation. Background Technology
[0002] Oxidative stress is one of the key factors leading to aging and chronic diseases. When the amount of reactive oxygen species (such as superoxide anion radicals and hydroxyl radicals) produced in the body exceeds the scavenging capacity of the antioxidant system, it will trigger a series of pathological reactions such as lipid peroxidation, protein oxidation, and DNA damage, which in turn can induce various health problems such as liver damage, cardiovascular disease, and neurodegenerative diseases.
[0003] As a traditional medicinal fungus, Ganoderma lucidum has been recorded in classics such as the Compendium of Materia Medica as "benefiting essence and qi, and strengthening tendons and bones". Modern pharmacological studies have confirmed that the active ingredients in Ganoderma lucidum, such as polysaccharides, triterpenoids, and sterols, have significant antioxidant and immunomodulatory effects. Among them, Ganoderma lucidum polysaccharides have become a key research object in the field of antioxidants in recent years due to their natural source, low toxicity, and outstanding antioxidant activity.
[0004] Currently, research on Ganoderma lucidum polysaccharides covers multiple areas, including extraction process optimization, activity evaluation, and mechanism of action analysis. In existing technologies, Ganoderma lucidum polysaccharides are mostly extracted from the fruiting bodies or mycelia of common Ganoderma lucidum strains and purified through processes such as water extraction, alcohol precipitation, and chromatography. However, common Ganoderma lucidum strains are limited by genetic characteristics, resulting in significant differences in their antioxidant activity. Their cultivation cycles are also long and yields are low, leading to high polysaccharide extraction costs, making it difficult to meet the needs of large-scale industrial production. Summary of the Invention
[0005] The purpose of this invention is to provide a method for preparing active polysaccharides from Ganoderma lucidum mutant strains and their application in antioxidant processes, thereby addressing the problems existing in the prior art. The active polysaccharides from these Ganoderma lucidum mutant strains exhibit high antioxidant activity, providing a new biomaterial resource for the preparation of antioxidant products.
[0006] To achieve the above objectives, the present invention provides the following solution:
[0007] This invention provides an active polysaccharide from a Ganoderma lucidum mutant strain, which is prepared by water extraction and alcohol precipitation using the mycelium of Ganoderma lucidum mutant strain CGMCC No.23283 as raw material.
[0008] Furthermore, the specific steps of the water extraction and alcohol precipitation method include:
[0009] The mycelium was mixed with water and then extracted in a water bath, followed by filtration to obtain a clear liquid;
[0010] After the clear liquid is concentrated, anhydrous ethanol is added to precipitate the precipitate.
[0011] The precipitate was subjected to dialysis to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.
[0012] Furthermore, the water bath extraction temperature is 90°C, and the time is 3 hours.
[0013] The present invention also provides a method for preparing active polysaccharides from Ganoderma lucidum mutant strains, comprising the steps of using the mycelium of Ganoderma lucidum mutant strain CGMCC No.23283 as raw material and preparing the active polysaccharides from the Ganoderma lucidum mutant strain by water extraction and alcohol precipitation method.
[0014] Furthermore, the specific steps of the water extraction and alcohol precipitation method include:
[0015] The mycelium was mixed with water and then extracted in a water bath, followed by filtration to obtain a clear liquid;
[0016] After the clear liquid is concentrated, anhydrous ethanol is added to precipitate the precipitate.
[0017] The precipitate was subjected to dialysis to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.
[0018] Furthermore, the water bath extraction temperature is 90°C, and the time is 3 hours.
[0019] This invention also provides the application of the above-mentioned Ganoderma lucidum mutant strain active polysaccharide in the preparation of antioxidant products.
[0020] Furthermore, the antioxidant product is a drug or health supplement.
[0021] The present invention also provides a drug that helps with antioxidation, the active ingredient of which includes the above-mentioned active polysaccharide from Ganoderma lucidum mutant strains.
[0022] The present invention also provides a health product that helps with anti-oxidation, the active ingredient of which includes the above-mentioned active polysaccharide from Ganoderma lucidum mutant strains.
[0023] The present invention discloses the following technical effects:
[0024] This invention develops a novel Ganoderma lucidum active polysaccharide, using a Ganoderma lucidum strain cultivated using space-induced mutagenesis technology. Compared to polysaccharides from common Ganoderma lucidum strains, the polysaccharide provided by this invention exhibits significantly enhanced antioxidant activity, demonstrating superior performance in key indicators such as total antioxidant capacity and free radical scavenging. This invention provides a new biomaterial resource for the preparation of antioxidant products.
[0025] This invention provides high-quality, highly active core raw materials for the innovative research and development of various Ganoderma lucidum derivative products, effectively filling the current gap in the research field of mycelial nutrient components of space-induced Ganoderma lucidum mutant strains. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0027] Figure 1 Line graph showing the total antioxidant capacity of active polysaccharides from Ganoderma lucidum mutant strains and Ganoderma lucidum original strains; where GLP1: active polysaccharides from Ganoderma lucidum mutant strains, and GLP2: active polysaccharides from Ganoderma lucidum original strains;
[0028] Figure 2 Line graphs showing the scavenging rates of superoxide anion free radicals by active polysaccharides from Ganoderma lucidum mutant strains and Ganoderma lucidum original strains; where GLP1: active polysaccharide from Ganoderma lucidum mutant strains, and GLP2: active polysaccharide from Ganoderma lucidum original strains.
[0029] Figure 3 Line graph showing the scavenging rates of hydroxyl radicals by active polysaccharides from Ganoderma lucidum mutant strains and Ganoderma lucidum original strains; where GLP1: active polysaccharide from Ganoderma lucidum mutant strains, and GLP2: active polysaccharide from Ganoderma lucidum original strains.
[0030] Figure 4 Line graphs showing the DPPH free radical scavenging rates of active polysaccharides from Ganoderma lucidum mutant strains and Ganoderma lucidum original strains; where GLP1: active polysaccharide from Ganoderma lucidum mutant strains, and GLP2: active polysaccharide from Ganoderma lucidum original strains. Detailed Implementation
[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0036] The Ganoderma lucidum mutagen strains involved in the following examples were deposited on September 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Ganoderma lucidum, with accession number CGMCC No. 23283.
[0037] Example 1
[0038] (1) Preparation of slant culture medium: Cut 500 g of potatoes into 1 cm cubes, add an appropriate amount of water, heat at 100℃ for 30 min, filter out potato residue, add 30 g / L glucose, 2 g / L peptone, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate and 20 g / L agar.
[0039] (2) Inoculation: After sterilizing the prepared culture medium solution in an autoclave at 121°C for 40 min, dispense it into glass petri dishes and place them in a clean bench for UV irradiation for 24 h. Inoculate the 3 mm Ganoderma lucidum mutant strain mother culture block into the petri dish and incubate it in an incubator at 25°C for 15 days.
[0040] (3) Polysaccharide preparation: The upper layer of mycelium was scraped from the culture dish covered with mycelium, and distilled water was added at a material-to-liquid mass ratio of 1:30. The mixture was placed in a water bath at 90°C and stirred for 3 hours. After extraction, the supernatant was filtered to obtain a clear liquid, which was concentrated to 1 / 4 of the original volume. Three times the volume of anhydrous ethanol was added and allowed to stand overnight to precipitate. The supernatant was then discarded. The precipitate was dissolved in purified water and dialyzed with running water for 24 hours using a 3500 Da cutoff dialysis bag. The retentate was then concentrated and freeze-dried to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.
[0041] Comparative Example 1
[0042] Same as Example 1, except that the Ganoderma lucidum mutagen strain is replaced with the original Ganoderma lucidum strain before mutagenesis.
[0043] The following experiments demonstrate the medicinal uses of the Ganoderma lucidum polysaccharide obtained by this invention.
[0044] Experimental Example 1
[0045] The total antioxidant capacity of the active polysaccharide (GLP1) from the Ganoderma lucidum mutant strain prepared in Example 1 and the active polysaccharide (GLP2) from the original Ganoderma lucidum strain prepared in Comparative Example 1 was determined:
[0046] FRAP buffer, Fe 3+ Mix the solution and TPTZ solution to prepare the FRAP working solution, and preheat it in a 37°C water bath for 10 minutes. Set up a blank group (170 μL FRAP working solution + 30 μL RO water) and a sample group (170 μL FRAP working solution + 5 μL pretreated sample + 25 μL RO water, with 3 replicates for each sample).
[0047] Add reagents sequentially according to the above groupings. Different concentrations of GLP1, GLP2, and positive control VC were added to the sample groups. The 96-well plates with added reagents were placed in a 37°C incubator for 30 minutes. Immediately after incubation, the absorbance (OD value) of each well was measured at 590 nm using a microplate reader, and the OD values of the blank and sample wells were recorded. The formula for calculating total antioxidant capacity is as follows:
[0048] Total antioxidant capacity (μmol Trolox / mL) = [(sample OD - blank OD - 0.0045) / 0.0969 × 10⁻⁶ -3 ] / 0.005×100%.
[0049] The results of the total antioxidant capacity test are as follows: Figure 1As shown, the total antioxidant capacity of the active polysaccharides from the Ganoderma lucidum mutant strain gradually increased with increasing concentration, reaching a maximum of 0.56 μmol Trolox / mL at 5 mg / mL. The concentration of active polysaccharides from the original Ganoderma lucidum strain had little effect on its antioxidant capacity, and did not exceed 0.1 μmol Trolox / mL in the range of 1-5 mg / mL.
[0050] Experimental Example 2
[0051] The superoxide anion free radical scavenging rate of the active polysaccharide (GLP1) from the Ganoderma lucidum mutant strain prepared in Example 1 and the active polysaccharide (GLP2) from the original Ganoderma lucidum strain prepared in Comparative Example 1 was determined:
[0052] Reagent preparation: Phosphate buffer: Weigh sodium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in distilled water, adjust the pH to 7.8, and bring to a final volume.
[0053] Xanthine solution: Weigh xanthine powder, dissolve it in a small amount of NaOH solution, then make up to volume with PBS, and store at 4°C protected from light.
[0054] Nitroblue tetrazolium (NBT) solution: Weigh NBT powder, dissolve in PBS, and store at 4°C protected from light.
[0055] Xanthine oxidase solution: Dilute with PBS to the working concentration according to the enzyme activity instructions and store on ice.
[0056] Sample solution: Dissolve the sample to be tested in distilled water and dilute to multiple concentration gradients (1, 2, 3, 4, 5 mg / mL).
[0057] The study included a control group (PBS 400 μL + xanthine 300 μL + NBT 200 μL), a sample group (sample 400 μL + xanthine 300 μL + NBT 200 μL), and a blank group (sample 400 μL + xanthine 300 μL + NBT 200 μL). The samples in the sample group contained different concentrations of GLP1, GLP2, and the positive control vitamin C.
[0058] Add reagents sequentially according to the above groupings, mix well, and preheat in a 37°C water bath for 5 minutes. Add 100 μL of xanthine oxidase solution (control group and sample group), and add 100 μL of PBS to the blank sample group. Mix immediately to start the reaction. Incubate in a 37°C water bath in the dark for 25 minutes. Immediately after the reaction, measure the absorbance of each tube at a wavelength of 550 nm. The formula for calculating the free radical scavenging rate is as follows:
[0059] Sweep rate (%) = [1 - (sample OD - blank OD) / control OD] × 100%.
[0060] The results of the superoxide anion free radical scavenging rate determination are as follows: Figure 2 As shown, at a polysaccharide concentration of 1 mg / mL, the scavenging rate of the active polysaccharide from the Ganoderma lucidum mutant strain against superoxide anion free radicals was 45%. With increasing polysaccharide concentration, the scavenging rate gradually increased, reaching a maximum of 86% at 5 mg / mL, indicating that high concentrations of polysaccharide have a strong scavenging ability against superoxide anion free radicals. Compared to the active polysaccharide from the original Ganoderma lucidum strain, its scavenging rate against superoxide anion free radicals was higher in the 1-5 mg / mL range.
[0061] Experimental Example 3
[0062] The hydroxyl radical scavenging rates of the active polysaccharide (GLP1) from the Ganoderma lucidum mutant strain prepared in Example 1 and the active polysaccharide (GLP2) from the original Ganoderma lucidum strain prepared in Comparative Example 1 were determined.
[0063] The following groups were set up: a control group (1 mL FeSO4 + 1 mL H2O2 + 1 mL salicylic acid solution), a sample group (1 mL FeSO4 + 1 mL sample solution + 1 mL salicylic acid solution, mixed and then 1 mL H2O2 was added, with gradient sample solutions of 1, 2, 3, 4, and 5 mg / mL), and a blank group (1 mL FeSO4 + 1 mL PBS + 1 mL salicylic acid solution). The samples in the sample group contained different concentrations of GLP1, GLP2, and the positive control VC.
[0064] Add reagents sequentially according to the above groupings, mix well, incubate in a 37℃ water bath for 30 min, centrifuge at 3000 rpm for 10 min, and measure the absorbance at 510 nm using the supernatant. The formula for calculating the free radical scavenging rate is as follows:
[0065] Clearance rate (%) = [blank OD - (sample OD - control OD)] / blank OD × 100%.
[0066] Results of hydroxyl radical scavenging rate determination are as follows Figure 3 As shown, in the concentration range of 1-4 mg / mL, the hydroxyl radical scavenging rate was less affected by the concentration of active polysaccharides from the Ganoderma lucidum mutant strain. At a concentration of 5 mg / mL, the scavenging rate increased significantly, reaching 57%, which was significantly higher than that of the active polysaccharides from the original Ganoderma lucidum strain.
[0067] Test Example 4
[0068] The DPPH free radical scavenging rates of the active polysaccharide (GLP1) from the Ganoderma lucidum mutant strain prepared in Example 1 and the active polysaccharide (GLP2) from the original Ganoderma lucidum strain prepared in Comparative Example 1 were determined.
[0069] Accurately weigh the DPPH reagent, dissolve it in anhydrous ethanol and dilute to volume to prepare a solution with a concentration of 0.1 mmol / L.
[0070] The following groups were set up: sample group (samples diluted with ethanol or distilled water to a series of concentration gradients (1, 2, 3, 4, 5 mg / mL)), sample group (2 mL sample solution + 2 mL DPPH solution), blank group (2 mL sample solution + 2 mL anhydrous ethanol), and control group (2 mL anhydrous ethanol + 2 mL DPPH solution). The samples in the sample groups contained different concentrations of GLP1, GLP2, and the positive control VC.
[0071] After adding the reagents to the groups as described above and mixing thoroughly, react for 30 minutes in the dark. After the reaction, using anhydrous ethanol as a reference, measure the absorbance of each group at 517 nm using a spectrophotometer. The formula for calculating the free radical scavenging rate is as follows:
[0072] Sweep rate (%) = [1 - (sample OD - control OD) / blank OD] × 100%.
[0073] The results of the DPPH free radical scavenging rate determination are as follows: Figure 4 As shown, there was a significant difference in the DPPH free radical scavenging rate between the active polysaccharides of the Ganoderma lucidum mutant strain and the active polysaccharides of the original Ganoderma lucidum strain. However, at 5 mg / mL, the active polysaccharides of the Ganoderma lucidum mutant strain showed a more significant DPPH free radical scavenging effect, reaching 76%, which was close to that of the VC group and 54% higher than that of the active polysaccharides of the original Ganoderma lucidum strain.
[0074] The above results indicate that the active polysaccharides of the Ganoderma lucidum mutant strain have a greater scavenging effect on the three oxidizing factors than the active polysaccharides of the original Ganoderma lucidum strain.
[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. An active polysaccharide derived from a Ganoderma lucidum mutant strain, characterized in that, The active polysaccharide of the Ganoderma lucidum mutant strain was prepared by water extraction and alcohol precipitation using the mycelium of Ganoderma lucidum mutant strain CGMCC No.23283 as raw material.
2. The active polysaccharide of the Ganoderma lucidum mutagen strain according to claim 1, characterized in that, The specific steps of the water extraction and alcohol precipitation method include: The mycelium was mixed with water and then extracted in a water bath, followed by filtration to obtain a clear liquid; After the clear liquid is concentrated, anhydrous ethanol is added to precipitate the precipitate. The precipitate was subjected to dialysis to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.
3. The active polysaccharide of the Ganoderma lucidum mutagenized strain according to claim 2, characterized in that, The water bath extraction was performed at a temperature of 90°C for 3 hours.
4. A method for preparing active polysaccharides from Ganoderma lucidum mutant strains, characterized in that, The method includes the step of preparing the active polysaccharide of the Ganoderma lucidum mutant strain CGMCC No. 23283 by water extraction and alcohol precipitation using mycelium as raw material.
5. The preparation method according to claim 4, characterized in that, The specific steps of the water extraction and alcohol precipitation method include: The mycelium was mixed with water and then extracted in a water bath, followed by filtration to obtain a clear liquid; After the clear liquid is concentrated, anhydrous ethanol is added to precipitate the precipitate. The precipitate was subjected to dialysis to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.
6. The preparation method according to claim 5, characterized in that, The water bath extraction was performed at a temperature of 90°C for 3 hours.
7. The use of the active polysaccharide of Ganoderma lucidum mutant strain as described in any one of claims 1-3 in the preparation of antioxidant products.
8. The application according to claim 7, characterized in that, The antioxidant product is a drug or health supplement.
9. A drug that helps with antioxidation, characterized in that, The active ingredient includes the active polysaccharide of Ganoderma lucidum mutant strain as described in any one of claims 1-3.
10. A health supplement that helps with antioxidation, characterized in that, The active ingredient includes the active polysaccharide of Ganoderma lucidum mutant strain as described in any one of claims 1-3.