Preparation method and application of ganoderma lucidum soluble fiber

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and relates to a preparation method and application of ganoderma lucidum soluble fibers. The ganoderma lucidum soluble fiber is prepared by combining an enzyme method with a semi-bionic extraction method, has the advantages of high yield, low cost, easiness in industrial production and the like, and meets the requirements of clinical use of traditional Chinese medicines; the prepared ganoderma lucidum soluble fiber has efficient anti-tumor, immune regulation and blood fat reduction activity, and has great medical potential.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a preparation method of ganoderma lucidum soluble fiber and application thereof. Technical Background

[0002] Ganoderma lucidum is the dried fruiting body of the Polyporaceae fungus Ganoderma lucidum or Ganoderma rubrum. It is a traditional rare and precious Chinese medicinal material and has been included in the catalog of medicinal and edible substances by the National Health Commission. Modern drug analysis studies have shown that soluble dietary fiber is one of the effective substances of Ganoderma lucidum, which has the effects of lowering blood sugar, anti-aging, anti-tumor and immune regulation.

[0003] For example, Chinese invention patent CN118104812A provides a method for extracting low molecular weight water-soluble dietary fiber from Ganoderma lucidum using a composite enzyme and hot water, comprising the following steps: (1) drying and crushing the fruiting bodies of Ganoderma lucidum to obtain Ganoderma lucidum powder; drying and crushing the fruiting bodies of Ganoderma lucidum to obtain Ganoderma lucidum powder; (2) mixing the Ganoderma lucidum powder and phosphate buffer, first adding papain for primary enzymolysis, boiling and killing the enzymes after the enzymolysis is completed, then adding cellulase and hemicellulase for secondary enzymolysis, boiling and killing the enzymes after the enzymolysis is completed, and obtaining an enzymolysis solution; (3) heating the enzymolysis solution for leaching, and performing post-treatment after the leaching is completed to obtain an extract; (4) mixing the extract with an ethanol solution, first precipitating the solution with alcohol overnight, and then performing post-treatment to obtain low molecular weight water-soluble dietary fiber from Ganoderma lucidum. The low molecular weight ganoderma lucidum water-soluble dietary fiber has an average molecular weight of 3300-3500Da, a weight average molecular weight of 3200-3400Da, and a molecular weight distribution index of 1.000-1.100. However, the hemicellulase used in step (2) is prone to cause the loss of effective components such as xylose, galactose, arabinose and mannose, which does not meet the strategic goal of sustainable utilization of traditional Chinese medicine resources.

[0004] Another example is that Chinese invention patents CN108159090A and CN108159089A disclose a method for preparing a dietary fiber extract of ganoderma lucidum spore powder, characterized in that the method comprises the steps of crushing the ganoderma lucidum spore powder, extracting the extract by ethanol reflux, extracting the filter residue by water reflux, and then enzymolyzing, precipitating, and eluting the extract to obtain the dietary fiber extract of ganoderma lucidum spore powder, wherein the enzymolysis is enzymolyzed by papain. However, papain is a mixed enzyme, which contains pectinase components in addition to papain, and extracting the dietary fiber of ganoderma lucidum spore powder by papain will cause the loss of pectin components.

[0005] Therefore, there is an urgent need to study a preparation method for Ganoderma lucidum soluble fiber with high extraction efficiency, less loss of effective ingredients, and the ability to fully utilize traditional Chinese medicine. Summary of the invention

[0006] The invention aims at solving the problems in the prior art and provides a preparation method and application of ganoderma lucidum soluble fiber.

[0007] To achieve the above object, the technical solution adopted in the present invention is as follows:

[0008] The present invention provides a method for preparing ganoderma lucidum soluble fiber, comprising the following steps:

[0009] (1) taking raw material Ganoderma lucidum, and crushing it to obtain Ganoderma lucidum powder;

[0010] (2) mixing the ganoderma lucidum powder obtained in step (1) with phosphate buffer, adding cellulase for enzymolysis, boiling to inactivate the enzyme after the enzymolysis is completed, and filtering under reduced pressure to obtain a residue;

[0011] (3) adding artificial gastric juice to the residue obtained in step (2) for extraction, filtering under reduced pressure, and collecting filtrate A and residue;

[0012] (4) adding artificial intestinal fluid to the filter residue obtained in step (3) for extraction, filtering under reduced pressure, and collecting filtrate B;

[0013] (5) mixing the filtrate A and the filtrate B obtained in step (3) and step (4), and concentrating under reduced pressure to obtain a concentrated solution;

[0014] (6) The concentrated solution obtained in step (5) is mixed with an ethanol solution, and after alcohol precipitation overnight, the mixture is centrifuged, and the precipitate is collected and dried to a constant weight to obtain a Ganoderma lucidum soluble fiber powder.

[0015] Preferably, in step (1), the ganoderma lucidum is selected from one of red ganoderma, purple ganoderma, cracked ganoderma spore powder or de-walled ganoderma spore powder.

[0016] Further preferably, the Ganoderma lucidum is selected from one of Ganoderma lucidum, Ganoderma lucidum or de-walled Ganoderma lucidum spore powder.

[0017] Preferably, in step (2), the solid-liquid ratio of the Ganoderma lucidum powder to the phosphate buffer is 1:20-60 g / mL, and the pH of the phosphate buffer is 5.5-6.0.

[0018] Further preferably, the solid-liquid ratio of the Ganoderma lucidum powder to the phosphate buffer is 1:40 g / mL, and the pH of the phosphate buffer is 5.7.

[0019] Preferably, in step (2), the mass percentage of the cellulase in the mixed solution of Ganoderma lucidum powder and phosphate buffer is 1-3%, the enzymolysis temperature is 45-60° C., the enzymolysis time is 60-120 min, and the enzyme is inactivated by boiling for 4-10 min.

[0020] Further preferably, the mass percentage of the cellulase in the mixed solution of Ganoderma lucidum powder and phosphate buffer is 2%, the enzymolysis temperature is 50° C., the enzymolysis time is 90 min, and the enzyme is inactivated by boiling for 8 min.

[0021] Preferably, in step (3), the material-liquid ratio of the residue to artificial gastric juice is 1:4-10 g / mL, and the pH of the artificial gastric juice is 1.5-4.0.

[0022] Further preferably, the solid-liquid ratio of the residue to artificial gastric juice is 1:8 g / mL, and the pH of the artificial gastric juice is 3.5.

[0023] Preferably, in step (4), the material-liquid ratio of the filter residue to the artificial intestinal fluid is 1:4-10 g / mL, and the pH of the artificial intestinal fluid is 7.5-9.0.

[0024] Further preferably, the material-liquid ratio of the filter residue to the artificial intestinal fluid is 1:6 g / mL, and the pH of the artificial intestinal fluid is 8.5.

[0025] Preferably, in step (6), the volume ratio of the concentrate to ethanol is 1:3-6, the volume concentration of the ethanol solution is 95%, the centrifugation is 5000 rpm for 10 min, and the drying temperature is 65°C.

[0026] Further preferably, the volume ratio of the concentrated solution to ethanol is 1:4.

[0027] The present invention also provides the use of the ganoderma lucidum soluble fiber prepared by the preparation method in preparing anti-tumor, blood lipid lowering and immunity regulating products.

[0028] Preferably, the dosage forms of the product include powder, granules, capsules, oral liquid, tablets, powders, and powder aerosols.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention adopts an enzyme method combined with a semi-bionic extraction method to prepare Ganoderma lucidum soluble fiber, which overcomes the disadvantage of the conventional extraction method that high-temperature decoction is easy to destroy the effective ingredients. In addition, the preparation process simulates the process of oral drug transport and absorption in the gastrointestinal tract, which is more in line with the clinical use characteristics of traditional Chinese medicine.

[0031] (2) The present invention combines artificial gastric juice acid extraction with artificial intestinal juice alkaline extraction to improve the extraction efficiency of Chinese medicinal ingredients, reduce the loss of effective ingredients, and achieve full utilization of Chinese medicinal materials.

[0032] (3) The preparation process of the present invention is simple, low in cost, and easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The effect of Ganoderma lucidum soluble fiber on the proliferation of colon cancer cells HCT116 (compared with the control group, *** P<0.001; compared with Ganoderma lucidum soluble fiber 3-5 group, ### P<0.001)

[0034] Figure 2 The effect of Ganoderma lucidum soluble fiber on the body weight of tumor-bearing mice;

[0035] Figure 3 The effect of Ganoderma lucidum soluble fiber on the volume of HCT116 colon cancer cell transplanted tumors in mice (compared with the model group, * P<0.05, *** P<0.001; compared with the Ganoderma lucidum soluble fiber 67 group, # P<0.05);

[0036] Figure 4 The effect of Ganoderma lucidum soluble fiber on the weight of HCT116 colon cancer cell transplanted tumors in mice (compared with the model group, * P<0.05, *** P<0.001; compared with the Ganoderma lucidum soluble fiber 67 group, # P<0.05);

[0037] Figure 5 The effect of Ganoderma lucidum soluble fiber on the subtypes and proportions of peripheral blood T cells in mice (compared with the control group, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01);

[0038] Figure 6 The effect of Ganoderma lucidum soluble fiber on the body weight of mice (compared with the control group, *** P<0.001; compared with the model group, ## P<0.01, ### P<0.001);

[0039] Figure 7 The effect of Ganoderma lucidum soluble fiber on the total cholesterol content in serum of mice (compared with the control group, *** P<0.001; compared with the model group, ## P<0.01, ### P<0.001);

[0040] Figure 8 The effect of Ganoderma lucidum soluble fiber on the total triglyceride content in serum of mice (compared with the control group, *** P<0.001; compared with the model group, ## P<0.01, ###P<0.001). DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these examples are merely exemplary and do not constitute any limitation to the scope of the present invention. The methods, ingredients and amounts involved in the following examples are all conventional methods known to those skilled in the art unless otherwise specified.

[0042] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited. Among them, Ganoderma lucidum fruiting body (red ganoderma), Ganoderma lucidum fruiting body (purity ganoderma), broken wall red ganoderma spore powder (purity 99.99%), broken wall purple ganoderma spore powder (purity 99.99%), wall-removed red ganoderma spore powder (purity 99.99%), and wall-removed purple ganoderma spore powder (purity 99.99%) are all purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd.

[0043] The preparation method of artificial gastric juice is as follows:

[0044] Take an appropriate amount of purified water, add 3.84 mL of concentrated hydrochloric acid, shake well, add purified water to adjust the pH to 1.5-4.0, then add pepsin (the amount of pepsin is 1% (g / mL) of the total volume), shake well, and you have it.

[0045] The preparation method of artificial intestinal fluid is as follows:

[0046] Take potassium dihydrogen phosphate, add appropriate amount of purified water to dissolve, shake well, add 0.1 mol / L sodium hydroxide to adjust the pH to 7.5-9.0, then add trypsin (the amount of trypsin is 1% (g / mL) of the total volume), shake well, and you have it.

[0047] Embodiment 1 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0048] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0049] (2) taking 5 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.8 at a solid-liquid ratio of 1:20, 1:30, 1:40, 1:50, and 1:60 g / mL, respectively, and then adding cellulase at a mass percentage of 2% of the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 120 min, boiling and deactivating the enzyme for 10 min after the enzymolysis is completed, and filtering under reduced pressure to obtain residues 1-5;

[0050] (3) adding artificial gastric juice having a pH value of 3 to the residue obtained in step (2) at a ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 1-5 and residue 1-5;

[0051] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue 1-5 obtained in step (3) at a ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 1′-5′;

[0052] (5) mixing the filtrate 1-5 obtained in step (3) and the filtrate 1'-5' obtained in step (4), respectively, and concentrating under reduced pressure to obtain a concentrated solution 1-5;

[0053] (6) The concentrated solution 1-5 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after alcohol precipitation overnight. The precipitate was collected and dried at 65° C. to constant weight, and Ganoderma lucidum soluble fiber powder 1, Ganoderma lucidum soluble fiber powder 2, Ganoderma lucidum soluble fiber powder 3, Ganoderma lucidum soluble fiber powder 4, and Ganoderma lucidum soluble fiber powder 5 were obtained. The weights were weighed to be 1.81 g, 1.88 g, 2.03 g, 2.05 g, and 2.01 g, respectively.

[0054] Embodiment 2 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0055] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0056] (2) taking 6 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, and adding phosphate buffer solutions having a pH value of 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0, respectively, at a solid-liquid ratio of 1:40 g / mL, and then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer solution, performing enzymolysis at 50° C. for 120 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 10 min, and filtering under reduced pressure to obtain residue 6-11;

[0057] (3) adding artificial gastric juice with a pH value of 3 to the residue obtained in step (2) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 6-11 and the residue 6-11;

[0058] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 6′-11′;

[0059] (5) The filtrate 6-11 obtained in step (3) and the filtrate 6'-11' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 6-11;

[0060] (6) The concentrated solution 6-11 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight alcohol precipitation. The precipitate was collected and dried at 65° C. to constant weight, and Ganoderma lucidum soluble fiber powder 6, Ganoderma lucidum soluble fiber powder 7, Ganoderma lucidum soluble fiber powder 8, Ganoderma lucidum soluble fiber powder 9, Ganoderma lucidum soluble fiber powder 10, and Ganoderma lucidum soluble fiber powder 11 were obtained. The weights were weighed to be 2.34 g, 2.51 g, 2.78 g, 2.13 g, 2.44 g, and 2.32 g, respectively.

[0061] Embodiment 3 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0062] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0063] (2) taking 5 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 1%, 1.5%, 2%, 2.5%, and 3% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 120 min, boiling and deactivating the enzyme for 10 min after the enzymolysis is completed, and filtering under reduced pressure to obtain residue 12-16;

[0064] (3) adding artificial gastric juice with a pH value of 3 to the residue obtained in step (2) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 12-16 and the residue 12-16;

[0065] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting 12′-16′ of the filtrate;

[0066] (5) The filtrate 12-16 obtained in step (3) and the filtrate 12'-16' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 12-16;

[0067] (6) The concentrated solution 12-16 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after alcohol precipitation overnight. The precipitate was collected and dried at 65° C. to constant weight, and Ganoderma lucidum soluble fiber powder 12, Ganoderma lucidum soluble fiber powder 13, Ganoderma lucidum soluble fiber powder 14, Ganoderma lucidum soluble fiber powder 15, and Ganoderma lucidum soluble fiber powder 16 were obtained. The weights were weighed to be 2.57 g, 2.73 g, 2.81 g, 2.67 g, and 2.64 g, respectively.

[0068] Embodiment 4 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0069] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0070] (2) 4 portions of the ganoderma lucidum powder obtained in step (1) were taken, each portion having a weight of 100 g, and a phosphate buffer having a pH value of 5.7 was added to the mixture at a solid-liquid ratio of 1:40 g / mL, and then cellulase was added at a mass percentage of 2% of the mixed solution of ganoderma lucidum powder and phosphate buffer, and enzymolysis was performed at 45° C., 50° C., 55° C., and 60° C. for 120 min, respectively. After the enzymolysis was completed, the mixture was boiled for 10 min to inactivate the enzyme, and filtered under reduced pressure to obtain residue 17-20;

[0071] (3) adding artificial gastric juice with a pH value of 3 to the residue obtained in step (2) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 17-20 and the residue 17-20;

[0072] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting 17′-20′ of the filtrate;

[0073] (5) The filtrate 17-20 obtained in step (3) and the filtrate and 17'-20' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 17-20;

[0074] (6) The concentrated solution 17-20 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 17, Ganoderma lucidum soluble fiber powder 18, Ganoderma lucidum soluble fiber powder 19, and Ganoderma lucidum soluble fiber powder 20. The weights of the powders were 2.57 g, 2.83 g, 2.67 g, and 2.74 g, respectively.

[0075] Embodiment 5 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0076] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0077] (2) 3 portions of the ganoderma lucidum powder obtained in step (1) were taken, each portion having a weight of 100 g, and a phosphate buffer having a pH value of 5.7 was added to the mixture at a solid-liquid ratio of 1:40 g / mL, and then cellulase was added at a mass percentage of 2% of the mixed solution of ganoderma lucidum powder and phosphate buffer, and enzymolysis was performed at 50° C. for 60, 90, and 120 min, respectively. After the enzymolysis was completed, the mixture was boiled for 10 min to inactivate the enzyme, and filtered under reduced pressure to obtain residues 21-23;

[0078] (3) adding artificial gastric juice with a pH value of 3 to the residue obtained in step (2) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 21-23 and the residue 21-23;

[0079] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 21′-23′;

[0080] (5) The filtrate 21-23 obtained in step (3) and the filtrate 21'-23' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 21-23;

[0081] (6) The concentrated solution 21-23 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 21, Ganoderma lucidum soluble fiber powder 22, and Ganoderma lucidum soluble fiber powder 23. The weights were weighed to be 2.58 g, 2.63 g, and 2.84 g, respectively.

[0082] Embodiment 6 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0083] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0084] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and inactivating the enzyme for 4, 6, 8, and 10 min, respectively, and filtering under reduced pressure to obtain a residue;

[0085] (3) adding artificial gastric juice with a pH value of 3 to the residue obtained in step (2) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 24-27 and the residue 24-27;

[0086] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting 24′-27′ of the filtrate;

[0087] (5) The filtrate 24-27 obtained in step (3) and the filtrate 24'-27' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 24-27;

[0088] (6) The concentrated solution 24-27 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight alcohol precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 24, Ganoderma lucidum soluble fiber powder 25, Ganoderma lucidum soluble fiber powder 26, and Ganoderma lucidum soluble fiber powder 27. The weights of the powders were 2.77 g, 2.83 g, 2.97 g, and 2.82 g, respectively.

[0089] Embodiment 7 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0090] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0091] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0092] (3) adding artificial gastric juice having a pH value of 3 to the residue obtained in step (2) at a solid-liquid ratio of 1:4, 1:6, 1:8, and 1:10 g / mL, respectively, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the filtrate 28-31 and the filter residue 28-31;

[0093] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting 28′-31′ of the filtrate;

[0094] (5) The filtrate 28-31 obtained in step (3) and the filtrate 28'-31' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 28-31;

[0095] (6) The concentrated solution 28-31 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 28, Ganoderma lucidum soluble fiber powder 29, Ganoderma lucidum soluble fiber powder 30, and Ganoderma lucidum soluble fiber powder 31. The weights were weighed to be 2.84 g, 3.03 g, 3.15 g, and 3.13 g, respectively.

[0096] Embodiment 8 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0097] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0098] (2) taking 6 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0099] (3) adding artificial gastric juice with pH values ​​of 1.5, 2, 2.5, 3, 3.5, and 4 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, respectively, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 32-37 and residue 32-37;

[0100] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:5 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 32′-37′;

[0101] (5) The filtrate 32-37 obtained in step (3) and the filtrate 32'-37' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 32-37;

[0102] (6) The concentrated solution 32-37 obtained in step (5) was mixed with 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after alcohol precipitation overnight. The precipitate was collected and dried at 65° C. to constant weight, and Ganoderma lucidum soluble fiber powder 32, Ganoderma lucidum soluble fiber powder 33, Ganoderma lucidum soluble fiber powder 34, Ganoderma lucidum soluble fiber powder 35, Ganoderma lucidum soluble fiber powder 36, and Ganoderma lucidum soluble fiber powder 37 were obtained. The weights were weighed to be 2.54 g, 2.66 g, 2.86 g, 3.13 g, 3.35 g, and 3.16 g, respectively.

[0103] Embodiment 9 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0104] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0105] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0106] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, extracting at 37° C. for 30, 60, 90, and 120 min, respectively, filtering under reduced pressure, and collecting filtrates 38-41 and residues 38-41;

[0107] (4) adding artificial intestinal fluid with a pH value of 8 to the residue obtained in step (3) at a solid-liquid ratio of 1:5 (g / mL), extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 38′-41′;

[0108] (5) The filtrate 38-41 obtained in step (3) and the filtrate 38'-41' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 38-41;

[0109] (6) The concentrated solution 38-41 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 38, Ganoderma lucidum soluble fiber powder 39, Ganoderma lucidum soluble fiber powder 40, and Ganoderma lucidum soluble fiber powder 41. The weights were weighed to be 2.74 g, 3.11 g, 3.31 g, and 3.29 g, respectively.

[0110] Embodiment 10 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0111] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0112] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0113] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, extracting at 37° C. for 90 min, filtering under reduced pressure, and collecting the corresponding filtrate and residue;

[0114] (4) adding artificial intestinal fluid with a pH value of 8 to the filter residue obtained in step (3) at a solid-liquid ratio of 1:4, 1:6, 1:8, and 1:10 g / mL, respectively, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 42-45;

[0115] (5) The filtrate obtained in step (3) is mixed with the filtrate 42-45 obtained in step (4) respectively, and concentrated under reduced pressure to obtain a concentrated solution 42-45;

[0116] (6) The concentrated solution 42-45 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight alcohol precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 42, Ganoderma lucidum soluble fiber powder 43, Ganoderma lucidum soluble fiber powder 44, and Ganoderma lucidum soluble fiber powder 45. The weights were weighed to be 3.14 g, 3.47 g, 3.49 g, and 3.47 g, respectively.

[0117] Embodiment 11 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0118] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0119] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0120] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, extracting at 37° C. for 90 min, filtering under reduced pressure, and collecting the corresponding filtrate and residue;

[0121] (4) adding artificial intestinal fluids with pH values ​​of 7.5, 8, 8.5, and 9 to the residue obtained in step (3) at a solid-liquid ratio of 1:6 g / mL, respectively, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrates 46-49;

[0122] (5) The filtrate obtained in step (3) is mixed with the filtrate 46-49 obtained in step (4), respectively, and concentrated under reduced pressure to obtain concentrated solution 46-49;

[0123] (6) The concentrated solutions 46-49 obtained in step (5) were mixed with 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight precipitation. The precipitate was collected and dried at 65° C. to constant weight, and Ganoderma lucidum soluble fiber powder 46, Ganoderma lucidum soluble fiber powder 47, Ganoderma lucidum soluble fiber powder 48, and Ganoderma lucidum soluble fiber powder 49 were obtained. The weights were 3.23 g, 3.45 g, 3.52 g, and 3.41 g, respectively.

[0124] Embodiment 12 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0125] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0126] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0127] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, extracting at 37° C. for 90 min, filtering under reduced pressure, and collecting the corresponding filtrate and residue;

[0128] (4) adding artificial intestinal fluid with a pH value of 8.5 to the residue obtained in step (3) at a solid-liquid ratio of 1:6 g / mL, extracting at 37° C. for 30, 60, 90, and 120 min, respectively, filtering under reduced pressure, and collecting filtrate 50-53;

[0129] (5) mixing the filtrate obtained in step (3) with the filtrate 50-53 obtained in step (4), respectively, and concentrating under reduced pressure to obtain a concentrated solution 50-53;

[0130] (6) The concentrated solution 50-53 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:3, and the mixture was centrifuged (5000 rpm, 10 min) after overnight precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 50, Ganoderma lucidum soluble fiber powder 51, Ganoderma lucidum soluble fiber powder 52, and Ganoderma lucidum soluble fiber powder 53. The weights of the powders were 3.12 g, 3.49 g, 3.51 g, and 3.52 g, respectively.

[0131] Embodiment 13 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0132] (1) taking raw material Ganoderma lucidum, crushing it to obtain Ganoderma lucidum powder;

[0133] (2) taking 4 portions of the ganoderma lucidum powder obtained in step (1), each portion having a weight of 100 g, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of ganoderma lucidum powder and phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and deactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0134] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, extracting at 37° C. for 90 min, filtering under reduced pressure, and collecting the corresponding filtrate A and residue;

[0135] (4) adding artificial intestinal fluid with a pH value of 8.5 to the residue obtained in step (3) at a solid-liquid ratio of 1:6 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting the corresponding filtrate B;

[0136] (5) mixing the filtrate A obtained in step (3) and the filtrate B obtained in step (4), and concentrating under reduced pressure to obtain a concentrated solution;

[0137] (6) The concentrated solution obtained in step (5) was mixed with 95% ethanol solution at a volume ratio of 1:3, 1:4, 1:5, and 1:6, respectively, and the mixture was centrifuged (5000 rpm, 10 min) after alcohol precipitation overnight. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 54, Ganoderma lucidum soluble fiber powder 55, Ganoderma lucidum soluble fiber powder 56, and Ganoderma lucidum soluble fiber powder 57. The weights of the powders were 3.50 g, 3.88 g, 3.90 g, and 3.87 g, respectively.

[0138] Embodiment 14 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0139] (1) respectively taking raw materials of Ganoderma lucidum, ... broken wall spore powder of Ganoderma lucidum, and wall-removed Ganoderma lucidum spore powder, and crushing them to obtain Ganoderma lucidum powder;

[0140] (2) taking 100 g of the Ganoderma lucidum powder obtained in step (1), respectively, adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:40 g / mL, then adding cellulase at a mass percentage of 2% in the mixed solution of Ganoderma lucidum powder and phosphate buffer, and performing enzymolysis at 50° C. for 90 min. After the enzymolysis was completed, the mixture was boiled for 8 min to inactivate the enzyme, and filtered under reduced pressure to obtain residues 58-61;

[0141] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:8 g / mL, extracting at 37° C. for 90 min, filtering under reduced pressure, and collecting filtrate 58-61 and residue 58-61;

[0142] (4) adding artificial intestinal fluid with a pH value of 8.5 to the residue obtained in step (3) at a solid-liquid ratio of 1:6 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate 58′-61′;

[0143] (5) The filtrate 58-61 obtained in step (3) and the filtrate 58'-61' obtained in step (4) are mixed respectively, and concentrated under reduced pressure to obtain a concentrated solution 58-61;

[0144] (6) The concentrated solution 58-61 obtained in step (5) was mixed with a 95% ethanol solution at a volume ratio of 1:4, and the mixture was centrifuged (5000 rpm, 10 min) after overnight alcohol precipitation. The precipitate was collected and dried at 65° C. to constant weight, thereby obtaining Ganoderma lucidum soluble fiber powder 58, Ganoderma lucidum soluble fiber powder 59, Ganoderma lucidum soluble fiber powder 60, and Ganoderma lucidum soluble fiber powder 61. The weights of the powders were 3.93 g, 4.01 g, 2.85 g, and 11.17 g, respectively.

[0145] Comparative Example 1 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0146] The only difference from Example 1 is that the cellulase in step (2) is replaced by papain to obtain Ganoderma lucidum soluble fiber powder 62, Ganoderma lucidum soluble fiber powder 63, Ganoderma lucidum soluble fiber powder 64, Ganoderma lucidum soluble fiber powder 65, and Ganoderma lucidum soluble fiber powder 66. The weights were weighed and were 1.66 g, 1.69 g, 1.87 g, 1.82 g, and 1.81 g, respectively.

[0147] Comparative Example 2 A method for preparing ganoderma lucidum soluble fiber comprises the following steps:

[0148] (1) crushing the raw material Ganoderma lucidum to obtain Ganoderma lucidum powder;

[0149] (2) taking 100 g of the Ganoderma lucidum powder obtained in step (1), adding a phosphate buffer having a pH value of 5.7 at a solid-liquid ratio of 1:10 g / mL, then adding cellulase at a mass percentage of 2% of the mixed solution of the Ganoderma lucidum powder and the phosphate buffer, performing enzymolysis at 50° C. for 90 min, and after the enzymolysis is completed, boiling and inactivating the enzyme for 8 min, filtering under reduced pressure, and obtaining a residue;

[0150] (3) adding artificial gastric juice with a pH value of 3.5 to the residue obtained in step (2) at a solid-liquid ratio of 1:3 g / mL, extracting at 37° C. for 90 min, filtering under reduced pressure, and collecting filtrate A and residue;

[0151] (4) adding artificial intestinal fluid with a pH value of 8.5 to the residue obtained in step (3) at a solid-liquid ratio of 1:2 g / mL, extracting at 37° C. for 60 min, filtering under reduced pressure, and collecting filtrate B and residue;

[0152] (5) mixing the filtrates A and B obtained in step (3) and step (4), and concentrating under reduced pressure to obtain a concentrated solution;

[0153] (6) The concentrated solution obtained in step (5) was mixed with 95% ethanol solution at a volume ratio of 1:4, and the mixture was precipitated overnight and then centrifuged (5000 rpm, 10 min). The precipitate was collected and dried at 65° C. to constant weight to obtain Ganoderma lucidum soluble fiber powder 67.

[0154] Effect Experiment

[0155] 1. Application of Ganoderma lucidum soluble fiber in anti-tumor (in vitro)

[0156] (1) Cell lines

[0157] Human colon cancer HCT116 cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were grown in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity, and the medium was changed every 2-3 days.

[0158] (2) Experimental samples

[0159] The Ganoderma lucidum soluble fiber powders 1-5 and 62-66 prepared in Example 1 and Comparative Example 1 were prepared into a 10 mg / mL sample solution using DMEM, sterilized by filtering through a 0.22 μm filter membrane, and stored at -20° C. for future use.

[0160] (3) Cell viability assay

[0161] HCT116 cells in the logarithmic growth phase were obtained and digested with trypsin to form a single cell suspension. The cell density was adjusted to 5 × 10 4 / mL, and the cells were evenly dispersed in a 96-well plate; when the cells grew to 60% confluence, the cells were divided into a control group and a soluble fiber group, the original DMEM was removed, 200 μm DMEM was added to the control group cells, and 200 μm 5 mg / mL sample solution was added to the Ganoderma lucidum soluble fiber group, and the culture was continued for 24 hours; after the culture was completed, the original culture medium was removed, 10 μm CCK-8 solution and 90 μm DMEM were added again, and 10 μm CCK-8 solution and 90 μm DMEM were added to the wells without cells as background controls, and the incubation was continued for 3 hours; after the incubation was completed, the light absorption value of each well at 570 nm was detected by a multifunctional microplate reader, and the relative cell viability was calculated according to the light absorption value of each well, and the relative cell viability = [(light absorption value of Ganoderma lucidum soluble fiber group-light absorption value of background group) / (light absorption value of control group-light absorption value of background group)] × 100%.

[0162] Depend on Figure 1It can be seen that each Ganoderma lucidum soluble fiber sample can significantly inhibit the proliferation of HCT116 cells; Ganoderma lucidum soluble fiber powder 3, Ganoderma lucidum soluble fiber powder 4 and Ganoderma lucidum soluble fiber powder 5 have the strongest inhibitory effect on HCT116 cells, and the effects are equivalent; the inhibitory effect of Ganoderma lucidum soluble fiber powders 3-5 on the proliferation of HCT116 cells is significantly stronger than that of Ganoderma lucidum soluble fiber powders 64-66.

[0163] 2. Application of Ganoderma lucidum soluble fiber in anti-tumor (in vivo)

[0164] (1) Cell lines

[0165] Human colon cancer HCT116 cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were grown in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured in an incubator at 37°C, 5% CO2, and saturated humidity, and the medium was changed every 2-3 days.

[0166] (2) Experimental samples

[0167] The Ganoderma lucidum soluble fiber powders 58-61 and Ganoderma lucidum soluble fiber powder 67 prepared in Example 14 and Comparative Example 2 were prepared into 10 mg / mL sample solutions with sterile water for later use.

[0168] (3) Experimental animals

[0169] Sixty male BALB / c nude mice, weighing 18-22 g, were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences and raised in the SPF-grade experimental animal environment of Zhejiang Institute of Traditional Chinese Medicine Co., Ltd. The optimal temperature was 20-26°C and the relative humidity was 50%-60%. The mice were allowed to eat freely.

[0170] (4) Tumor-bearing mouse model

[0171] Adjust the density of HCT116 cells to 2 × 10 7 After disinfecting the right axillary skin of the mouse with an alcohol cotton ball, 0.2 mL of cell suspension was drawn with a sterile syringe and inoculated subcutaneously in the right axillary skin. The tumor growth was checked every day. When the tumor grew to 60 mm 3 Start administering the sample solution.

[0172] (5) Grouping and processing

[0173] Tumor-bearing mice were randomly divided into the model group, Ganoderma lucidum soluble fiber powder 58 group (200 mg / kg / day), Ganoderma lucidum soluble fiber powder 59 group (200 mg / kg / day), Ganoderma lucidum soluble fiber powder 60 group, Ganoderma lucidum soluble fiber powder 61 group and Ganoderma lucidum soluble fiber powder 67 group (200 mg / kg / day), 10 mice in each group. The samples were continuously gavaged once a day for 4 weeks, and the model group was given an equal amount of sterile water once a day.

[0174] (6) Mouse body weight and tumor volume detection

[0175] Use a vernier caliper to measure and record the longest diameter L (cm) and the shortest diameter S (cm) of the tumor according to the formula: V = LS 2 / 2 Calculate the tumor volume twice a week, and record the weight of the mice for 4 consecutive weeks. After the treatment, the nude mice were killed, the tumor tissues were removed and weighed, and the tumor inhibition rate was calculated. Tumor inhibition rate = (1-average tumor volume of the sample group / average tumor volume of the model group) × 100%.

[0176] Depend on Figure 2-4 It can be seen that there was no significant change in the body weight of mice in each Ganoderma soluble fiber group; compared with the model group, the tumor volume and tumor weight of mice in each Ganoderma soluble fiber group were significantly reduced; and the inhibitory effect of Ganoderma soluble fiber 58-61 on tumor volume and tumor weight was significantly stronger than that of Ganoderma soluble fiber 67.

[0177] 3. Application of Ganoderma lucidum soluble fiber in regulating immunity

[0178] (1) Experimental samples

[0179] The Ganoderma lucidum soluble fiber powder 58-61 prepared in Example 14 was prepared into a 10 mg / mL sample solution with sterile water for later use.

[0180] (2) Experimental animals

[0181] ICR mice, weighing 18-22 g, were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences and raised in the SPF-grade experimental animal environment of Zhejiang Institute of Traditional Chinese Medicine Co., Ltd. The optimal temperature was 20-26°C and the relative humidity was 50%-60%. The mice were allowed to eat freely.

[0182] (3) Experimental methods

[0183] Grouping: 50 ICR mice, half male and half female, including 10 3-month-old mice and 40 14-month-old mice, were adaptively fed for 7 days. The 14-month-old mice were randomly divided into the elderly model group, the Ganoderma lucidum soluble fiber powder 58 group (200 mg / kg / day), the Ganoderma lucidum soluble fiber powder 59 group (200 mg / kg / day), the Ganoderma lucidum soluble fiber powder 60 group and the Ganoderma lucidum soluble fiber powder 61 group (200 mg / kg / day) according to their body weight, and the 3-month-old mice were used as the young control group, with 10 mice in each group.

[0184] Treatment: The sample group was given a designated dose of sample solution by gavage for 8 weeks, once a day, and the control group and model group were given an equal amount of sterile water, once a day. The mice were weighed 24 hours after the last gavage treatment. The mice were then killed, and blood was collected from the abdominal aorta. Flow cytometry was used to detect the subtypes and proportions of circulating blood T lymphocytes.

[0185] (4) Experimental results

[0186] Depend on Figure 5 It can be seen that compared with the control group, the peripheral blood CD3 + CD4 + and CD3 + CD8 + Compared with the model group, the proportion of CD3 T cells in the peripheral blood of mice in each Ganoderma lucidum soluble fiber group was significantly reduced. + CD4 + and CD3 + CD8 + The proportion of T cells in the four groups of Ganoderma lucidum soluble fiber mice increased significantly, and the peripheral blood CD3 + CD4 + and CD3 + CD8 + There was no significant difference in the proportion of T cells.

[0187] 4. Application of Ganoderma lucidum soluble fiber in lowering blood lipids

[0188] (1) Experimental samples

[0189] The Ganoderma lucidum soluble fiber powder 58-61 prepared in Example 14 was prepared into a 10 mg / mL sample solution with sterile water for later use.

[0190] (2) Experimental animals

[0191] ICR mice, weighing 18-22 g, were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences and raised in the SPF-grade experimental animal environment of Zhejiang Institute of Traditional Chinese Medicine Co., Ltd. The optimal temperature was 20-26°C and the relative humidity was 50%-60%. The mice were free to drink water and eat ordinary feed.

[0192] (3) Experimental methods

[0193] Grouping: 50 ICR mice, male, were adaptively raised for 7 days and fed with a normal diet (4% fat, 20% carbohydrates and 20% protein). The mice were randomly divided into a control group, a model group, a sample low-dose group (100 mg / kg / day), a sample medium-dose group (200 mg / kg / day) and a sample high-dose group (400 mg / kg / day) according to their body weight, with 10 mice in each group.

[0194] Feed replacement: The mice in the control group continued to eat ordinary feed, while the mice in the model group and sample group were replaced with a high-fat feed (60% fat, 20% carbohydrates and 20% protein).

[0195] Treatment: The sample group was given a designated dose of sample solution by gavage for 8 weeks, once a day, and the control group and model group were given an equal amount of sterile water, once a day. The mice were weighed 24 hours after the last gavage treatment. The mice were then killed, blood was collected from the abdominal aorta, and serum was separated to detect the content of total cholesterol and total triglyceride.

[0196] (4) Experimental results

[0197] Depend on Figure 6-8 The results showed that compared with the control group, the body weight, serum TC and TG contents of the mice in the model group were significantly increased; compared with the model group, the body weight, serum TC and TG contents of the mice in each Ganoderma lucidum soluble fiber group were significantly decreased, and there was no significant difference in the body weight, serum TC and TG contents of the mice in the four Ganoderma lucidum soluble fiber groups.

[0198] 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 preparing ganoderma lucidum soluble fiber, characterized in that: The following steps are involved: (1) taking raw material Ganoderma lucidum, and crushing it to obtain Ganoderma lucidum powder; (2) mixing the ganoderma lucidum powder obtained in step (1) with phosphate buffer, adding cellulase for enzymolysis, boiling to inactivate the enzyme after the enzymolysis is completed, and filtering under reduced pressure to obtain a residue; (3) adding artificial gastric juice to the residue obtained in step (2) for extraction, filtering under reduced pressure, and collecting filtrate A and residue; (4) adding artificial intestinal fluid to the filter residue obtained in step (3) for extraction, filtering under reduced pressure, and collecting filtrate B; (5) mixing the filtrate A and the filtrate B obtained in step (3) and step (4), and concentrating under reduced pressure to obtain a concentrated solution; (6) The concentrated solution obtained in step (5) is mixed with an ethanol solution, and after alcohol precipitation overnight, the mixture is centrifuged, and the precipitate is collected and dried to a constant weight to obtain a Ganoderma lucidum soluble fiber powder.

2. The preparation method according to claim 1, characterized in that: The ganoderma lucidum in step (1) is selected from one of red ganoderma lucidum, purple ganoderma lucidum, broken wall ganoderma lucidum spore powder and de-walled ganoderma lucidum spore powder.

3. The preparation method according to claim 1, characterized in that: The solid-liquid ratio of the ganoderma lucidum powder to the phosphate buffer in step (2) is 1:20-60 g / mL, and the pH of the phosphate buffer is 5.5-6.

0.

4. The preparation method according to claim 1, characterized in that: In step (2), the mass percentage of the cellulase in the mixed solution of ganoderma lucidum powder and phosphate buffer is 1-3%, the enzymolysis temperature is 45-60° C., the enzymolysis time is 60-120 min, and the boiling inactivation time is 5-10 min.

5. The preparation method according to claim 1, characterized in that: In step (3), the material-liquid ratio of the residue to the artificial gastric juice is 1:5-10 g / mL, and the pH of the artificial gastric juice is 1.5-4.

0.

6. The preparation method according to claim 1, characterized in that: In step (4), the material-liquid ratio of the filter residue to the artificial intestinal fluid is 1:5-10 g / mL, and the pH of the artificial intestinal fluid is 7.5-9.

0.

7. The preparation method according to claim 1, characterized in that: In step (6), the volume ratio of the concentrated solution to ethanol is 1:3-6, the volume concentration of the ethanol solution is 95%, the centrifugation is performed at a speed of 5000 rpm for 10 min, and the drying temperature is 65°C.

8. A Ganoderma lucidum soluble fiber prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the ganoderma lucidum soluble fiber prepared by the preparation method according to any one of claims 1 to 7 in the preparation of anti-tumor, blood lipid lowering and immunity regulating products.

10. The use according to claim 9, characterized in that: The dosage form of the product is powder, granule, capsule, tablet, oral solution or pill.

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