Ganoderma sinensis fermented mycelium extract and preparation method thereof

Abstract

The invention provides a preparation method of a ganoderma sinensis fermented mycelium extract. The method comprises the following steps: (1) activating a strain; (2) primary culture; (3) secondary seed culture; (4) mycelium pretreatment; (5) dynamically extracting with hot water; and (6) heating, concentrating and freeze-drying to obtain the required ganoderma sinensis fermented mycelium extract. According to the preparation method of the ganoderma sinensis fermented mycelium extract, the fermentation process is stable, the preparation method is simple and convenient, the cost is low, and the method has the advantage of industrial production. The Ganoderma sinensis fermented mycelium extract prepared by the invention can provide a raw material source with remarkable functions for cosmetics, health care products and food industries.

Description

Technical Field

[0001] The invention belongs to the field of extraction and preparation of edible and medicinal fungi, and specifically relates to a Ganoderma lucidum fermentation mycelium extract and a preparation method thereof. Background Art

[0002] Ganoderma spp. is a collective term for fungi belonging to the phylum Basidiomycota, class Agaricomycetes, orders Polyporales, family Ganodermataceae, and genus Ganoderma P. Karst. It is a well-known medicinal fungus in my country and East Asia, with a folk medicinal history of over 2,000 years. Ganoderma sinense, Ganoderma lucidum, and Ganoderma tsugae are among the more famous species of Ganoderma with a long history of application and development. Modern research confirms that polysaccharides and triterpenes are the primary active substances in Ganoderma, exhibiting numerous physiological functions, including immune-enhancing, anti-tumor, anti-inflammatory, and hepatoprotective properties. Ganoderma extracts rich in polysaccharides and triterpenes are used in the development of pharmaceuticals, health foods, and functional foods. In recent years, Ganoderma extracts have also begun to be used as functional ingredients in cosmetics.

[0003] Ganoderma mycelium is a dikaryotic hyphae formed by plasmogamy after the germination of basidiospores. Fermentation of Ganoderma mycelium is typically carried out under controlled conditions, including culture medium formulation, temperature, lighting, and rotational speed. This allows for scalable production and produces consistently high-quality mycelium. Furthermore, Ganoderma mycelium obtained through specific fermentation conditions metabolizes to produce active products such as triterpenes, polysaccharides, and proteins with specific structures and exhibits excellent biological activity.

[0004] Given the particularity and convenience of Ganoderma lucidum fermentation, searching for components with antioxidant functions from the fermented mycelium of different Ganoderma lucidum and optimizing the preparation process for use in product development in cosmetics, food, health products, medicines, etc. will have broad application prospects. Summary of the Invention

[0005] The present invention aims to provide a method for preparing a Ganoderma lucidum fermentation mycelium extract, comprising the following steps:

[0006] (1) Activation of strains: Inoculate the Ganoderma lucidum strain on potato dextrose agar (PDA) medium, and when the mycelium is about to fill the plate, start taking the mycelium for the next step of cultivation;

[0007] (2) Primary seed culture: Ganoderma lucidum mycelium was inoculated into the primary culture medium and cultured with shaking;

[0008] (3) Secondary seed culture: transfer the primary seed solution into the secondary culture medium, first shake culture, then static culture;

[0009] (4) Mycelium pretreatment: collect the upper mycelium, dry it, extract it with anhydrous ethanol, remove the ethanol and dry it naturally;

[0010] (5) Hot water dynamic extraction: Mix the mycelium obtained in (4) with deionized water, boil, heat, and stir to extract, and filter to obtain the filtrate;

[0011] (6) concentrating the filtrate obtained in (5) and freeze-drying to obtain the desired Ganoderma lucidum fermentation mycelium extract;

[0012] Furthermore, the strain in step (1) is Ganoderma sinense.

[0013] Furthermore, the formula of the primary culture medium in step (2) is: 3% soluble starch, 1.2% glucose, 1% yeast powder, 0.15% MgSO4·7H2O, 0.3% KH2PO4, pH 5-7 (all weight percentages); the inoculation method is: take 5-10 pieces of Ganoderma lucidum mycelium with a diameter of 0.5 cm and place them in the primary culture medium; the culture conditions are: 25-30°C, 120-150r / min, and shaking culture in the dark for 7-10 days.

[0014] Furthermore, the formula of the secondary culture medium in step (3) is the same as the formula of the primary culture medium; the inoculation method is: take the primary culture medium containing the Ganoderma lucidum mycelium, and pour it into the secondary culture medium at an inoculation ratio of 10%-15% (volume percentage); the culture conditions are: 25-30°C, 120-150r / min, shaking culture under dark conditions for 3-5d, and then transferred to static culture for 12-21d.

[0015] Furthermore, the mycelium pretreatment in step (4) is specifically carried out as follows: taking the upper mycelium of the fermented Ganoderma lucidum in step (3), drying it at 40-50° C., adding anhydrous ethanol at a material-liquid ratio of 1:10 to 1:30 (mass-to-volume ratio g / ml), ultrasonically extracting it for 1 h-2 h, repeating the extraction twice, filtering to remove the ethanol, and naturally drying the remaining raw material.

[0016] Furthermore, the solid-liquid ratio of the Ganoderma lucidum fermentation mycelium and deionized water in step (5) is 1:30 to 1:40 (mass-to-volume ratio g / ml), the temperature of the heating and stirring extraction is 75°C to 100°C, the stirring speed is 30 to 60 rpm, and the extraction time is 1 to 2.5 h.

[0017] Furthermore, the extract of the fermented mycelium of Ganoderma lucidum described in step (6) is filtered to remove the medicinal residue, and is concentrated by heating under reduced pressure so that the concentrated solution has a volume of 1 / 20 to 1 / 10 of the original extract.

[0018] Furthermore, the drying of the Ganoderma lucidum fermentation mycelium extract concentrate in step (6) is carried out by vacuum freeze drying.

[0019] The present invention also provides the Ganoderma lucidum fermentation mycelium extract prepared by the above method.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The preparation method of the present invention adopts a process of first pre-treating with ethanol and then decocting and extracting with deionized water to extract the fermented mycelium of Ganoderma lucidum, so as to obtain the Ganoderma lucidum fermented mycelium extract. The polysaccharide content of the extract is 10-15% (W / W), and the extract has good comprehensive DPPH free radical scavenging ability, ABTS free radical scavenging ability, hydroxyl free radical scavenging ability, total reducing power and in vitro tyrosinase inhibition ability, reflecting potential excellent antioxidant activity application value. The preparation method is simple to operate and low in cost, can be widely used in the fields of cosmetics, food, medicine, etc., and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The DPPH free radical scavenging ability of Ganoderma lucidum fermented mycelium extract;

[0023] Figure 2 The ABTS free radical scavenging ability of Ganoderma lucidum fermented mycelium extract;

[0024] Figure 3 The scavenging ability of Ganoderma lucidum fermented mycelium extract on hydroxyl radicals;

[0025] Figure 4 Total reducing power scavenging capacity of Ganoderma lucidum fermented mycelium extract;

[0026] Figure 5 Inhibitory ability of Ganoderma lucidum fermented mycelium extract on tyrosinase activity. Specific implementation methods

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Ganoderma sinense strain: Institute of Microbiology, College of Ecology and Nature Conservation, Beijing Forestry University (strain collection number: Dai20076).

[0031] Potato agar (PDA) with glucose: purchased from Beijing Solebow Technology Co., Ltd.

[0032] Soluble starch, glucose, yeast powder, MgSO4·7H2O, KH2PO4 and food-grade ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0033] Reagents: 1,1-diphenyl-2-picrylhydrazyl (DPPH), analytical grade, Jiangsu Anhuai Chemical Technology Co., Ltd.

[0034] 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt, ABTS) and bovine serum albumin (analytical grade) were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0035] Glucose, phenol, concentrated sulfuric acid, ferrous sulfate, potassium ferrocyanide, and anhydrous ethanol were all analytically pure and were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0036] Potassium persulfate, sodium dihydrogen phosphate, and disodium hydrogen phosphate were of analytical grade and were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0037] Ferric chloride and trichloroacetic acid, analytical grade, were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0038] Coomassie Brilliant Blue G-250, analytical grade, Shanghai Jinsui Biotechnology Co., Ltd.;

[0039] Salicylic acid, hydrogen peroxide (H2O2), and phosphoric acid were of analytical grade and purchased from Shanghai Titan Technology Co., Ltd.

[0040] Tyrosinase, Nanjing Dulai Biotechnology Co., Ltd.;

[0041] Levodopa (L-DOPA), analytically pure, obtained from Shenggong (Bioengineering) Co., Ltd.

[0042] ZWY-2102 Constant Temperature Oscillator, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.

[0043] LRH250 biochemical incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.

[0044] UV1901PC UV spectrophotometer, Shanghai Aoxi Scientific Instrument Co., Ltd.

[0045] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0046] Example 1 Ganoderma lucidum mycelium fermentation

[0047] Inoculate the Ganoderma lucidum strain on a glucose potato agar (PDA) medium, and when the mycelium is about to fill the plate, start taking out the mycelium for the next step of culture;

[0048] Take 5-10 pieces of Ganoderma mycelium, each 0.5 cm in diameter, and place them in a primary culture medium. The primary culture medium contains: 3% soluble starch, 1.2% glucose, 1% yeast extract, 0.15% MgSO4·7H2O, 0.3% KH2PO4, and a pH of 5-7 (all percentages by weight). Culture conditions: 25°C, 150 rpm, and shaking in the dark for 7 days.

[0049] Take the primary culture medium containing Ganoderma lucidum mycelium and pour it into the secondary culture medium at a volume percentage inoculation ratio of 10%. The formula of the secondary culture medium is the same as that of the primary culture medium. Shake and culture for 5 days under dark conditions at 25°C, 150r / min, and then transfer to static culture for 21 days.

[0050] Example 2 Extraction of Ganoderma lucidum fermentation mycelium

[0051] The upper mycelium of Ganoderma lucidum fermentation was taken, dried at 50°C, and anhydrous ethanol was added at a solid-liquid ratio of 1:30 (g / ml). Ultrasonic extraction was performed for 2 hours, and the extraction was repeated twice. The ethanol was filtered out, and the remaining mycelium was dried naturally.

[0052] Take the naturally dried Ganoderma lucidum fermentation mycelium, add deionized water, the material-liquid ratio of the raw material to deionized water is 1:30 (g / ml), heat and stir to extract, the temperature is 100℃, the stirring speed is 50rpm, and the extraction time is 2.5h. The extract is filtered to remove the medicinal residue, and the remaining material liquid is concentrated by heating under reduced pressure to make the concentrate 1 / 20 of the original extract. The obtained Ganoderma lucidum fermentation mycelium extract concentrate is frozen at -20℃ overnight and vacuum freeze-dried to obtain the desired Ganoderma lucidum fermentation mycelium extract. The calculation formula for the extract yield is as follows:

[0053] Extract yield (%) = extract weight / extraction raw material weight × 100% (1)

[0054] Example 3 Determination of polysaccharide content in the extract of Ganoderma lucidum fermented mycelium

[0055] Prepare a 5% phenol aqueous solution (weight percentage), accurately measure 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of a 100 μg / mL glucose standard solution (as a reference solution) and place them in a 25 mL colorimetric tube, then add deionized water to 1 mL, add 1 mL of 5% phenol solution, mix thoroughly, and then slowly add 5 mL of concentrated sulfuric acid along the inner wall. Shake quickly and thoroughly, and place at room temperature for 30 minutes. Use deionized water as a blank group, measure the absorbance of each group of solutions at 490 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0056] Take the Ganoderma lucidum fermentation mycelium extract prepared in Example 2 and prepare a 1 mg / mL test solution with deionized water (1 mg of the Ganoderma lucidum fermentation mycelium extract prepared in Example 2 and 1 mL of deionized water). Pipette 0.2 mL of the Ganoderma lucidum fermentation mycelium extract test solution, add deionized water to 1 mL, place in a 25 mL colorimetric tube, add 1 mL of 5% phenol solution, mix thoroughly, then slowly add 5 mL of concentrated sulfuric acid along the inner wall, shake quickly and thoroughly, and place at room temperature for 30 minutes. Then measure the absorbance, read the glucose content in the test solution from the standard curve, and calculate the polysaccharide content.

[0057] Example 4 Determination of protein content of Ganoderma lucidum fermentation mycelium extract

[0058] Prepare Coomassie Brilliant Blue G-250 solution: Accurately weigh 100 mg of Coomassie Brilliant Blue G-250 and dissolve it in 50 mL of 95% ethanol (by weight). Add 100 mL of 85% phosphoric acid (by weight), and dilute to 1000 mL with deionized water for later use.

[0059] Accurately pipette 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 100 μg / mL (weight percentage) bovine serum albumin standard solution (as a reference solution) into a test tube and place them in a 25 mL colorimetric tube. Add deionized water to 1.0 mL, then add 5 mL of Coomassie Brilliant Blue G-250 solution, shake well, and react at room temperature for 3 minutes. Measure the absorbance of each group of solutions at a wavelength of 595 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0060] Take the Ganoderma lucidum fermentation mycelium extract prepared in Example 2 and prepare it into a 1 mg / mL test solution with deionized water (1 mg of the Ganoderma lucidum fermentation mycelium extract prepared in Example 2 and 1 mL of deionized water). Pipette 0.2 mL of the Ganoderma lucidum fermentation mycelium extract test solution, add deionized water to 1 mL, place it in a 25 mL colorimetric tube, add 5.0 mL of Coomassie Brilliant Blue G-250 solution, mix thoroughly, react at room temperature for 3 minutes, measure the absorbance, read the bovine serum albumin content in the test solution from the standard curve, and calculate the protein content.

[0061] The yield, polysaccharide content and protein content of the Ganoderma lucidum fermentation mycelium extract obtained after measurement are shown in Table 1 below:

[0062] Table 1 Yield and polysaccharide content of Ganoderma lucidum fermentation mycelium extract

[0063]

[0064] Example 5 Determination of DPPH free radical scavenging ability of Ganoderma lucidum fermented mycelium extract

[0065] Accurately weigh 20 mg of DPPH, dissolve it with anhydrous ethanol, and dilute it to a volume in a 250 mL brown volumetric flask to obtain a DPPH solution with a concentration of 0.2 mmol / L. Store it in a refrigerator at 4°C. Add water to the Ganoderma lucidum fermentation mycelium extract prepared in Example 2 to prepare sample solutions with a mass concentration of 0.2 to 1.0 mg / mL. Take 2 mL of sample solutions of different concentrations and mix them with 2 mL of 0.2 mmol / L DPPH-ethanol solution. After standing in the dark for 30 minutes, measure the absorbance of the mixed solution at a wavelength of 517 nm using an ultraviolet spectrophotometer and adjust the value to zero with deionized water. The DPPH free radical scavenging rate of the Ganoderma lucidum fermentation mycelium extract can be calculated according to formula (2). Repeat the experiment three times and take the average value.

[0066]

[0067] Where: A i A is the absorbance of 2 mL of water extract sample solution mixed with 2 mL of DPPH solution; j is the absorbance of an equal volume of anhydrous ethanol instead of DPPH solution; A0 is the absorbance of an equal volume of deionized water instead of Ganoderma lucidum fermentation mycelium extract solution.

[0068] See the results Figure 1 It can be seen that with the increase of concentration, the scavenging ability of Ganoderma lucidum fermentation mycelium extract on DPPH gradually increased.

[0069] Example 6 Determination of ABTS free radical scavenging ability of Ganoderma lucidum fermented mycelium extract

[0070] Prepare 7.4 mmol / L ABTS stock solution and 2.6 mmol / L potassium persulfate stock solution, respectively. Mix the ABTS stock solution and potassium persulfate stock solution in a volume ratio of 1:1, let stand in the dark for 12 to 16 hours, and then dilute with anhydrous ethanol until the absorbance at 734 nm is in the range of 0.70 ± 0.02. This is the ABTS working solution. Take 1 mL of each 0.2 to 1.0 mg / mL aqueous solution of Ganoderma lucidum fermentation mycelium extract (prepared in Example 2) in a test tube, add 6 mL of ABTS working solution, mix well, let stand at room temperature for 6 minutes, and measure the absorbance at 734 nm using an ultraviolet spectrophotometer. Deionized water is used as a blank control. Calculate the ABTS cation radical scavenging rate according to formula (3). Set up three parallel tests and take the average value.

[0071]

[0072] Where: A i A is the absorbance of 1 mL of water extract sample solution mixed with 6 mL of ABTS working solution; j is the absorbance of an equal volume of deionized water instead of the ABTS working solution; A0 is the absorbance of an equal volume of deionized water instead of the aqueous solution of Ganoderma lucidum fermentation mycelium extract.

[0073] See attached for the results Figure 2 It can be seen that with the increase of the concentration of Ganoderma lucidum fermentation mycelium extract, its ABTS scavenging ability gradually increased.

[0074] Example 7 Determination of the Hydroxyl Radical Scavenging Ability of Ganoderma Lucidum Fermented Mycelium Extract

[0075] 1 mL of 6 mmol / L H2O2 solution, 1 mL of 0.2-1.0 mg / mL aqueous solution of Ganoderma lucidum fermented mycelium extract (prepared in Example 2), 1 mL of 6 mmol / L ferrous sulfate aqueous solution, and 1 mL of 6 mmol / L salicylic acid ethanol solution were added to a test tube in sequence, mixed evenly, and allowed to stand in the dark for 30 min. The absorbance was measured at a wavelength of 510 nm. Three parallel experiments were performed, and the clearance rate was calculated according to formula (4):

[0076]

[0077] Where: A i Indicates the absorbance of the sample solution after adding water extract; A j A0 represents the absorbance of an equal volume of anhydrous ethanol instead of salicylic acid ethanol solution; A0 represents the absorbance of an equal volume of deionized water instead of the sample solution.

[0078] See the results Figure 3 It can be seen that with the increase of concentration, the ability of Ganoderma lucidum fermentation mycelium extract to scavenging hydroxyl free radicals gradually increases.

[0079] Example 8 Determination of the total reducing power of the Ganoderma lucidum fermentation mycelium extract

[0080] Take 1 mL of each aqueous solution of Ganoderma lucidum fermentation mycelium extract (prepared as in Example 2) with a mass concentration of 0.2-1.0 mg / mL, add 2.5 mL of 0.2 mol / L pH 6.6 phosphate buffer and 1.5 mL of 1% potassium ferricyanide (weight percentage), and mix well. Then transfer to a 50°C water bath to react for 20 minutes, quickly cool to room temperature, add 2 mL of 10% trichloroacetic acid (weight percentage) solution dropwise, mix thoroughly, centrifuge at 3800 r / min for 10 minutes, take 2 mL of the supernatant respectively and place in a new test tube, add 2 mL of deionized water and 0.5 mL of 0.1% ferric chloride solution (weight percentage), shake well, stand in the dark for 10 minutes, and measure the absorbance at a wavelength of 700 nm. Repeat the experiment three times.

[0081] See the results Figure 4 It can be seen that with the increase of concentration, the total reducing power of Ganoderma lucidum fermented mycelium extract gradually increases.

[0082] Example 8 Determination of Tyrosinase Inhibitory Ability of Ganoderma Lucidum Fermented Mycelium Extract

[0083] 1.5mmol / L L-DOPA was used as the substrate, and PBS (pH=6.6, 0.03mmol / L) and aqueous solution of Ganoderma lucidum fermentation mycelium extract (as the test solution, with concentrations of 2, 4, and 10mg / mL, respectively) (prepared in Example 2) were added, and the mixture was placed in a 28°C constant temperature box for reaction for 10 minutes. After 10 minutes, tyrosinase aqueous solution (enzyme activity 70U / mL) was added and the reaction was continued for 15 minutes. After 15 minutes, the absorbance at a wavelength of 475nm was measured on a microplate reader, and the tyrosinase inhibition rate was calculated according to formula (4). The experiment was repeated three times and the average value was taken. The experimental system is shown in Table 2, A is blank zero adjustment, B is used for blank control, C is sample zero adjustment, and D is used for sample group. The experimental results are shown in Appendix. Figure 5 When the concentration of the aqueous solution of Ganoderma lucidum fermentation mycelium extract was 10 mg / mL, its inhibition rate on tyrosinase exceeded 20%.

[0084]

[0085] Table 2 Reaction system of tyrosinase inhibition experiment

[0086]

Claims

1. A method for preparing a Ganoderma lucidum fermented mycelium extract, characterized in that The steps include: (1) Activation of strains: Inoculate the Ganoderma lucidum strain on potato dextrose agar medium, and when the mycelium is about to fill the plate, remove the mycelium for the next step of cultivation; (2) Primary seed culture: Ganoderma lucidum mycelium is inoculated into primary culture medium and cultured by shaking; (3) Secondary seed culture: transfer the primary seed solution into the secondary culture medium, first shake culture, then static culture; (4) Mycelium pretreatment: Take the upper mycelium of the fermented Ganoderma lucidum in step (3) and dry it at 40-50° C., add anhydrous ethanol at a material-liquid ratio of 1:10 to 1:30 and a mass-to-volume ratio of g / ml, perform ultrasonic extraction for 1 h to 2 h, filter to remove the ethanol, and dry the remaining raw material naturally; (5) Hot water dynamic extraction: the mycelium obtained in (4) is mixed with deionized water, boiled, heated, stirred and extracted, and the filtrate is filtered and collected; (6) heating and concentrating the filtrate obtained in (5) to make the volume of the concentrate 1 / 20 to 1 / 10 of the original extract; and then freeze-drying to obtain the desired Ganoderma lucidum fermented mycelium extract; The formula of the primary culture medium in step (2) is: 3% soluble starch, 1.2% glucose, 1% yeast powder, 0.15% MgSO4·7H2O, 0.3% KH2PO4, and pH value 5-7; the inoculation method is: take 5-10 pieces of Ganoderma lucidum mycelium with a diameter of 0.5 cm and place them in the primary culture medium; the culture conditions are: 25-30°C, 120-150r / min, and shake culture in the dark for 7-10 days; The formula of the secondary culture medium in step (3) is the same as that of the primary culture medium; the inoculation method is: take the primary culture medium containing Ganoderma lucidum mycelium, and pour it into the secondary culture medium at an inoculation ratio of 10%-15% by volume; the culture conditions are: 25-30°C, 120-150r / min, shaking culture under dark conditions for 3-5d, and then transferred to static culture for 12-21d; The solid-liquid ratio of the Ganoderma lucidum fermented mycelium and deionized water in step (5) is 1:30-1:40, the mass-to-volume ratio is g / ml, the temperature of the heating and stirring extraction is 75°C-100°C, the stirring speed is 30-60rpm, and the extraction time is 1-2.5h.

2. The Ganoderma lucidum fermented mycelium extract prepared by the method according to claim 1.

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