A method for separating and utilizing all components of ganoderma lucidum spores and its application

Abstract

The invention discloses a method and application for separating and utilizing all components of ganoderma lucidum spores, and belongs to the field of processing technology. The method includes S1 preparing an emulsion and a spore wall shell; S2 emulsion concentration and alcohol precipitation to obtain a supernatant and a precipitate; S3 respectively drying the supernatant and the precipitate to obtain a small molecule peptide component and a polysaccharide component; S4 spore wall shell is enzymolyzed by composite enzyme A and composite enzyme B, solid-liquid separation, and an enzymolysis solution and a residue are obtained; composite enzyme A includes cellulase and chitinase, and composite enzyme B includes papain, bromelain and α-amylase; S5 enzymolysis solution is used to prepare soluble dietary fiber; S6 residue is dried and extracted to obtain ganoderma lucidum spore oil and residue; S7 residue is used to prepare chitosan. The present invention can complete the separation of multiple products at one time, and realizes the separation and utilization of all components of ganoderma lucidum spores. Compared with the prior art, the yield of spore oil and soluble dietary fiber of the present invention is higher, and the purity of soluble dietary fiber can also be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of ganoderma lucidum spore powder processing, and specifically relates to a method and application of separating and utilizing all components of ganoderma lucidum spores. Background Art

[0002] Ganoderma lucidum, also known as Zhicao, is the dried fruiting body of Ganoderma lucidum (Leyss. exFr.) Karst or Ganoderma sinense Zhao, Xu et Zhang, a Basidiomycetes, family Polyporaceae. It is sweet and mild in nature, has the effects of strengthening the body, nourishing and strengthening the body, and has high medicinal value. During the growth and maturity period, Ganoderma lucidum will eject extremely tiny oval reproductive cells from the gills, which are called Ganoderma lucidum spores or Ganoderma lucidum spore powder. In recent years, researchers have conducted a lot of research on it and found that Ganoderma lucidum spore powder contains a variety of active ingredients, such as polysaccharides, triterpenes, amino acids, alkaloids, sterols and inorganic elements, and confirmed a series of pharmacological effects such as anti-tumor, anti-epileptic, immune enhancement, and regulation of blood sugar and blood lipids from different levels such as genes, proteins or metabolic pathways. At present, corresponding drugs have entered the market and achieved good clinical efficacy.

[0003] CN104013652A discloses a refining process and comprehensive utilization method of ganoderma lucidum spore powder, which comprises the following steps: removing the wall of the broken ganoderma lucidum spore powder after the wall is broken, and then soaking the broken ganoderma lucidum spore powder in an ethanol solution with a volume fraction of 20% to 95% and water, separating the wall shell, obtaining a wall-removed ganoderma lucidum spore powder filtrate and a ganoderma lucidum spore powder wall shell, concentrating and drying the wall-removed ganoderma lucidum spore powder filtrate to obtain a refined ganoderma lucidum spore powder, and subjecting the ganoderma lucidum spore powder wall shell to a comprehensive recycling process to obtain a wall shell alcohol extract solid powder, a dichloromethane extract solid powder, an ethyl acetate extract solid powder, a wall shell water extract solid powder, a n-butanol extract solid powder, an aqueous phase powder or chitosan. The invention realizes the separation of the ineffective components (wall shell) of the spore powder and the enrichment of the effective components (polysaccharides and triterpenes, etc.) through wall removal and refining, realizes the comprehensive utilization of the wall shell, and improves the economic benefits.

[0004] CN106692213A discloses a spore oil, which is directly prepared by breaking the wall of Ganoderma lucidum spores, granulating, drying and supercritical CO2 extraction followed by two-stage separation, without any conventional refining process such as deacidification and deodorization. The Ganoderma lucidum spore oil is rich in monounsaturated fatty acids mainly composed of oleic acid, and retains the effective ingredients unique to Ganoderma lucidum such as triterpene acid, ergofluidol, fat-soluble vitamins, etc., and has good anti-tumor activity.

[0005] CN108159090A discloses the use of a dietary fiber extract of ganoderma lucidum spore powder in preparing a preparation for treating and / or preventing diseases related to intestinal flora imbalance, and discloses a method for preparing the dietary fiber extract of ganoderma lucidum spore powder, comprising extracting the ganoderma lucidum spore powder by reflux extraction with ethanol, extracting the filter residue by reflux extraction with water, and then enzymolyzing, precipitating and eluting the extract to obtain the ganoderma lucidum spore powder dietary fiber extract.

[0006] At present, the separation process of the active ingredients of Ganoderma lucidum spore powder is complicated, the compatibility between processes is poor, and no research has been found that can separate spore polysaccharides, spore oil and chitosan at one time. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for separating and utilizing all components of Ganoderma lucidum spores. The present invention has good compatibility and can complete the separation of small molecule peptides, spore polysaccharides, spore oil, soluble dietary fiber and chitosan at one time, thereby realizing the separation and utilization of all components of Ganoderma lucidum spores.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In one aspect, the present invention provides a method for separating and utilizing all components of Ganoderma lucidum spores, comprising the following steps:

[0010] S1, softening the ganoderma spores, emulsifying and breaking the spore wall, and separating the solid and liquid to obtain an emulsion and spore wall shell;

[0011] S2, concentrating the emulsion obtained in step S1, cooling it, adding ethanol and precipitating it to obtain a supernatant and a precipitate;

[0012] S3, drying the supernatant and the precipitate obtained in step S2 respectively to obtain a small molecule peptide component and a polysaccharide component;

[0013] S4, hydrolyzing the spore wall shell obtained in step S1 with compound enzyme A, adding compound enzyme B after the initial hydrolysis, performing secondary hydrolysis, inactivating the enzyme, obtaining a hydrolysis mixture, and obtaining a hydrolyzed liquid and a residue after solid-liquid separation;

[0014] S5, drying the enzymatic hydrolyzate obtained in step S4 to obtain soluble dietary fiber;

[0015] S6, drying the residue obtained in step S4 and then extracting it to obtain Ganoderma lucidum spore oil and residue;

[0016] S7, treating the residue obtained in step S6 with acid or alkali to obtain chitosan.

[0017] Preferably, the softening in step S1 is soaking in water overnight, and the amount of water added is 3-20 times the mass of the Ganoderma lucidum spores.

[0018] Preferably, the emulsification and wall breaking in step S1 is: using water as solvent and high pressure homogenization.

[0019] Further preferably, the high-pressure homogenization is: homogenization pressure 60-120 MPa, homogenization temperature 30-70° C., and homogenization at least once.

[0020] Preferably, the solid-liquid separation in step S1 is at least one of centrifugation, filtration and filter pressing.

[0021] Preferably, the concentration in step S2 is performed at 30-80° C. to 25-75% of the mass of the Ganoderma lucidum spores.

[0022] Preferably, the alcohol precipitation in step S2 is: adding ethanol to a concentration of 50-85 wt%, and standing at 0-10° C. for 8-48 h. More preferably, adding ethanol to a concentration of 55-65 wt%.

[0023] Preferably, in step S4, the complex enzyme A comprises cellulase and chitinase, and the complex enzyme B comprises papain, bromelain and α-amylase.

[0024] Further preferably, the weight ratio of the cellulase to the chitinase is (1-2):(2-3), and the weight ratio of the papain, bromelain and α-amylase is (2-4):(2-4):1.

[0025] Preferably, in step S4, the compound enzyme A is 0.3-0.5% of the mass of the spore wall shell, and the compound enzyme B is 0.8-1% of the mass of the spore wall shell.

[0026] Preferably, the conditions for the initial enzymatic hydrolysis in step S4 are: temperature 40-60°C, time 1-5h, pH 5-5.5; more preferably, temperature 40-50°C, time 1-3h.

[0027] Preferably, the conditions for the secondary enzymatic hydrolysis in step S4 are: temperature 30-50°C, time 1-5h, pH 6-7; more preferably, temperature 40-50°C, time 1-3h.

[0028] Preferably, the solid-liquid separation in step S4 is at least one of centrifugation, filtration and filter pressing.

[0029] Preferably, the extraction in step S6 is CO 2 Supercritical extraction, further preferably, the extraction conditions are: extraction pressure 10-45MPa, temperature 25-50°C.

[0030] The drying methods involved in the present invention are all conventional drying methods in the art, and the purpose is to control the water content to not exceed 10%, such as spray drying, freeze drying, vacuum drying, oven drying or air drying. Preferably, the drying is spray drying at a temperature of 30-70°C.

[0031] In another aspect, the present invention provides the use of the product prepared by the above method in the preparation of cosmetics, food or medicine.

[0032] The present invention has the following beneficial effects:

[0033] The invention has good compatibility and can complete the separation of small molecule peptides, spore polysaccharides, spore oil, soluble dietary fiber and chitosan at one time, thereby realizing the separation and utilization of all components of Ganoderma lucidum spores.

[0034] Compared with the prior art, the yield of the ganoderma lucidum spore oil and soluble dietary fiber prepared by the present invention is higher, and the purity of the soluble dietary fiber can also be improved. DETAILED DESCRIPTION

[0035] The present invention is described below through specific embodiments to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto, and the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0036] The endpoints and any value of the scope disclosed in this article are not limited to the accurate scope or value, and these scopes or values ​​should be understood to include values ​​close to these scopes or values. For numerical ranges, between the endpoint values ​​of each scope, between the endpoint values ​​of each scope and a separate point value, and between separate point values, one or more new numerical ranges can be combined with each other, and these numerical ranges should be considered as specifically disclosed in this article. Unless the context clearly indicates otherwise, the singular form "one", "a kind of" and "described" as used herein include singular and plural indicators. The numerical range described by the endpoint is included in all numerical values ​​and fractions within the corresponding range, as well as the described endpoint.

[0037] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should belong to the scope of protection of the present invention. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified. Cellulase CAS: 9012-54-8, enzyme activity 500,000 U / g, chitinase CAS: 9001-06-3, enzyme activity 100,000 U / g, papain CAS: 9001-73-4, enzyme activity 1 million U / g, bromelain CAS: 9001-00-7, enzyme activity 200,000 U / g, α-amylase CAS: 9000-90-2, enzyme activity 1 million U / g, unless otherwise specified, the temperature is room temperature, 20-25°C, and the moisture content of the dried product in this embodiment is 5-8%.

[0038] Example 1 Method for separating and utilizing all components of Ganoderma lucidum spores

[0039] Here are the steps:

[0040] S1, adding 7 times the mass of water to the spores of Ganoderma lucidum to soak overnight, emulsifying (pressure 80MPa, temperature 50°C, homogenization 2 times), breaking the spore wall, and filtering to obtain an emulsion and spore wall shell;

[0041] S2, concentrating the emulsion obtained in step S1 at 50° C. to 50% of the mass of the Ganoderma lucidum spores, adding ethanol to make the concentration of ethanol 60wt%, cooling to 5° C., and subjecting to alcohol precipitation for 24 hours to obtain a supernatant and a precipitate;

[0042] S3, spray drying the supernatant and precipitate obtained in step S2 (50° C.) to obtain a small molecule peptide component and a polysaccharide component;

[0043] S4, mixing the spore wall shell obtained in step S1, complex enzyme A (the weight ratio of cellulase and chitinase is 1.5:2.5) and water, the amount of complex enzyme added is 0.4% of the mass of the spore wall shell, adjusting the concentration of the spore wall shell to 5%, enzymolysis at pH 5-5.5, 45°C for 2h, after the initial enzymolysis, adding complex enzyme B (the weight ratio of papain, bromelain and α-amylase is 3:3:1), the amount of complex enzyme B added is 0.9% of the mass of the spore wall shell, at pH 6-7, 45°C for 1.5h, after the secondary enzymolysis, at a temperature of 140°C and a pressure of 0.2MPa, the enzyme is inactivated for 10min to obtain an enzymolysis mixture, and after filter pressing, an enzymolysis solution and a residue are obtained;

[0044] S5, concentrating the enzymatic hydrolyzate obtained in step S4 and spray drying (50° C.) to obtain soluble dietary fiber;

[0045] S6, drying the slag obtained in step S4,2 Supercritical extraction (extraction pressure 22MPa, temperature 32°C) to obtain Ganoderma lucidum spore oil and residue;

[0046] S7, treating the residue obtained in step S6 with acid or alkali to obtain chitosan.

[0047] Example 2 Method for separating and utilizing all components of Ganoderma lucidum spores

[0048] Here are the steps:

[0049] S1, adding 5 times the mass of water to the spores of Ganoderma lucidum to soak overnight, emulsifying (pressure 60MPa, temperature 70°C, homogenization 3 times), breaking the spore wall, and filtering to obtain an emulsion and spore wall shell;

[0050] S2, concentrating the emulsion obtained in step S1 at 70° C. to 25% of the mass of the Ganoderma lucidum spores, adding ethanol to make the concentration of ethanol 65wt%, cooling to 0° C. and performing alcohol precipitation for 12 hours to obtain a supernatant and a precipitate;

[0051] S3, spray drying the supernatant and precipitate obtained in step S2 (50° C.) to obtain a small molecule peptide component and a polysaccharide component;

[0052] S4, mixing the spore wall shell obtained in step S1, complex enzyme A (the weight ratio of cellulase and chitinase is 1:2) and water, the amount of complex enzyme added is 0.3% of the mass of the spore wall shell, adjusting the concentration of the spore wall shell to 5%, enzymolysis at pH 5-5.5, 40°C for 3h, after the initial enzymolysis, adding complex enzyme B (the weight ratio of papain, bromelain and α-amylase is 4:2:1), the amount of complex enzyme B added is 0.8% of the mass of the spore wall shell, secondary enzymolysis at pH 6-7, 40°C for 3h, after the secondary enzymolysis, inactivate the enzyme at a temperature of 140°C and a pressure of 0.2MPa for 10min to obtain an enzymolysis mixture, and obtain an enzymolysis solution and a residue after filter pressing;

[0053] S5, concentrating the enzymatic hydrolyzate obtained in step S4 and spray-drying the concentrated enzymatic hydrolyzate to obtain soluble dietary fiber;

[0054] S6, drying the slag obtained in step S4, 2 Supercritical extraction (extraction pressure 10MPa, temperature 50°C) to obtain Ganoderma lucidum spore oil and residue;

[0055] S7, treating the residue obtained in step S6 with acid or alkali to obtain chitosan.

[0056] Example 3 Method for separating and utilizing all components of Ganoderma lucidum spores

[0057] Here are the steps:

[0058] S1, adding 15 times the mass of water to the spores of Ganoderma lucidum to soak overnight, emulsifying (pressure 120MPa, temperature 30°C, homogenization once), breaking the spore wall, and filtering to obtain an emulsion and spore wall shell;

[0059] S2, concentrating the emulsion obtained in step S1 at 30° C. to 75% of the mass of the Ganoderma lucidum spores, adding ethanol to make the concentration of ethanol 55wt%, cooling to 10° C. and performing alcohol precipitation for 48h to obtain a supernatant and a precipitate;

[0060] S3, spray drying the supernatant and precipitate obtained in step S2 (50° C.) to obtain a small molecule peptide component and a polysaccharide component;

[0061] S4, mixing the spore wall shell obtained in step S1, compound enzyme A (the weight ratio of cellulase and chitinase is 2:3) and water, wherein the amount of compound enzyme added is 0.5% of the mass of the spore wall shell, adjusting the concentration of the spore wall shell to 5%, performing enzymolysis at pH 5-5.5 and 50° C. for 1 h, adding compound enzyme B (the weight ratio of papain, bromelain and α-amylase is 2:4:1) after the initial enzymolysis, wherein the amount of compound enzyme B added is 1% of the mass of the spore wall shell, performing secondary enzymolysis at pH 6-7 and 50° C. for 1 h, and after the secondary enzymolysis, inactivating the enzyme at a temperature of 140° C. and a pressure of 0.2 MPa for 10 min to obtain an enzymolysis mixture, and obtaining an enzymolysis solution and a residue after filter pressing;

[0062] S5, concentrating the enzymatic hydrolyzate obtained in step S4 and spray drying (50° C.) to obtain soluble dietary fiber;

[0063] S6, drying the slag obtained in step S4, 2 Supercritical extraction (extraction pressure 45MPa, temperature 25°C) to obtain Ganoderma lucidum spore oil and residue;

[0064] S7, treating the residue obtained in step S6 with acid or alkali to obtain chitosan.

[0065] Comparative Example 1: Method for separating and utilizing all components of Ganoderma lucidum spores

[0066] The difference between this comparative example and Example 1 is that the compound enzyme A is cellulase, and the compound enzyme B is bromelain and α-amylase in a weight ratio of 3:1.

[0067] The rest are the same as in Example 1.

[0068] Comparative Example 2: Method for separating and utilizing all components of Ganoderma lucidum spores

[0069] The difference between this comparative example and Example 1 is that the compound enzyme A is cellulase and pectinase in a weight ratio of 3:5, and the compound enzyme B is papain.

[0070] The rest are the same as in Example 1.

[0071] Comparative Example 3: Method for separating and utilizing all components of Ganoderma lucidum spores

[0072] The difference between this comparative example and Example 1 is that the compound enzyme A is cellulase and chitinase in a weight ratio of 3:1; the compound enzyme B is papain, bromelain and α-amylase in a weight ratio of 6:1:3.

[0073] The rest are the same as in Example 1.

[0074] The yield and purity of the products prepared in the examples and comparative examples are shown in Table 1 below.

[0075] in,

[0076] Small molecule peptide components are tested according to the testing method of GB / T22492-2008;

[0077] The polysaccharide component is tested according to the test method in Appendix C.2 of T / ZZB 0474-2018;

[0078] Soluble dietary fiber is tested according to the test method of GB5009.88-20236.6;

[0079] Ganoderma lucidum spore oil is tested according to the following method:

[0080] Oleanolic acid standard solution: Accurately weigh 10 mg of oleanolic acid reference substance dried to constant weight at 105°C, dissolve it in anhydrous ethanol and make up to volume in a 10 mL volumetric flask.

[0081] Sample treatment: Take 0.250g of the sample content, weigh accurately, add 40mL of anhydrous ethanol, and oscillate ultrasonically for 10 minutes. If necessary, heat in a hot water bath to completely dissolve the Ganoderma lucidum spore oil, then adjust the volume to 50mL, filter, discard the initial filtrate, and collect the filtrate. Accurately pipette 0.1mL of the filtrate and evaporate to dryness for later use.

[0082] Determination: Accurately pipette 0, 20, 40, 60, 80, 100 μL of standard solution and evaporate to dryness. Add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid to the sample tube and standard tube, respectively, seal, shake well, place in a 70°C water bath for 15 min, take out and cool to room temperature, accurately add 4 mL of glacial acetic acid, shake well, and measure the absorbance value at a wavelength of 550±1 nm.

[0083] Calculation results:

[0084]

[0085] Where:

[0086] X-total triterpenes in the sample (calculated as oleanolic acid), g / 100g;

[0087] M 1 -The amount of colorimetrically equivalent oleanolic acid in the sample assay solution, μg;

[0088] M 2 - Sample weight, g;

[0089] V 1 - Sample volume, mL;

[0090] V 2 - Sample volume of the sample constant solution, mL.

[0091] Table 1

[0092]

[0093] The results show that the present invention has good compatibility and can complete the separation of components such as small molecule peptides, spore polysaccharides, spore oil and soluble dietary fiber at one time, thereby achieving the separation and utilization of all components of Ganoderma lucidum spores.

[0094] Compared with the prior art, the yield of the ganoderma lucidum spore oil and soluble dietary fiber prepared by the present invention is higher, and the purity of the soluble dietary fiber can also be improved.

[0095] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for separating and utilizing all components of Ganoderma lucidum spores, characterized in that: The steps include: S1, softening the ganoderma spores, emulsifying and breaking the spore wall, and separating the solid and liquid to obtain an emulsion and spore wall shell; S2, concentrating the emulsion obtained in step S1, cooling it, adding ethanol and precipitating it to obtain a supernatant and a precipitate; S3, drying the supernatant and the precipitate obtained in step S2 respectively to obtain a small molecule peptide component and a polysaccharide component; S4, hydrolyzing the spore wall shell obtained in step S1 with compound enzyme A, adding compound enzyme B after the initial hydrolysis, performing secondary hydrolysis, inactivating the enzyme, obtaining a hydrolysis mixture, and obtaining a hydrolyzed liquid and a residue after solid-liquid separation; In step S4, the complex enzyme A comprises cellulase and chitinase, and the complex enzyme B comprises papain, bromelain and α-amylase; S5, drying the enzymatic hydrolyzate obtained in step S4 to obtain soluble dietary fiber; S6, drying the residue obtained in step S4 and then extracting it to obtain Ganoderma lucidum spore oil and residue; S7, the residue obtained in step S6 was treated with acid and alkali to obtain chitosan; In step S4, the weight ratio of cellulase to chitinase is (1-2): (2-3), and the weight ratio of papain, bromelain and α-amylase is (2-4): (2-4): 1; In step S4, the compound enzyme A is 0.3-0.5% of the mass of the spore wall shell, and the compound enzyme B is 0.8-1% of the mass of the spore wall shell.

2. The method according to claim 1, characterized in that The softening in step S1 is to soak in water overnight.

3. The method according to claim 2, characterized in that The emulsification and wall breaking in step S1 is: using water as solvent and high pressure homogenization.

4. The method according to claim 1, characterized in that: The concentration in step S2 is to concentrate at 30-80° C. to 25-75% of the mass of the Ganoderma lucidum spores.

5. The method according to claim 4, characterized in that The alcohol precipitation in step S2 is as follows: adding ethanol to make the concentration of ethanol 50-85wt%, and standing at 0-10°C for 8-48h.

6. The method according to claim 1, characterized in that The conditions for the initial enzymatic hydrolysis in step S4 are: temperature 40-65° C., time 1-5 h, pH 5-5.

5.

7. The method according to claim 1, characterized in that The conditions of the secondary enzymatic hydrolysis in step S4 are: temperature 30-50° C., time 1-5 h, pH 6-7.

8. The method according to claim 1, characterized in that The extraction in step S6 is CO2 supercritical extraction.

9. Use of the method according to any one of claims 1 to 8 in the preparation of cosmetics, foods or medicines.

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