Ganoderma lucidum polypeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to ganoderma lucidum polypeptide and a preparation method and application thereof. The preparation method comprises the following steps: (1) taking ganoderma lucidum powder, adding water, and cooking to obtain a mixture 1; (2) adding bacillus subtilis liquid into the mixture 1, and fermenting to obtain ganoderma lucidum polypeptide fermentation liquid bacteria liquid; (3) adding neutral protease into the ganoderma lucidum polypeptide fermentation liquid for primary enzymolysis, and then adding aminopeptidase for secondary enzymolysis to obtain ganoderma lucidum polypeptide enzymatic hydrolysate; and (4) filtering, concentrating and spray-drying the lucid ganoderma polypeptide enzymatic hydrolysate to obtain the lucid ganoderma polypeptide. The molecular weight of the ganoderma lucidum polypeptide ranges from 200 Da to 5000 Da, molecular weight distribution is uniform, the polypeptide content can reach 31.11% at most, the ganoderma lucidum polypeptide can be applied to functional cosmetics, and the application range of the ganoderma lucidum polypeptide is widened.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological fermentation, and particularly relates to a Ganoderma lucidum polypeptide, a preparation method and application thereof. BACKGROUND

[0002] Ganoderma lucidum belongs to the kingdom of fungi and is a precious edible and medicinal fungus. Its medicinal value is recorded in detail in the ancient Chinese book Shennong Bencao Jing. There are six types of Ganoderma lucidum, and its growth is affected by the climate. The most suitable temperature for growth is 25-28℃. Ganoderma lucidum is rich in active ingredients such as polysaccharides, triterpenes, sterols, adenosine and amino acids, and has biological activities such as anti-tumor, anti-aging, hypoglycemic and hypolipidemic, and immune regulation. The commonly referred Ganoderma lucidum refers to its fruiting body, which is characterized by cork structure and handle shape. The application of Ganoderma lucidum extract in cosmetics can scavenge free radicals, delay aging, whiten, fade color spots, moisturize, anti-inflammatory, anti-allergic, and improve the cuticle layer of the skin, promote skin renewal, etc. Therefore, the appropriate addition of Ganoderma lucidum extract is gradually becoming a new trend in the research and development of cosmetics.

[0003] Chinese invention patent application CN119039389A discloses a Ganoderma lucidum oligopeptide with anti-tumor efficacy and a preparation method thereof. The present application takes Ganoderma lucidum as the natural source of innovative drugs, and successfully separates a heptapeptide with anti-tumor efficacy through crude extraction, enzymatic hydrolysis and ultrafiltration of the fermentation broth of Ganoderma lucidum. The polypeptide has good killing effect on human HCT116 cells, can effectively prevent and treat colon cancer, and has broad application prospects.

[0004] Chinese invention patent application CN118638891A discloses a Ganoderma lucidum oligopeptide with anti-tumor efficacy and a preparation method thereof. The present application first separates and purifies three oligopeptides from Ganoderma lucidum. The oligopeptides have broad-spectrum anti-tumor activity, can effectively inhibit tumor cell proliferation, have good biological compatibility, and have great application prospects.

[0005] Chinese invention patent application CN1634537A discloses a Ganoderma lucidum extract, a preparation method and use thereof. The present application reports that the Ganoderma lucidum extract is obtained by using Ganoderma lucidum broken spore powder as raw material, degreasing with petroleum ether, extracting with ethanol reflux, then extracting with chloroform and ethyl acetate, and finally recovering and drying under reduced pressure to obtain a powder-like Ganoderma lucidum extract. Although the preparation method can obtain a Ganoderma lucidum extract with excellent solubility, the steps are complicated, harmful solvents are introduced in large quantities, the cost is high, and it is difficult to produce on a large scale.

[0006] Chinese invention patent application CN106924110A discloses a preparation method and application method of Ganoderma lucidum extract. Water, ethanol or butanediol is used as an extraction solvent, and then a Ganoderma lucidum extract is obtained by coarse filtration. The method is relatively simple, but the extract is only filtered simply, resulting in a large amount of insoluble substances in the prepared extract.

[0007] The existing preparation method of ganoderma lucidum extract has many defects, and is not suitable for the extraction of polypeptide. Therefore, it is urgent to develop a method for rapidly and large-scale preparation of ganoderma lucidum peptide extract which can be widely used in the research and development of various ordinary foods, health foods and cosmetics. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a ganoderma lucidum polypeptide and a preparation method and application thereof. The present application uses multiple proteases for enzymolysis to prepare the ganoderma lucidum polypeptide by screening the fermentation mode and the protease, and the enzymolysis efficiency is high, the obtained ganoderma lucidum polypeptide has high content, and has the effects of antioxidant, anti-aging, anti-inflammatory soothing, autophagy and self-healing, and can be used as a functional cosmetic raw material.

[0009] In order to achieve the above-mentioned purposes of the present application, the specific technical scheme adopted by the present application is as follows: A preparation method of a ganoderma lucidum polypeptide, comprising the following steps: (1) taking ganoderma lucidum powder, cooking with water, cooling to obtain a mixture 1; (2) adding bacillus subtilis liquid to the mixture 1, and fermenting to obtain a ganoderma lucidum polypeptide fermentation broth; (3) adding neutral protease to the ganoderma lucidum polypeptide fermentation broth for primary enzymolysis, and then adding amino peptidase for secondary enzymolysis to obtain a ganoderma lucidum polypeptide enzymolysis liquid; (4) filtering the ganoderma lucidum polypeptide enzymolysis liquid, concentrating, and spray drying to obtain a ganoderma lucidum polypeptide.

[0010] Preferably, in step (1), the mass ratio of the ganoderma lucidum powder to water is 1:30-50, the temperature of the cooking is 80-100℃, and the time of the cooking is 1.5-2.5h, and the cooling is to 40-55℃.

[0011] Preferably, in step (2), the adding amount of the bacillus subtilis liquid is 8%-12% of the volume of the mixture 1, and the preparation method of the bacillus subtilis liquid comprises: adding bacillus subtilis powder, proteose peptone, anhydrous glucose and yeast powder into water and mixing to obtain the bacillus subtilis liquid.

[0012] Further preferably, the mass ratio of the bacillus subtilis powder, the proteose peptone, the anhydrous glucose, the yeast powder and the water is 2-4:12-18:8-12:8-12:1000, and the fermentation condition is aerobic fermentation, the pH is 6.5-7.5, and the fermentation time is 2.5-3.5 days.

[0013] Still more preferably, the preparation method of the bacillus subtilis liquid comprises: taking 3g of bacillus subtilis powder, 15g of proteose peptone, 10g of anhydrous glucose and 10g of yeast powder, and adding them into 1L of pure water to obtain the bacillus subtilis liquid.

[0014] Preferably, the amount of the neutral protease added in step (3) is 0.8%-2% of the mass of the Ganoderma lucidum powder, the temperature of the first enzymolysis is 43-52℃, the pH of the first enzymolysis is 6.8-7.6, and the time of the first enzymolysis is 1.5-2.5h.

[0015] Preferably, the amount of the aminopeptidase added in step (3) is 0.8%-2% of the mass of the Ganoderma lucidum powder, the temperature of the second enzymolysis is 55-65℃, the pH of the second enzymolysis is 8.0-9.0, and the time of the second enzymolysis is 1.5-2.5h.

[0016] Preferably, the filtration in step (4) comprises: first filtering with a 0.4-0.5μm filter plate, then ultrafiltrating with a 4000-6000Da membrane core, and finally nanofiltrating with a 150-250Da membrane core.

[0017] Preferably, the temperature of the concentration in step (4) is 55-65℃, the concentration is to 20%-25% of the volume of the filtrate, the conditions of the spray drying are: the inlet air temperature is 140-160℃, the outlet air temperature is 80-90℃, the flow rate is 180-220mL / h, and 0.8%-1.2% of hydroxypropyl methyl cellulose by mass of the concentrated solution is added before the spray drying.

[0018] The present application also relates to the Ganoderma lucidum polypeptide prepared by the preparation method.

[0019] The present application also relates to the application of the Ganoderma lucidum polypeptide prepared by the preparation method in cosmetics.

[0020] Compared with the prior art, the present application has the following beneficial effects: (1) The present application uses Ganoderma lucidum as the main raw material, performs liquid fermentation, and performs enzymolysis on the Ganoderma lucidum polypeptide fermentation liquor after the fermentation, so that the specific neutral protease and aminopeptidase are used to perform double-enzymolysis method enzymolysis, and the macromolecular protein can be selectively cut to obtain polypeptides with smaller molecular weight.

[0021] (2) The Ganoderma lucidum polypeptide prepared by the present application has a molecular weight of 200-5000 Da, a uniform molecular weight distribution, and a polypeptide content of up to 31.11%, which can be applied to functional cosmetics, improves the application range of the Ganoderma lucidum polypeptide, and is helpful for the development and utilization of Ganoderma lucidum resources. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the standard curve diagram of the bovine serum albumin in test example 1 of the present application. DETAILED DESCRIPTION

[0023] The application will be further described in detail below with specific examples. The following examples are not intended to limit the application, but only to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] Zilingzhi meets the standard requirements of the People's Republic of China Pharmacopoeia (2020 edition) and is purchased from Wupingshengda Agricultural Development Co., Ltd.; hydroxypropyl methylcellulose, type E5mpa.s, is purchased from Shandong Liaocheng Ahua Pharmaceutical Co., Ltd.; Bacillus subtilis powder, batch number KC20250609, execution standard NY / T 2131, is purchased from Henan Xinyangshao Biological Technology Co., Ltd.

[0025] Alkaline protease (product number: B6360C), neutral protease (product number: NM6362C), acid protease (product number: P6361C), papain (product number: G6430C), flavor protease (product number: FM0125C), and complex protease (M0127C) are purchased from Beijing Boaotuo Technology Co., Ltd. Aminopeptidase (execution standard: GB1886174) is purchased from Cangzhou Xiasheng Enzyme Biological Technology Co., Ltd.

[0026] Preparation of Bacillus subtilis seed liquid: liquid medium configuration: 15 g of proteose peptone, 10 g of anhydrous glucose, and 10 g of yeast powder are added to 1 L of water, and sterilized at 121℃ for 20 min. A good colony is selected on the flat medium with an inoculation loop, inoculated into the liquid medium, and fermented until the OD600 is 1.0. When the OD600 is 1.0, the liquid is inoculated into a new culture medium at a ratio of 2%.

[0027] Bacillus subtilis liquid (bacterial powder configuration): 3 g of Bacillus subtilis powder, 15 g of proteose peptone, 10 g of anhydrous glucose, and 10 g of yeast powder are added to 1 L of pure water to obtain Bacillus subtilis liquid (bacterial powder configuration).

[0028] Example 1 A preparation method of a Ganoderma lucidum polypeptide, comprising the following steps: (1) After the Zilingzhi (dried body) is crushed, 100 g of Ganoderma lucidum powder is added to 3,000 mL of pure water, and 2 h of 90℃ cooking is performed to obtain a mixture 1.

[0029] (2) The pH of the mixture 1 is adjusted to 7.0, and 10% of the Bacillus subtilis liquid (bacterial powder configuration) of the volume of the mixture 1 is added, and aerobic fermentation is performed for 3 days to obtain a Ganoderma lucidum polypeptide fermentation broth.

[0030] (3) To the Ganoderma lucidum polypeptide fermentation broth, 2% of neutral protease by weight of Ganoderma lucidum powder was added, and the mixture was enzymolyzed at 50°C and pH 7.0 for 2 hours. Then, 1% of amino peptidase by weight of Ganoderma lucidum powder was added, and the mixture was enzymolyzed at 65°C and pH 8.5 for 2 hours. After the enzymolysis, the enzyme was inactivated by keeping the mixture at 90°C for 10 minutes, and a Ganoderma lucidum polypeptide enzymolysis solution was obtained.

[0031] (4) After the Ganoderma lucidum polypeptide enzymolysis solution was cooled to room temperature, it was filtered through a 0.45-μm filter plate, and then ultrafiltration was performed using a 5000-Da membrane core. The dialysate was collected, and nanofiltration was performed using a 200-Da membrane core. The concentrated solution was collected (the original volume was maintained during the circulation process, and purified water was added). The concentrated solution was concentrated using a rotary evaporator at a temperature of 60°C until the volume was 400 mL. 1% of hydroxypropyl methyl cellulose by weight of the concentrated solution was added. The inlet air temperature was set to 150°C, the outlet air temperature was set to 85°C, and the flow rate was set to 200 mL / h. Spray drying was performed, and a Ganoderma lucidum polypeptide was obtained.

[0032] Example 2 A method for preparing a Ganoderma lucidum polypeptide, comprising the following steps: (1) 100 g of Ganoderma lucidum powder was obtained by crushing dried Ganoderma lucidum (Ganoderma lucidum (dry)). The powder was added to 4000 mL of purified water, and the mixture was boiled at 100°C for 1.5 hours to obtain a mixture 1.

[0033] (2) The pH of the mixture 1 was adjusted to 7.2, and 8% of Bacillus subtilis liquid (bacterial powder was configured) by volume of the mixture 1 was added. Fermentation was performed for 3.5 days to obtain a Ganoderma lucidum polypeptide fermentation broth.

[0034] (3) To the Ganoderma lucidum polypeptide fermentation broth, 1.5% of neutral protease by weight of Ganoderma lucidum powder was added, and the mixture was enzymolyzed at 52°C and pH 7.2 for 1.5 hours. Then, 1.5% of amino peptidase by weight of Ganoderma lucidum powder was added, and the mixture was enzymolyzed at 60°C and pH 8.7 for 2.5 hours. After the enzymolysis, the enzyme was inactivated by keeping the mixture at 90°C for 10 minutes, and a Ganoderma lucidum polypeptide enzymolysis solution was obtained.

[0035] (4) After the Ganoderma lucidum polypeptide enzymolysis solution was cooled to room temperature, it was filtered through a 0.40-μm filter plate, and then ultrafiltration was performed using a 4000-Da membrane core. The dialysate was collected, and nanofiltration was performed using a 150-Da membrane core. The concentrated solution was collected (the original volume was maintained during the circulation process, and purified water was added). The concentrated solution was concentrated using a rotary evaporator at a temperature of 65°C until the volume was 500 mL. 1% of hydroxypropyl methyl cellulose by weight of the concentrated solution was added. The inlet air temperature was set to 150°C, the outlet air temperature was set to 85°C, and the flow rate was set to 200 mL / h. Spray drying was performed, and a Ganoderma lucidum polypeptide was obtained.

[0036] Example 3 A method for preparing a Ganoderma lucidum polypeptide, comprising the following steps: (1) The Ganoderma lucidum (dry body) is crushed and 100 g of Ganoderma lucidum powder is added to 5000 mL of pure water, and then boiled at 100°C for 2.5 hours to obtain a mixture 1.

[0037] (2) The pH of the mixture 1 is adjusted to 6.8, and 12% of the Bacillus subtilis liquid (bacterial powder configuration) of the volume of the mixture 1 is added, and then fermented for 2.5 days to obtain a Ganoderma lucidum polypeptide fermentation broth.

[0038] (3) 1% of the neutral protease of the mass of the Ganoderma lucidum powder is added to the Ganoderma lucidum polypeptide fermentation broth, and then enzymolysis is carried out at pH 6.8 and 43°C for 2.5 hours, and then 2% of the aminopeptidase of the mass of the Ganoderma lucidum powder is added, and then enzymolysis is carried out at pH 8.3 and 55°C for 1.5 hours, and then the enzyme is inactivated by keeping at 90°C for 10 minutes after the enzymolysis is completed, and then a Ganoderma lucidum polypeptide enzymolysis liquid is obtained.

[0039] (4) After the Ganoderma lucidum polypeptide enzymolysis liquid is cooled to room temperature, it is filtered by using a 0.5 μm filter plate, and then ultrafiltration is carried out by using a 6000 Da membrane core, and then the dialysate is collected, and then nanofiltration is carried out by using a 250 Da membrane core, and then the concentrated liquid is collected (the original volume is maintained during the circulation process, and pure water is supplemented); the concentrated liquid is concentrated by using a rotary evaporator, the temperature is 60°C, and then the concentrated liquid is concentrated to 500 mL, and then 1% of the hydroxypropyl methyl cellulose of the mass of the concentrated liquid is added, and then the inlet air temperature is set to 150°C, the outlet air temperature is set to 85°C, and the flow rate is set to 200 mL / h, and then spray drying is carried out, and then a Ganoderma lucidum polypeptide is obtained.

[0040] Comparative Example 1 The difference between this comparative example and Example 1 is only that step (1) is different, and specifically, the Ganoderma lucidum (dry body) is crushed and 100 g of Ganoderma lucidum powder is added to 3000 mL of pure water, and then stirred uniformly to obtain a mixture 1.

[0041] Comparative Example 2 The difference between this comparative example and Example 1 is only that step (1) is different, and specifically, the Ganoderma lucidum (dry body) is crushed and 100 g of Ganoderma lucidum powder is added to 3000 mL of pure water, and then high-pressure treatment is carried out by using a high-pressure kettle for 3 hours (working pressure 80 kPa) to obtain a mixture 1.

[0042] Comparative Example 3 The difference between this comparative example and Example 1 is only that step (2) is different, and specifically, the pH of the mixture 1 is adjusted to 7.0, 1 wt.% of peptone, 1 wt.% of glucose, and 0.5 wt.% of yeast powder are added, 121°C sterilization is carried out for 20 minutes, and then after cooling, 2% (volume percentage) of the Bacillus subtilis liquid (bacterial powder configuration) is inoculated, and then liquid fermentation is carried out for 3 days to obtain a Ganoderma lucidum polypeptide fermentation broth.

[0043] Comparative Example 4 The difference between this comparative example and Example 1 is only that the fermentation method is different, and specifically, (1) After the Ganoderma lucidum (dry body) is crushed, 10 g of Ganoderma lucidum powder is mixed with 1 g of water, and then is placed in a 250 ml conical flask. After being stirred uniformly, the mixture is sealed with a preservative film and sterilized at 121°C for 20 minutes. After being cooled, a solid-state fermentation medium is obtained. Bacillus subtilis is added at an addition amount of 20%, and then is placed in a constant-temperature incubator at 28°C for solid-state fermentation for 5 days. Finally, the mixture is sterilized at 121°C for 20 minutes.

[0044] (2) 10 g of the fermented powder is added to 300 mL of pure water, and the pH of the liquid is adjusted to 7.0. Enzymolysis is performed, and the subsequent steps are the same as those in Example 1.

[0045] Comparative Example 5 The difference between this comparative example and Example 1 is only that the fermentation step in step (2) is not included.

[0046] Comparative Example 6 The difference between this comparative example and Example 1 is only that step (2) is different, specifically as follows: (2) The pH of mixture 1 is adjusted to 7.0, and 10% of Bacillus subtilis liquid (seed liquid) of the volume of mixture 1 is added. Fermentation is performed for 3 days to obtain a Ganoderma lucidum polypeptide fermentation broth liquid.

[0047] Comparative Example 7 The difference between this comparative example and Example 1 is only that the protease in step (3) is different, specifically as follows: the neutral protease is replaced by alkaline protease.

[0048] Comparative Example 8 The difference between this comparative example and Example 1 is only that the protease in step (3) is different, specifically as follows: the neutral protease is replaced by acid protease.

[0049] Comparative Example 9 The difference between this comparative example and Example 1 is only that the protease in step (3) is different, specifically as follows: the neutral protease is replaced by papain.

[0050] Comparative Example 10 The difference between this comparative example and Example 1 is only that the protease in step (3) is different, specifically as follows: the neutral protease is replaced by flavor protease.

[0051] Comparative Example 11 The difference between this comparative example and Example 1 is only that the protease in step (3) is different, specifically as follows: the aminopeptidase is replaced by flavor protease.

[0052] Comparative Example 12 The difference between this comparative example and Example 1 is only that the protease in step (3) is different, specifically as follows: the aminopeptidase is replaced by complex protease.

[0053] Comparative Example 13 The only difference between this comparative example and Example 1 is that the filtration process in step (4) is different, specifically: after the Ganoderma lucidum polypeptide enzymatic hydrolysate is brought to room temperature, 11 μm filter paper is used for filtration.

[0054] Comparative Example 14 The only difference between this comparative example and Example 1 is that the order of fermentation and enzymolysis is different, specifically: (1) After the Ganoderma lucidum (dry body) is crushed, 100 g of Ganoderma lucidum powder is added to 3000 mL of pure water, and 2 h of cooking at 100°C is performed to obtain a mixture 1.

[0055] (2) The pH of the mixture 1 is adjusted to 7.0, 2% of neutral protease is added, and 2 h of enzymolysis at pH 7.0 and 50°C is performed, then 1% of aminopeptidase is added, and 2 h of enzymolysis at pH 8.5 and 65°C is performed, after the enzymolysis is completed, 10 min of enzyme inactivation at 90°C is performed, and a Ganoderma lucidum polypeptide enzymatic hydrolysate is obtained.

[0056] (3) After the Ganoderma lucidum polypeptide enzymatic hydrolysate is brought to room temperature, 10% of Bacillus subtilis liquid (bacterial powder configuration) is added, and 3 days of fermentation is performed, and a Ganoderma lucidum polypeptide fermentation broth bacterial liquid is obtained.

[0057] (4) The Ganoderma lucidum polypeptide fermentation broth bacterial liquid is filtered with a 0.45 μm filter plate, then ultrafiltration is performed with a 5000 Da membrane core, the dialysate is collected, then nanofiltration is performed with a 200 Da membrane core, the concentrated liquid is collected (the original volume is maintained during the circulation process, and pure water is supplemented), and the above concentrated liquid is concentrated using a rotary evaporator, the temperature is 60°C, and the concentrated liquid is concentrated to 400 mL, 1% hydroxypropylmethyl cellulose is added, the inlet air temperature is set to 150°C, the outlet air temperature is set to 85°C, and the flow rate is set to 200 mL / h, and spray drying is performed, and a product is obtained.

[0058] Effect test Test Example 1 Detection of polypeptide content by biuret reagent method Biuret reagent: reagent A: 0.15 g of CuSO4, 0.6 g of potassium sodium tartrate, and 50 mL of distilled water are uniformly mixed; reagent B: 30 mL of 10% NaOH solution (A and B are mixed uniformly when used).

[0059] Preparation of bovine serum albumin standard curve: (standard curve range 0-5 mg / mL) first prepare an appropriate amount of 10 mg / mL bovine serum albumin solution (water as solvent), take 0, 50, 100, 150, 200, and 250 μL into centrifuge tubes, supplement water to 0.5 mL, add 2 mL of biuret reagent (A and B are mixed and used), react at room temperature for 30 min, measure the absorbance at 540 nm, and prepare a standard curve. The standard curve is shown in Figure 1 .

[0060] Determination of sample solution: Take sample, add 1.0 mL of 10% trichloroacetic acid to dissolve, centrifuge, precipitate protein, take 0.5 mL of supernatant, add 2 mL of biuret reagent (A and B are mixed and used), react at room temperature for 30 min, measure the absorbance at 540 nm, and calculate the polypeptide content of the sample according to the standard curve.

[0061] Polypeptide content determination calculation formula: Polypeptide content standard curve: y = Ax + B (R 2 >0.99); Polypeptide concentration (mg / mL) = (y-B) / A; Polypeptide content (%) = protein or polypeptide concentration (mg / mL) / sample solution concentration (mg / mL) x 100%.

[0062] Test results are shown in Table 1.

[0063]

[0064] Test Example 2 Anti-inflammatory efficacy evaluation The inhibition rate of different samples on COX-2 was tested by COX-2 kit, and the specific operation was according to the use instruction provided by the manufacturer.

[0065] Sample detection: a. According to Table 2, set control holes and sample holes using 96-well black plates, and add samples and solutions in turn according to the table below. After adding the sample to be tested, mix well, and incubate at 37°C for 10 minutes.

[0066]

[0067] Note: # Sample solvent refers to the solvent used for preparation and dilution of the inhibitor to be tested.

[0068] b. Add COX-2 Probe 5 μL to each well.

[0069] c. Quickly add 5 μL of COX-2 Substrate working solution to each well, and mix well. Note: After adding COX-2 Substrate working solution, the reaction will start. If the number of wells is large, low-temperature operation or the use of a row gun can be used to reduce the error caused by the time difference in adding COX-2 Substrate working solution between wells, and mixing can also be done on a plate shaker.

[0070] d. After incubation at 37°C in the dark for 5 min, fluorescence determination was performed. The excitation wavelength was 560 nm, and the emission wavelength was 590 nm. When the fluorescence reading is low, the incubation time can also be appropriately extended to 10-20 minutes.

[0071] e.The average fluorescence value of each sample well and blank control well was calculated, which could be recorded as RFUblank control, RFU100% enzyme activity control, RFUpositive inhibitor control and RFU sample, respectively. Wherein, RFU was relative fluorescence unit RFU (Relative Fluorescence Unit).

[0072] The inhibition percentage of each sample was calculated, and the calculation formula was as follows: Inhibition rate (%) = (RFU100% enzyme activity control - RFU sample) / (RFU100% enzyme activity control - RFU blank control) × 100%.

[0073] The test results are shown in Table 3.

[0074]

[0075] Elastase Anti-aging efficacy evaluation Tris-HCl solution: 2 mmol / L Tris-HCl solution was prepared, and the pH was adjusted to 8.0 with 1 mol / L and 0.1 mol / L NaOH solution. After use, it was stored at 4°C.

[0076] Elastase solution: 1 U / mL elastase solution was prepared with 2 mmol / L Tris-HCl solution. If there is any surplus after use, it is stored at -20°C.

[0077] Substrate solution: 0.5 mg / mL substrate solution was prepared with 2 mmol / L Tris-HCl solution. If there is any surplus after use, it is stored at -4°C.

[0078] Test solution: Take the water-soluble sample, and prepare the test solution by dissolving it with 2 mmol / L Tris-HCl solution.

[0079] Experimental procedure: In the 96-well plate, each reagent was added according to Table 4.

[0080] First, incubate the enzyme solution and test solution (or Tris-HCl solution) for 15 min, then add the substrate solution and buffer and incubate for 50 min. Measure the absorbance value at 410 nm.

[0081] Elastase inhibition rate calculation formula: Inhibition rate (%) = [1 - (A - B) / C] × 100%.

[0082] The test results are shown in Table 5.

[0083]

[0084]

[0085] The above results show that, using Ganoderma lucidum as raw material, liquid fermentation method inoculated with Bacillus subtilis is used to extract Ganoderma lucidum polypeptide, which is subjected to enzymatic hydrolysis by neutral protease and amino peptidase, and then subjected to ultrafiltration of 5000 Da and nanofiltration of 200 Da to intercept the molecular weight, and finally concentrated and spray dried, to obtain Ganoderma lucidum polypeptide with ideal yield, higher polypeptide content, and more uniform molecular weight distribution, and with antioxidant, anti-aging and other effects, which can be developed and applied in functional cosmetic raw materials.

[0086] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical solutions of the present application.

Claims

1. A method for preparing Ganoderma lucidum polypeptide, characterized in that, The preparation method comprises the following steps: (1) taking Ganoderma lucidum powder, cooking with water, cooling to obtain mixture 1; (2) adding Bacillus subtilis liquid to the mixture 1, fermenting to obtain Ganoderma lucidum polypeptide fermentation broth liquid; (3) adding neutral protease to the Ganoderma lucidum polypeptide fermentation broth liquid for primary enzymolysis, then adding amino peptidase for secondary enzymolysis to obtain Ganoderma lucidum polypeptide enzymolysis liquid; (4) filtering the Ganoderma lucidum polypeptide enzymolysis liquid, concentrating, spray drying to obtain Ganoderma lucidum polypeptide.

2. The production method according to claim 1, characterized by, In step (1), the mass ratio of Ganoderma lucidum powder to water is 1:30-50, the cooking temperature is 80-100 DEG C, the cooking time is 1.5-2.5 h, and the cooling temperature is 40-55 DEG C.

3. The preparation method according to claim 1, characterized in that, In step (2), the adding amount of Bacillus subtilis liquid is 8%-12% of the volume of mixture 1, and the preparation method of the Bacillus subtilis liquid comprises the following steps: adding Bacillus subtilis powder, proteose peptone, anhydrous glucose and yeast powder into water and mixing to obtain the Bacillus subtilis liquid.

4. The production method according to claim 3, characterized by, The mass ratio of the Bacillus subtilis powder, proteose peptone, anhydrous glucose, yeast powder and water is 2-4:12-18:8-12:8-12:1000, and the fermentation conditions are aerobic fermentation, pH is 6.5-7.5, and the fermentation time is 2.5-3.5 days.

5. The preparation method according to claim 1, characterized in that, In step (3), the adding amount of the neutral protease is 0.8%-2% of the mass of Ganoderma lucidum powder, the primary enzymolysis temperature is 43-52 DEG C, the primary enzymolysis pH is 6.8-7.6, and the primary enzymolysis time is 1.5-2.5 h.

6. The method of claim 1, wherein, In step (3), the adding amount of the amino peptidase is 0.8%-2% of the mass of Ganoderma lucidum powder, the secondary enzymolysis temperature is 55-65 DEG C, the secondary enzymolysis pH is 8.0-9.0, and the secondary enzymolysis time is 1.5-2.5 h.

7. The preparation method according to claim 1, characterized in that, In step (4), the filtering comprises the following steps: first filtering through a 0.4-0.5 μm filter plate, then ultrafiltrating through a 4000-6000 Da membrane core, and finally nanofiltrating through a 150-250 Da membrane core.

8. The method of claim 1, wherein, In step (4), the concentration temperature is 55-65 DEG C, the concentration is performed to 20%-25% of the volume of the filtrate, the spray drying conditions are as follows: the inlet air temperature is 140-160 DEG C, the outlet air temperature is 80-90 DEG C, the flow rate is 180-220 mL / h, and 0.8%-1.2% of hydroxypropylmethyl cellulose is added to the concentrated liquid before spray drying.

9. Ganoderma lucidum polypeptide prepared by the preparation method in any one of claims 1-8.

10. Application of Ganoderma lucidum polypeptide prepared by the preparation method in any one of claims 1-8 in cosmetics.

Images