Method for improving yield and antioxidant activity of ganoderma lucidum exopolysaccharides
By adding Clinacanthus leaf powder to the liquid fermentation medium of Ganoderma lucidum, the problem of insufficient yield and activity of Ganoderma lucidum extracellular polysaccharides was solved, and high-yield and high-activity extracellular polysaccharide production was achieved, especially with a significant improvement in antioxidant capacity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-02
- Publication Date
- 2026-03-31
AI Technical Summary
Traditional liquid fermentation of Ganoderma lucidum for the production of extracellular polysaccharides suffers from limited yield, single active ingredient, and unsatisfactory molecular weight distribution. Furthermore, there are currently no reports of using Gynostemma pentaphyllum for bidirectional fermentation to achieve synergistic effects.
A specific concentration of Clinacanthus nutans leaf powder was added to the liquid fermentation medium of Ganoderma lucidum to prepare a fermentation medium consisting of glucose, peptone, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and vitamin B. After inoculation with Ganoderma lucidum suspension, liquid fermentation culture was carried out.
It significantly improved the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides. The extracellular polysaccharides were mainly composed of galactose, and the molecular weight distribution showed a bimodal or multimodal pattern. The high molecular weight components were significantly increased, and the antioxidant capacity was significantly enhanced.
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Figure CN121759386A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides. Background Technology
[0002] Ganoderma lucidum ( Ganoderma lucidum Extracellular polysaccharides (EPS) have been extensively studied due to their various biological activities, including immunomodulation and antioxidant effects, and are promising raw materials for microbial-derived nutritional supplements. Traditional methods for producing EPS through liquid fermentation of Ganoderma lucidum suffer from limited yield, limited active ingredients, and unsatisfactory molecular weight distribution. Conventional optimization methods primarily focus on adjusting physicochemical parameters such as carbon and nitrogen sources, pH, and temperature. While these methods can increase yield to some extent, they are insufficient for targeted modification of the polysaccharide structure to enhance its specific functions.
[0003] Two-way liquid fermentation technology offers a novel approach to addressing these issues, potentially regulating fungal metabolic pathways by adding specific plant substrates to the fungal culture medium. However, not all plant substrates produce positive effects. Screening for specific plant substrates that can synergistically enhance Ganoderma lucidum EPS yield and significantly improve its structure-function profile remains a pressing technical challenge in this field. Currently, there are no reports of using Clinacanthus nutans for two-way fermentation of Ganoderma lucidum to produce highly active EPS in a synergistic manner. Summary of the Invention
[0004] The purpose of this invention is to provide a method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides. By adding a specific concentration of Clinacanthus nutans leaf powder to the liquid fermentation medium of Ganoderma lucidum, the yield of extracellular polysaccharides is synergistically increased and its chemical structure is changed. The change in structure also significantly enhances the antioxidant activity of the product.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of Clinacanthus nutans leaf powder in the preparation of fermentation culture medium that improves the yield of Ganoderma lucidum extracellular polysaccharides and antioxidant activity, wherein the concentration of Clinacanthus nutans leaf powder in the fermentation culture medium is 2-10 g / L.
[0006] As a preferred embodiment, the preparation method of the Clinacanthus nutans leaf powder includes the following steps: drying the harvested Clinacanthus nutans leaves, pulverizing them, passing them through an 80-120 mesh sieve, and collecting the sieve-passing material to obtain the powder.
[0007] Preferably, the drying temperature is 45-55°C.
[0008] The present invention provides a fermentation culture medium containing the aforementioned Clinacanthus nutans leaf powder, comprising the following components at the following concentrations: glucose 20-60 g / L, peptone 2-6 g / L, potassium dihydrogen phosphate 1.0-3.0 g / L, magnesium sulfate heptahydrate 1.0-3.0 g / L, vitamin B 0.005-0.05 g / L, and the aforementioned Clinacanthus nutans leaf powder 2-10 g / L.
[0009] Preferably, the components include the following concentrations: glucose 30-50 g / L, peptone 3-5 g / L, potassium dihydrogen phosphate 1.2-2.5 g / L, magnesium sulfate heptahydrate 1.2-2.5 g / L, vitamin B 0.008-0.03 g / L, and the *Clinacanthus nutans* leaf powder 4-8 g / L.
[0010] The present invention also provides a method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides, comprising the following steps: inoculating a Ganoderma lucidum suspension into the fermentation medium for liquid fermentation culture.
[0011] Preferably, the inoculum amount of the Ganoderma lucidum suspension is 1.5-2.5%, and the concentration of the Ganoderma lucidum suspension is 10-100 g / L.
[0012] The present invention also provides Ganoderma lucidum extracellular polysaccharide obtained by the method, wherein the monosaccharide composition of the Ganoderma lucidum extracellular polysaccharide is mainly composed of galactose and contains uronic acid.
[0013] Preferably, the molecular weight distribution of the Ganoderma lucidum extracellular polysaccharide is bimodal or multimodal, and it contains high molecular weight components with a molecular weight greater than 10 kDa.
[0014] By adopting the above technical solution, the present invention has the following beneficial effects: The technical solution of this invention improves the yield of extracellular polysaccharides and changes their chemical structure by adding a specific concentration of Clinacanthus nutans leaf powder to the liquid fermentation medium of Ganoderma lucidum. The structural change also significantly enhances the antioxidant activity of the product.
[0015] Experimental examples of this invention also show that, compared with the control group without the addition of *Ganoderma lucidum*, the method of this invention can increase the biomass of *Ganoderma lucidum* mycelium by more than 60% and the yield of extracellular polysaccharides by more than 110%. The simple glucan, mainly composed of glucose (>90%) in the control group, is transformed into a complex heteropolysaccharide mainly composed of galactose (>50%), containing rhamnose, arabinose, glucuronic acid, galacturonic acid, glucosamine, and other monosaccharides. The distribution changes from a single low molecular weight (approximately 4.7 kDa) to a bimodal distribution containing significantly high molecular weight components (approximately 38.5 kDa), with a significantly increased molecular weight and wider distribution. Infrared spectroscopy analysis shows that the glycosidic bond configuration of the polysaccharide changes, which may affect its spatial conformation and biological recognition. Due to the structural changes, the prepared *Ganoderma lucidum* extracellular polysaccharide exhibits significantly enhanced in vitro antioxidant activity. At a concentration of 2 mg / mL, the *Ganoderma lucidum* extracellular polysaccharide exhibits enhanced antioxidant activity against DPPH free radicals, hydroxyl free radicals, and ABTS. + The free radical scavenging rates reached 55.5%, 35.1% and 88.0% respectively, and the overall antioxidant capacity was significantly better than that of Ganoderma lucidum extracellular polysaccharides produced by traditional methods. Attached Figure Description
[0016] Figure 1 The figure shows the effect of different concentrations of *Clinacanthus cunea* on the biomass and extracellular polysaccharide yield of *Ganoderma lucidum* in Experiment Example 1. Figure 1 In this context, A represents biomass and B represents yield. Figure 2 This is a comparison of the infrared spectra of Ganoderma lucidum extracellular polysaccharides in the experimental group and the control group in Experiment Example 1; Figure 3 The chromatograms show the monosaccharide composition of the extracellular polysaccharides in the experimental and control groups of Ganoderma lucidum in Experiment Example 1. Figure 3 In the diagram, A represents the ion chromatogram of the mixed standard, B represents the ion chromatogram of the group without the addition of Clinacanthus nutans (control group), and C represents the ion chromatogram of the group with the addition of 6 g / L Clinacanthus nutans leaf powder (experimental group). Figure 4 The high-performance liquid chromatography gel permeation chromatograms show the molecular weights of extracellular polysaccharides from Ganoderma lucidum in the experimental and control groups in Example 1. Figure 4 In the diagram, A represents the blank phase chromatogram, B represents the chromatogram of the group without the addition of Clinacanthus nutans (control group), and C represents the chromatogram of the group with the addition of 6 g / L Clinacanthus nutans leaf powder (experimental group). Figure 5 This is a comparison of the in vitro antioxidant activity of Ganoderma lucidum extracellular polysaccharides in the experimental group and the control group in Experiment Example 1. Figure 5 In the diagram, A represents the scavenging effect of hydroxyl radicals, B represents the scavenging effect of vitamin C (positive control) on hydroxyl radicals, C represents the scavenging effect of DPPH radicals, D represents the scavenging effect of vitamin C (positive control) on hydroxyl radicals, and E represents ABTS. +The free radical scavenging effect is shown in the figure. F represents the scavenging effect of hydroxyl free radicals in the positive control vitamin C. OH-CG represents the antioxidant activity of hydroxyl radicals in the control group, OH-EG represents the antioxidant activity of hydroxyl radicals in the experimental group, OH-VC represents the antioxidant activity of hydroxyl radicals in the positive control vitamin C, DPPH-CG represents the antioxidant activity of DPPH in the control group, DPPH-EG represents the antioxidant activity of DPPH in the experimental group, DPPH-VC represents the antioxidant activity of DPPH in the positive control vitamin C, ABTS-CG represents the antioxidant activity of ABTS in the control group, ABTS-EG represents the antioxidant activity of ABTS in the experimental group, and ABTS-VC represents the antioxidant activity of ABTS in the positive control vitamin C. Detailed Implementation
[0017] This invention provides the application of Clinacanthus nutans leaf powder in the preparation of fermentation culture medium that improves the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides.
[0018] In this invention, *Clinacanthus nutans*, a plant belonging to the genus *Clinacanthus* in the family Acanthaceae, is also known as *Qianlizhui*, *Roucicao*, *Handicao*, *Ezuihua*, and *Shabashecao*. It is a traditional medicinal plant with potential medicinal value. It possesses various biological activities, including anti-inflammatory, antiviral, antioxidant, immunomodulatory, and skin repair and protection effects.
[0019] In this invention, the concentration of Clinacanthus nutans leaf powder in the fermentation medium is preferably 2-10 g / L, more preferably 4-8 g / L, and even more preferably 6 g / L.
[0020] In this invention, the preparation method of the Clinacanthus nutans leaf powder includes the following steps: drying the harvested Clinacanthus nutans leaves, pulverizing them, passing them through an 80-120 mesh sieve, and collecting the sieve residue to obtain the powder.
[0021] In this invention, freshly picked Clinacanthus leaves are preferably used. The drying method described in this invention is preferably oven drying; the drying temperature is preferably 45-55℃, more preferably 48-52℃, and even more preferably 50℃; the mesh size of the sieve is preferably 80-120 mesh, more preferably 90-110 mesh, and even more preferably 100 mesh.
[0022] This invention provides a fermentation medium containing the powdered leaves of *Clinacanthus nutans*, comprising the following components at the following concentrations: glucose 20-60 g / L, peptone 2-6 g / L, potassium dihydrogen phosphate 1.0-3.0 g / L, magnesium sulfate heptahydrate 1.0-3.0 g / L, vitamin B1 0.005-0.05 g / L, and the *Clinacanthus nutans* leaf powder 2-10 g / L; more preferably, it comprises glucose 30-50 g / L, peptone 3-5 g / L, potassium dihydrogen phosphate 1.2-2.5 g / L, magnesium sulfate heptahydrate 1.2-2.5 g / L, vitamin B1 0.008-0.03 g / L, and the *Clinacanthus nutans* leaf powder 4-8 g / L; even more preferably, it comprises glucose 40 g / L, peptone 4 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, and vitamin B1 0.01 g / L. g / L and the aforementioned Clinacanthus leaf powder 6 g / L.
[0023] The present invention also provides a method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides, comprising the following steps: inoculating a Ganoderma lucidum suspension into the fermentation medium for liquid fermentation culture.
[0024] In this invention, Ganoderma lucidum is first inoculated into a seed culture medium and cultured at 28°C and 150 rpm for 7 days. The seed culture medium includes 30 g / L glucose, 3 g / L yeast powder, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, and 0.01 g / L vitamin B. After obtaining mycelial balls, the mycelial balls are broken up to obtain a Ganoderma lucidum suspension. The Ganoderma lucidum suspension is then inoculated into the fermentation medium for liquid fermentation. The concentration of the Ganoderma lucidum suspension in this invention is preferably 10-100 g / L, more preferably 30-50 g / L, and even more preferably 40 g / L. The inoculation amount (v / v) of the Ganoderma lucidum suspension in this invention is preferably 1.5-2.5%, more preferably 1.8-2.2%, and even more preferably 2%.
[0025] In this invention, the preferred temperature for liquid fermentation culture is 25-30℃, more preferably 27-29℃, and even more preferably 28℃; the preferred fermentation time is 7-11 days, more preferably 8-10 days, and even more preferably 9 days. The liquid fermentation culture process is accompanied by shaking, with a shaking frequency of 120-180 rpm, more preferably 140-160 rpm, and even more preferably 150 rpm.
[0026] The present invention also provides Ganoderma lucidum extracellular polysaccharide obtained by the method, wherein the monosaccharide composition of the Ganoderma lucidum extracellular polysaccharide is mainly composed of galactose and contains uronic acid.
[0027] In this invention, the molecular weight distribution of the Ganoderma lucidum extracellular polysaccharide is bimodal or multimodal, and it contains high molecular weight components with a molecular weight greater than 10 kDa.
[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0029] Example 1
[0030] A fermentation medium containing Clinacanthus nutans leaf powder has the following components: 40 g / L glucose, 4 g / L peptone, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 0.01 g / L vitamin B, and 6 g / L of the Clinacanthus nutans leaf powder.
[0031] Example 2
[0032] A fermentation medium containing Clinacanthus nutans leaf powder has the following components: 20 g / L glucose, 6 g / L peptone, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 0.03 g / L vitamin B, and 2 g / L of the Clinacanthus nutans leaf powder.
[0033] Example 3
[0034] A fermentation medium containing Clinacanthus nutans leaf powder has the following components: 60 g / L glucose, 2 g / L peptone, 2.5 g / L potassium dihydrogen phosphate, 2.5 g / L magnesium sulfate heptahydrate, 0.008 g / L vitamin B, and 8 g / L of the Clinacanthus nutans leaf powder.
[0035] Example 4
[0036] A method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides includes the following steps: The Ganoderma lucidum mycelium was activated for 7 days and then crushed to obtain a 40 g / L Ganoderma lucidum mycelium suspension. The Ganoderma lucidum mycelium suspension was inoculated into the fermentation medium described in Example 1 at an inoculation rate of 2% (v / v) for liquid fermentation culture, and fermented in a shaker at 28°C and 150 rpm for 9 days.
[0037] Example 5
[0038] A method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides includes the following steps: The Ganoderma lucidum mycelium was activated for 7 days and then crushed to obtain a 30 g / L Ganoderma lucidum mycelium suspension. The Ganoderma lucidum mycelium suspension was inoculated into the fermentation medium described in Example 1 at an inoculation rate of 1.5% (v / v) for liquid fermentation culture, and fermented in a shaker at 25°C and 120 rpm for 7 days.
[0039] Example 6
[0040] A method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides includes the following steps: The Ganoderma lucidum mycelium was activated for 7 days and then crushed to obtain a 50 g / L Ganoderma lucidum mycelium suspension. The Ganoderma lucidum mycelium suspension was inoculated into the fermentation medium described in Example 1 at an inoculation rate of 2.5% (v / v) for liquid fermentation culture, and fermented in a shaker at 29°C and 180 rpm for 11 days.
[0041] Example 7
[0042] A method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides includes the following steps: The Ganoderma lucidum mycelium was activated for 7 days and then crushed to obtain a 40 g / L Ganoderma lucidum mycelium suspension. The Ganoderma lucidum mycelium suspension was inoculated into the fermentation medium described in Example 2 at an inoculation rate of 2% (v / v) for liquid fermentation culture, and fermented in a shaker at 28°C and 150 rpm for 9 days.
[0043] Example 8
[0044] A method for increasing the yield and antioxidant activity of Ganoderma lucidum extracellular polysaccharides includes the following steps: The Ganoderma lucidum mycelium was activated for 7 days and then crushed to obtain a 40 g / L Ganoderma lucidum mycelium suspension. The Ganoderma lucidum mycelium suspension was inoculated into the fermentation medium described in Example 3 at an inoculation rate of 2% (v / v) for liquid fermentation culture, and fermented in a shaker at 28°C and 150 rpm for 9 days.
[0045] Experiment Example 1: Preparation of Ganoderma lucidum extracellular polysaccharides
[0046] Freshly harvested leaves of *Clinacanthus nutans* were completely dried in a 50°C oven, then pulverized using a pulverizer. The pulverized leaves were passed through a 100-mesh sieve, and the sieve residue was collected to obtain *Clinacanthus nutans* leaf powder. A fermentation medium was then prepared using this powder, comprising: 40 g / L glucose, 4 g / L peptone, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 0.01 g / L vitamin B, and 6 g / L of the *Clinacanthus nutans* leaf powder. *Ganoderma lucidum* mycelium balls were activated for 7 days, then crushed to obtain a 40 g / L *Ganoderma lucidum* mycelium suspension. This suspension was inoculated into the fermentation medium at a 2% (v / v) inoculation rate for liquid fermentation at 28°C and 150 rpm for 9 days. The fermentation broth was filtered through gauze to remove mycelium, and the filtrate was centrifuged at 12000 g for 15 minutes at 4°C. The supernatant was dialyzed against deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. After concentration, it was freeze-dried to obtain a white flocculent polysaccharide product (experimental group EPS).
[0047] A control group was set up, which was exactly the same as the above treatment except that Clinacanthus nutans powder was not added, and EPS of the control group was obtained. (one)
[0049] The yield of EPS in the experimental group and the control group was measured: After fermentation, the fermentation broth was filtered through gauze to remove mycelium. The mycelium was then washed with distilled water and freeze-dried to constant weight. Biomass was determined by weighing. The fermentation broth after mycelium removal was centrifuged at 12000g for 10 minutes at 4°C to remove insoluble particles. The resulting supernatant was dialyzed thoroughly at 4°C using a dialysis membrane with a molecular weight cutoff of 1000 Da for 24 hours. After dialysis, the sample was freeze-dried for 48 hours to constant weight. The extracellular polysaccharide (EPS) content was determined using the phenol-sulfuric acid method.
[0050] The results show (e.g.) Figure 1 The EPS yield in the experimental group was 3.14 g / L, which was 116.6% higher than that in the control group (1.45 g / L). (two)
[0052] The functional groups of polysaccharides in the experimental group EPS and the control group EPS were analyzed by FT-IR.
[0053] Methods: 2 mg of polysaccharide sample and 200 mg of potassium bromide were accurately weighed and compressed into tablets. A blank control was prepared by compressing potassium bromide powder into tablets. The tablets were scanned and recorded using a Fourier transform infrared spectrometer (FT-IR650, Tianjin Gangdong Technology Development Co., Ltd.), with a scanning range of 4000–400 cm⁻¹. -1 .
[0054] The results are as follows Figure 2 As shown, the EPS infrared spectra of both the experimental group (with 6 g / L Clinacanthus leaf powder) and the control group (without Clinacanthus leaf powder) exhibited typical polysaccharide characteristic absorption peaks: the common broad band was attributed to the O–H stretching vibration (experimental group: 3407 cm⁻¹). - ¹; Control group: 3396 cm - ¹) and C–H stretching vibrations (experimental groups: 2973, 2940, 2890 cm⁻¹) - ¹; Control group: 2935 cm - ¹), confirming that the sample is a polysaccharide.
[0055] However, significant differences exist between the two sets of spectra. The experimental group showed differences at 1741 cm⁻¹. - The additional absorption peak at ¹ is likely attributed to the C=O stretching vibration, indicating the presence of a carboxyl or ester group, while this peak is not prominent in the control group. The C=O stretching vibration peak also appeared at 1648 cm⁻¹ in the experimental group.- ¹ at this location, the control group had a height of 1639 cm. - ¹. Furthermore, the experimental group was at 862 cm⁻¹ - The characteristic peak of the α-configuration of the pyranose ring appears at ¹, while the control group shows a peak at 898 cm⁻¹. - The peak at position ¹ exhibits a typical β-configuration absorption peak. These peak shifts suggest that the addition of *Clinacanthus nutans* leaf powder may have altered the glycosidic bond configuration of EPS or introduced structural modifications. Both groups showed absorption bands related to C–O stretching and O–H bending vibrations, but with slight differences in wavenumbers. In conclusion, the addition of *Clinacanthus nutans* powder to the fermentation medium affects the functional group characteristics of EPS, particularly potentially introducing carbonyl groups and altering the anomeric carbon configuration of the sugar ring, thereby possibly influencing the bioactivity and physicochemical properties of the polysaccharide. (three)
[0057] The monosaccharide composition of EPS in the experimental group and the control group was analyzed by ion chromatography.
[0058] First, 5 mg of each of the 16 monosaccharide standards were placed in ampoules, and 2 mL of 3M TFA was added. The solutions were hydrolyzed at 120°C for 3 hours. The acid-hydrolyzed solution was accurately pipetted into a tube and dried under nitrogen. 5 mL of water was added and vortexed to prepare a standard stock solution. Precise concentrations of each monosaccharide standard solution were then prepared as a mixed standard. The mass of different monosaccharides was determined using an absolute quantification method, and the molar ratio was calculated based on the molar mass of the monosaccharides.
[0059] Accurately weigh 5 mg of lyophilized polysaccharide sample and place it in an ampoule. Add 2 mL of 3M TFA and hydrolyze at 120℃ for 3 h. Accurately pipette the acid-hydrolyzed solution into a tube and dry under nitrogen. Add 5 mL of water and vortex to mix. Add 50 µL of the experimental group polysaccharide solution to 950 µL of deionized water, and add 20 µL of the control group polysaccharide solution to 980 µL of deionized water. Centrifuge at 12000 rpm for 5 min. Use the supernatant for IC analysis. Column: Dionex Carbopac TM PA20 (3 150 mm); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaOAc; Flow rate: 0.3 ml / min; Injection volume: 5 µL; Column temperature: 30℃; Detection was performed using an electrochemical detector.
[0060] The results show (e.g.) Figure 3 The experimental group's EPS consisted of heteropolysaccharides rich in galactose (55.6 mol%), while the control group's EPS consisted of dextran mainly composed of glucose (93.6 mol%). (Four)
[0062] The molecular weight of EPS in the experimental group and the control group was determined and analyzed by high performance gel permeation chromatography.
[0063] Weigh 58.44 g of NaCl, dissolve it in purified water, and transfer it to a 2 L volumetric flask. Sonicate for 10 min, then filter through a 0.22 μm filter membrane to prepare the mobile phase solution. Accurately weigh 5 mg of each standard (dextran), dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, and transfer the sample to a 1.8 mL vial to prepare the standard solution. Accurately weigh 5 mg of the polysaccharide sample, dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, vortex to dissolve, and centrifuge at 12000 rpm for 10 min. Collect the supernatant, filter it through a 0.22 μm aqueous microporous membrane, and then transfer the sample to a 1.8 mL vial to prepare the sample solution.
[0064] Mobile phase: 0.5M NaCl solution; Column: BRT105-103-101 tandem gel column (8×300 mm); Flow rate: 0.7 ml / min; Column temperature: 40℃; Injection volume: 25 μL; Detection was performed using a RID-20A differential detector. Standards were placed in the injection tray and analyzed using the above chromatographic method. Retention times were obtained, and standard curves were plotted for LgMp-RT (Mp peak molecular weight), LgMw-RT (Mw weight-average molecular weight), and LgMn-RT (Mn number-average molecular weight). The molecular weight calculation formula was derived. Polysaccharide sample solutions were placed in the injection tray and analyzed using the above chromatographic method. Chromatograms and retention times were obtained. Substituting the retention times into the formula yielded the molecular weights (Mp, Mw, Mn).
[0065] The results show (e.g.) Figure 4 The EPS in the experimental group showed a bimodal distribution, containing two components: a low molecular weight component of 4.4 kDa and a high molecular weight component of 38.5 kDa; the EPS in the control group was a uniform low molecular weight component of 4.7 kDa. (five)
[0067] DPPH, ·OH, and ABTS levels in the experimental group EPS and the control group EPS + The half-maximal clearance concentration (IC50) 50 Antioxidant activity analysis was performed.
[0068] method: DPPH: Prepare 1 mL of each of the following polysaccharide sample solutions (0.25, 0.5, 1.0, 1.5, 2.0 mg / mL) using deionized water. Place 50 μL of each sample solution into a 96-well plate and add 150 μL of 0.05 mmol / L DPPH solution (prepared with methanol). Incubate at room temperature, in the dark, and mix for 30 min. Measure the absorbance (As) at 517 nm. For the sample control group, methanol was used instead of the DPPH solution, and the absorbance (Aj) was measured. For the blank group, deionized water was used instead of the sample solution, and the absorbance (A0) was measured. Vitamin C was used as a positive control. DPPH free radical scavenging rate (%) = [(A0 - (As - Aj)) / A0] × 100%.
[0069] • OH: Prepare 1 ml of each of the polysaccharide sample solutions (0.25, 0.5, 1.0, 1.5, 2.0 mg / ml) using deionized water. Add 2 mL of 9 mmol / L ferrous sulfate and 2 mL of 9 mmol / L hydrogen peroxide, mix thoroughly, and react at room temperature for 10 min. Then add 2 ml of 9 mmol / L salicylic acid and react at room temperature for 10 min. Measure the absorbance (As) at 510 nm. For the sample control group, use deionized water instead of hydrogen peroxide and measure the absorbance (Aj). For the blank group, use deionized water instead of the sample solution and measure the absorbance (A0). Use vitamin C as a positive control. • OH free radical scavenging rate (%) = [(A0 - (As - Aj)) / A0] × 100%.
[0070] ABTS + Prepare 1 ml of each of the following polysaccharide sample solutions (0.25, 0.5, 1.0, 1.5, and 2.0 mg / mL) using deionized water. Prepare an equal mixture of 7 mmol / L ABTS and 1.4 mmol / L potassium persulfate, and incubate overnight at room temperature in the dark. Before use, dilute with distilled water to a absorbance of 0.7 ± 0.02 at 734 nm. Mix 100 μL of the polysaccharide solution and 100 μL of the ABTS solution and react at room temperature for 5 min. Measure the absorbance (As) at 734 nm. For the sample control group, use distilled water instead of the ABTS solution and measure the absorbance (Aj). For the blank group, use deionized water instead of the sample solution and measure the absorbance (A0). Vitamin C was used as a positive control. ABTS + Free radical scavenging rate (%) = [(A0-(As-Aj)) / A0] × 100%.
[0071] The results show (e.g.) Figure 5 ), DPPH, ·OH, ABTS of EPS in the experimental group + The half-maximal clearance concentration (IC50) 50 The levels of all samples were significantly lower than those in the control group, indicating that their antioxidant capacity was comprehensively enhanced.
[0072] In summary, this invention, by adding a specific concentration of Clinacanthus nutans leaf powder to the Ganoderma lucidum liquid fermentation medium, synergistically increased the yield of extracellular polysaccharides and altered their chemical structure. The structural changes also significantly enhanced the antioxidant activity of the product.
[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. The use of a powder of leaves of Gynostemma pentaphyllum in the preparation of a fermentation medium for increasing the yield of Ganoderma lucidum extracellular polysaccharides and antioxidant activity, characterized in that, The concentration of the leaf powder of the plant is 2-10 g / L.
2. Use according to claim 1, characterized in that, The preparation method of the leaf powder of the plant comprises the following steps: drying and crushing the picked leaves, sieving through a 80-120 mesh screen, and collecting the undersize, thereby obtaining the leaf powder.
3. Use according to claim 1, characterized in that, The drying temperature is 45-55℃.
4. A fermentation medium comprising the powder of leaves of Desfontainia spinosa according to any one of claims 1 to 3, characterized in that, The components include the following concentrations: glucose 20-60 g / L, peptone 2-6 g / L, potassium dihydrogen phosphate 1.0-3.0 g / L, magnesium sulfate heptahydrate 1.0-3.0 g / L, vitamin B 0.005-0.05 g / L, and the leaf powder of the plant according to any one of claims 1-3 2-10 g / L.
5. The fermentation medium of claim 4, wherein, The components include the following concentrations: glucose 30-50 g / L, peptone 3-5 g / L, potassium dihydrogen phosphate 1.2-2.5 g / L, magnesium sulfate heptahydrate 1.2-2.5 g / L, vitamin B 0.008-0.03 g / L, and the leaf powder of the plant according to any one of claims 1-3 4-8 g / L.
6. A method for improving the yield and antioxidant activity of Ganoderma lucidum exopolysaccharide, characterized in that, The method comprises the following steps: The Ganoderma lucidum suspension is inoculated into the fermentation medium according to claim 4 for liquid fermentation culture.
7. The method of claim 6, wherein, The inoculation amount of the Ganoderma lucidum suspension is 1.5-2.5%, and the concentration of the Ganoderma lucidum suspension is 10-100 g / L.
8. The Ganoderma lucidum exopolysaccharide obtained by the method of claim 6 or 7, characterized in that, The monosaccharide composition of the Ganoderma lucidum exopolysaccharide mainly contains galactose and also contains uronic acid.
9. Ganoderma lucidum exopolysaccharide according to claim 8, characterized in that, The molecular weight distribution of the Ganoderma lucidum exopolysaccharide is bimodal or multimodal, and contains high molecular weight components with a molecular weight greater than 10 kDa.