Application of beta-1, 3-glucan in preparation of product for regulating intestinal flora balance

Abstract

The invention provides an application of beta-1, 3-glucan in preparation of a product for regulating intestinal flora balance. The invention finds that the beta-1, 3-glucan can inhibit the growth of intestinal harmful bacteria such as desulfatovibrio, Coriobacteria UCG-002, Colidextribacter and the like, and can promote and / or not inhibit the growth of intestinal probiotics such as clostridium sensricitum 1, Bacteroides, Lactobacillus, Clostridium praeparatum and the like, which indicates that the beta-1, 3-glucan is suitable for preparing a product for regulating the balance of intestinal flora, and also shows that the beta-1, 3-glucan is suitable for preparing a product for regulating the balance of intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to the use of β-1,3-glucan in the preparation of products for regulating the balance of intestinal flora. Background Art

[0002] An imbalance in the intestinal flora may cause a variety of problems in the human body: (1) Digestive system problems: diarrhea, constipation, bloating, abdominal pain, nausea, vomiting, etc., which affect nutrient absorption; (2) Decreased immunity: weakening immune function, increasing the risk of infection, and even causing autoimmune diseases; (3) Metabolic disorders: inducing metabolic diseases such as obesity, diabetes, and cardiovascular disease; (4) Emotional and cognitive disorders: intestinal flora affects neurotransmitters through the "gut-brain axis", which may lead to anxiety, depression, or decreased cognitive function; (5) Chronic inflammation: may cause chronic intestinal and systemic inflammation, increasing the risk of cancer. In addition, the causes of intestinal flora imbalance are also very common: (1) Unreasonable diet structure: A diet high in sugar, fat, and fiber will inhibit the growth of beneficial bacteria and promote the reproduction of harmful bacteria; (2) Stress and lifestyle: Long-term mental stress, staying up late, lack of exercise, etc. will weaken intestinal immunity and affect the balance of flora; (3) Disease and infection: Intestinal inflammation, infectious diseases (such as gastroenteritis) or immune system abnormalities may directly interfere with the flora ecology; (4) Antibiotic abuse: While antibiotics kill pathogenic bacteria, they will also destroy beneficial bacteria in the intestines, leading to a disorder in the flora structure. Therefore, it is very important to regulate the balance of intestinal flora.

[0003] β-1,3-glucan is a polysaccharide composed of glucose molecules connected by β-1,3 glycosidic bonds. It is widely found in organisms such as fungi, bacteria, yeast, oats, and barley. Due to its unique biological activity, β-1,3-glucan has shown broad application potential in many fields such as fat substitutes, skin moisturizing, and hair care. However, there are currently no reports on its role in regulating the balance of intestinal flora. Summary of the Invention

[0004] The present invention addresses the deficiencies of the existing technology and aims to provide an application of β-1,3-glucan in the preparation of products for regulating the balance of intestinal flora, providing a new raw material for products for regulating the balance of intestinal flora and also providing a new application for β-1,3-glucan.

[0005] The first object of the present invention is to provide a use of β-1,3-glucan in the preparation of a product for regulating the balance of intestinal flora.

[0006] The second object of the present invention is to provide the use of β-1,3-glucan in the preparation of products for enhancing immunity.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides the use of β-1,3-glucan in the preparation of a product for regulating the balance of intestinal flora, wherein the regulating the balance of intestinal flora is to inhibit the growth of harmful intestinal bacteria and promote and / or not inhibit the growth of probiotics in the intestine; wherein the harmful intestinal bacteria are one or more of Desulfovibrio, Coriobacteriaceae_UCG-002, and Colidextribacter, and the probiotics are one or more of Clostridium sensu stricto 1, Bacteroides, Lactobacillus, and Faecalibacterium.

[0009] Preferably, the Bacteroides genus is Bacteroides plebeius.

[0010] Preferably, the Faecalibacterium is Faecalibacterium prausnitzii.

[0011] The present invention also provides the use of beta-1,3-glucan in preparing products for enhancing immunity.

[0012] Preferably, the β-1,3-glucan is extracted from Ganoderma lucidum.

[0013] Preferably, the weight average molecular weight of the β-1,3-glucan is greater than 80 KDa.

[0014] More preferably, the weight average molecular weight of the β-1,3-glucan is 300 KDa.

[0015] Preferably, the monosaccharide composition ratio of the β-1,3-glucan is as follows, in terms of molar percentage: glucose: glucuronic acid: mannose: fucose: xylose = (90-92): (0-1): (4-6): (0-1): (0-2). Most preferably, it is 91.7: 1.0: 4.5: 1.0: 1.8.

[0016] Preferably, the linkage of the β-1,3-glucan includes 1,3-Glcp, 1,3,6-Glcp, and T-Glcp, and is branched by a single Glc side chain at position O-6. The glycosidic bond composition of the β-1,3-glucan, calculated in molar percentage, is as follows: 1,3-Glcp: 1,3,6-Glcp: T-Glcp: 1,6-Glcp: 1,4-Glcp: 1,3,4-Manp = (45.3-48.1): (22.1-23.5): (20.5-21.3): (2.2-2.4): (4.7-4.9): (2.4-2.6). Most preferably, it is 48.1: 22.1: 20.5: 2.2: 4.7: 2.4.

[0017] Preferably, the preparation method of β-1,3-glucan comprises the following steps:

[0018] S1. Extracting Ganoderma lucidum at 95-100°C for 6-8 hours, and separating the residue and the extract;

[0019] S2. extracting the residue obtained in S1 with alkali at 75-85°C for 5-7h, separating the residue and the extract, and neutralizing the extract to obtain a neutralized extract;

[0020] S3. The extract obtained in S1 and the neutralized extract obtained in S2 are combined, and microfiltration, alcohol precipitation, and ultrasound are performed in sequence to obtain the β-1,3-glucan.

[0021] Further preferably, in the water extraction in S1, the mass ratio of Ganoderma lucidum to water is 1.5 kg: 64-72 L.

[0022] Further preferably, the water extraction in S1 is a two-step water extraction; wherein, during the first water extraction, the mass ratio of Ganoderma lucidum to water is 1.5 kg: 36-40 L, and the time is 3.5-4.5 h; during the second water extraction, the mass ratio of Ganoderma lucidum to water is 1.5 kg: 28-32 L, and the time is 2.5-3.5 h.

[0023] Further preferably, in the alkaline extraction in S2, the extraction reagent used is a NaOH solution, such as a 0.2-0.6 mol / L NaOH solution.

[0024] More preferably, the ratio of the residue to the NaOH solution is 1 kg: 36-44 L.

[0025] More preferably, the alkaline extraction in S2 is a two-step alkaline extraction; wherein, during the first alkaline extraction, the mass ratio of the residue to the NaOH solution is 1 kg: 18-22 L, and the time is 2.5-3.5 h; during the second alkaline extraction, the mass ratio of the residue to the NaOH solution is 1 kg: 18-22 L, and the time is 2.5-3.5 h.

[0026] Further preferably, the reagent used for neutralization in S2 is an HCl solution, such as an HCl solution with a concentration of 36% (v / v) to 38% (v / v).

[0027] Further preferably, the pore size of the microfiltration membrane used in the microfiltration in S3 is 0.2-0.5 μm, the operating pressure is 0.2-0.3 MPa, and the temperature is 20-30°C.

[0028] Further preferably, the alcohol used in the alcohol precipitation in S3 is ethanol.

[0029] More preferably, the alcohol precipitation time in S3 is 1.8 to 2.2 hours.

[0030] Further preferably, the power of the ultrasound in S3 is 2000-4000W.

[0031] Preferably, the product is one or more of food, medicine, and health care products.

[0032] Preferably, the product is a product for treating or assisting in the treatment of any one of irritable bowel syndrome, functional constipation, neonatal necrotizing enterocolitis, intestinal bacterial overgrowth syndrome and inflammatory bowel disease.

[0033] The present invention has the following beneficial effects:

[0034] The present invention provides the use of β-1,3-glucan in the preparation of products that regulate the balance of intestinal flora. The present invention finds that β-1,3-glucan can inhibit the growth of harmful intestinal bacteria such as Desulfovibrio, Coriobacteriaceae_UCG-002, and Colidextribacter, and promote and / or do not inhibit the growth of probiotic intestinal bacteria such as Clostridium sensu stricto 1, Bacteroides, Lactobacillus, and Faecalibacterium, indicating that β-1,3-glucan is suitable for preparing products that regulate the balance of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the molecular weight distribution diagram of the product obtained in Example 1.

[0036] Figure 2 This is the UV-visible scanning spectrum of the product obtained in Example 1.

[0037] Figure 3 This is the infrared absorption spectrum of the product obtained in Example 1.

[0038] Figure 4The product obtained in Example 1 1 H-NMR spectrum.

[0039] Figure 5 The product obtained in Example 1 13 C-NMR spectrum.

[0040] Figure 6 HSQC spectrum of the product obtained in Example 1.

[0041] Figure 7 The HMBC spectrum of the product obtained in Example 1 is shown in FIG.

[0042] Figure 8 A is a schematic diagram of the animal experiment scheme. Figure 8 B is a photo of each immune organ. Figure 8 C is the calculation result of spleen index, Figure 8 D is the result of spleen lymphocyte proliferation activity assay, Figure 8 E is the result of phagocytic ability determination of peritoneal macrophages.

[0043] Figure 9 A is the slice image, Figure 9 B is the statistical result of the total number of spleen cells in mice. Figure 9 C is mouse CD3 + T cells and CD19 + B cell count results.

[0044] Figure 10 The results are for the determination of β-1,3-glucan by high performance gel exclusion chromatography.

[0045] Figure 11 These are the results of determination of NO, TNF-α, and IL-6 contents.

[0046] Figure 12 Figure 2 shows the difference in microbial composition among the groups at the OTU level. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048] Unless otherwise specified, the reagents and materials used in the following examples were all commercially available, and the Ganoderma lucidum used in the following examples was Ganoderma lucidum (Leyss. ex Fr.) Karst.

[0049] Example 1 Preparation and Structural Analysis of β-1,3-Glucan

[0050] 1. Preparation method

[0051] S1. Primary water extraction: Weigh 1.5 kg of pest-free Ganoderma lucidum fruiting bodies, wash, dry, and slice. Add to 38 L of water and soak for 12 h. Then, heat and extract at 100°C for 4 h. Filter the resulting residue (residue 1) and the filtrate (extract 1).

[0052] S2. Secondary water extraction: Residue 1 was added to 30 L of water, heated at 100 ° C for 3 h, and the resulting residue was filtered to obtain residue 2, and the filtrate was obtained as extract 2;

[0053] S3. Primary alkaline extraction: Residue 2 was added to 30 L of 0.5 mol / L NaOH solution (so that the ratio of residue 2 to NaOH solution was 1 kg:20 L), and the mixture was heated at 80°C for 3 h. The resulting residue was filtered to obtain residue 3, and the filtrate was obtained as extract 3.

[0054] S4. Secondary alkaline extraction: Residue 3 was added to 30 L of 0.5 mol / L NaOH solution (so that the ratio of residue 3 to NaOH solution was 1 kg:20 L), and the mixture was heated at 80°C for 3 h. The resulting residue was filtered to obtain residue 4, and the filtrate was obtained as extract 4.

[0055] S5. Combined extracts: After neutralizing extract 3 and extract 4 with 37% (v / v) HCl, extract 1 and extract 2 were combined to obtain a total extract;

[0056] S6. Microfiltration: The total extract was subjected to microfiltration membrane treatment, and the microfiltration non-permeate was collected; wherein the pore size of the microfiltration membrane was 0.2 μm, the operating pressure was controlled at 0.2 to 0.3 MPa, and the temperature was controlled at 20 to 30 ° C;

[0057] S7. Alcohol precipitation: The microfiltration retentate was lyophilized and dissolved in distilled water at a concentration of 20 mg / mL. 95% (v / v) ethanol was then added (to a final ethanol concentration of 50% (v / v)). The mixture was allowed to stand for 2 h to complete the alcohol precipitation. The resulting precipitate was centrifuged and lyophilized to obtain a lyophilized precipitate.

[0058] S8. Ultrasonication: The lyophilized precipitate was dissolved in distilled water at a concentration of 5 mg / mL and sonicated at 3000 W. Samples were taken during the sonication process for real-time molecular weight detection. After degradation to the target molecular weight of 200-400 kDa, the precipitate was centrifuged and lyophilized to obtain 46.0 g of product (polysaccharide content of the product was 95.0% as determined by the phenol-sulfuric acid method).

[0059] The steps of real-time molecular weight detection are as follows:

[0060] The TSK-gel G-4000PWXL column was calibrated using 80kDa, 50kDa, 25kDa, 12kDa, 5kDa and 1kDa dextran standards, and a dextran standard curve was drawn.

[0061] After dissolving 1 mg of sample in 200 μL of distilled water, the sample was filtered through a 0.22 μm aqueous filter membrane. The weight-average molecular weight of the sample was determined by high-performance gel permeation chromatography (HPGPC) using a dextran standard curve. The instrument used for HPGPC included a high-performance liquid chromatograph (Shimadzu LC-20Ai), a differential refractive index detector (RID-20A), an autosampler, and a TSK-gel G-3000PWXL (7.8×300 mm) column. The detection conditions were as follows: the mobile phase was 0.2 mol / L NaCl solution, the column oven temperature was 40°C, the flow rate was 0.6 mL / min, and the injection volume was 20 μL.

[0062] 2. Structural Analysis

[0063] (1) Monosaccharide composition analysis

[0064] The product obtained in Example 1 was hydrolyzed and derivatized, and then its monosaccharide composition was determined by high performance liquid chromatography. The hydrolysis method was based on Chambers RE, Clamp J R. An Assessment of Methanolysis and Other Factors Used in the Analysis of Carbohydrate-Containing Materials [J]. The biochemical journal, 1971, 125: 1009-1018. The derivatization method was based on Strydom DJ. Chromatographic separation of 1-phenyl-3-methyl-Spyrazolone-derivatized neutral, acidic and basic aldoses [J]. Journal of Chromatography A, 1994, 678: 17-23. The high performance liquid chromatography conditions were: Shimadzu LC-20AT high performance liquid chromatograph, chromatographic column COSMOSIL 5C18-PAQ (4.6×250 mm), mobile phase: 81.9% (v / v) phosphate (PBS, 0.1 mol / L, pH=7) and 18.1% (v / v) acetonitrile, column temperature: 35°C, flow rate: 1.0 mL / min, sample injection volume: 10 μL, UV detection wavelength: 245 nm.

[0065] The results are shown in Table 1.

[0066] Table 1

[0067]

[0068] It can be seen that the product obtained in Example 1 has the highest Glc content of 91.7%, proving that it is glucan.

[0069] (2) Molecular weight determination

[0070] The TSK-gel G-3000PWXL column was calibrated using 80kDa, 50kDa, 25kDa, 12kDa, 5kDa and 1kDa dextran standards, and a dextran standard curve was drawn.

[0071] 1 mg of the product obtained in Example 1 was dissolved in 200 μL of distilled water and filtered through a 0.22 μm aqueous filter. The weight-average molecular weight of the product obtained in Example 1 was determined using a standard curve for HPGPC and dextran. The instrument used for HPGPC included a high-performance liquid chromatograph (Shimadzu LC-20Ai), a differential refractive index detector (RID-20A), an autosampler, and a TSK-gel G-3000PWXL (7.8 × 300 mm) chromatographic column. The detection conditions were as follows: the mobile phase was a 0.2 mol / L NaCl solution, the column oven temperature was 40°C, the flow rate was 0.6 mL / min, and the injection volume was 20 μL.

[0072] The results are as follows Figure 1 As shown, it can be seen that its weight average molecular weight is 300KDa.

[0073] (3) UV-visible spectroscopy analysis

[0074] The product obtained in Example 1 was added to distilled water to a final concentration of 1 mg / mL, and distilled water was used as a blank control. UV-visible spectroscopy analysis was performed using a Shimadzu UV-2700 UV-visible full wavelength scanner in the wavelength range of 200-900 nm.

[0075] The results are as follows Figure 2 As shown, there is no obvious absorption peak in the spectrum, indicating that the product obtained in Example 1 does not contain protein or nucleic acid substances and is relatively pure.

[0076] (4) Infrared spectroscopy analysis

[0077] 1.5 mg of the product obtained in Example 1 and 200 mg of potassium bromide were weighed and ground in a mortar until the mixture was uniform. The mixture was pressed into tablets using a mold. The potassium bromide tablets were used as a blank control. The infrared spectrometer PerkinElmer Spectrum Two was used to analyze the mixture at 4000-400 cm -1 Scan within the wavenumber range.

[0078] The results are as follows Figure 3 As shown, it can be seen that there is 891.0cm -1 The absorption peak at , indicates that the product obtained in Example 1 has β configuration.

[0079] (5) Methylation analysis

[0080] The methylation analysis of the product obtained in Example 1 was performed using an Agilent Technologies 7890B-5977B gas chromatograph-mass spectrometer. The analysis procedure was as follows: maintain at 120°C for 1 min; increase the temperature to 210°C at a rate of 3°C / min and maintain this temperature for 2 min; then increase the temperature to 260°C at a rate of 10°C / min and maintain this temperature for 4 min.

[0081] The results are shown in Table 2.

[0082] Table 2

[0083]

[0084] As can be seen, the polysaccharide in the product obtained in Example 1 is primarily composed of 1,3-Glcp glycosidic bonds, with a content as high as 48.1%. In addition, it also contains a certain amount of 1,3,6-Glcp (22.1%) and T-Glcp (20.5%), indicating that its main chain is 1,3-linked and has a terminal branch at the O-6 position. Based on the molar ratio, the β-1,3-glucan content in the polysaccharide was determined to be 90.7%. In addition, since the polysaccharide content in the product obtained in Example 1 is 95.0%, combined with the β-1,3-glucan content in the polysaccharide, it can be calculated that the purity of the β-1,3-glucan in the product obtained in Example 1 is 86.17%.

[0085] (6) Nuclear magnetic resonance analysis

[0086] Weigh 20 mg of the product obtained in Example 1, add 500 μL of 99.8% D₂O, and thoroughly shake to dissolve. Centrifuge at 12,000 rpm for 5 minutes. The supernatant is scanned using a Bruker Avance 600 MHz NMR spectrometer at 20°C. The data are analyzed using MestReNova software.

[0087] The results are as follows Figures 4 to 7As shown, Figure 4 The product obtained in Example 1 1 H-NMR spectrum, Figure 5 The product obtained in Example 1 13 C-NMR spectrum, Figure 6 is the HSQC spectrum of the product obtained in Example 1, Figure 7 The HMBC spectrum of the product obtained in Example 1 is shown in FIG. Figures 4 to 7 It can be seen that the product obtained in Example 1 is β-1,3-glucan, and its structural formula is shown below:

[0088]

[0089] Example 2: Activity test of β-1,3-glucan in enhancing immunity

[0090] 1. Animal husbandry

[0091] Six- to seven-week-old female C57BL / 6N mice were housed in a clean animal care facility. All animal husbandry and experimental procedures adhered to the guidelines in the Laboratory Animal Care and Use Manual. Mice were acclimated for 7 days, maintained at 25°C, and maintained on a 12-hour light / dark cycle. All mice received adequate maintenance feed and drinking water.

[0092] 2. Experimental Grouping and Drug Administration

[0093] The mice were randomly divided into 3 groups, with 10 mice in each group. The drug administration cycle was 28 days. The specific treatments were as follows:

[0094] (1) Control group: gavage with 200 μL of dH2O daily;

[0095] (2) CTX group: 200 μL of dH O was gavaged daily, and 4 mg / mL of cyclophosphamide (CTX) solution was intraperitoneally injected on days 23 and 24 (the dose of cyclophosphamide was 40 mg / kg mouse);

[0096] (3) CTX+β-1,3-glucan group (specific treatments as follows Figure 8 A): 200 mg / kg of β-1,3-glucan obtained in Example 1 was orally administered daily, and 4 mg / mL of cyclophosphamide (CTX) solution was intraperitoneally injected on days 23 and 24 (the cyclophosphamide dose was 40 mg / kg mouse).

[0097] The mice were killed by cervical dislocation on the 29th day after the first oral administration.

[0098] 3. Immune organ separation and organ index detection

[0099] Mice were immersed in 75% (v / v) ethanol for 2 minutes, and their limbs were fixed to a mouse dissection table with a syringe. Sterile scissors and forceps were used to cut the mouse skin, bluntly peel the skin, and use sterile scissors to cut the mouse endothelium. The mesenteric lymph nodes (MLNs), spleen, and thymus of the mouse were removed in sequence. The fat was thoroughly removed and photographed. The spleen was weighed and recorded, and the spleen index was calculated according to the formula "Spleen Index (mg / g) = spleen weight / mouse body weight".

[0100] The results are as follows Figure 8 B to 8C, where Figure 8 B is a photo of each immune organ. Figure 8 C represents the spleen index calculation result. As can be seen, after CTX injection, the volume of the thymus, spleen, and mesenteric lymph nodes, as well as the spleen index, decreased significantly, indicating immunosuppression. However, after oral administration of β-1,3-glucan, the size of each immune organ and the spleen index recovered, indicating that β-1,3-glucan can counteract the immunosuppression caused by CTX and effectively enhance immunity.

[0101] 4. Splenic lymphocyte proliferation assay

[0102] After soaking mice in 75% (v / v) ethanol for 2 minutes, the spleen was removed using sterile scissors and forceps in a laminar flow hood, weighed, and placed in a Petri dish containing 4 mL of sterile D-Hanks solution. The spleen was thoroughly ground using a syringe plunger on a 50-mesh sterile copper mesh. The cell suspension was filtered through a 200-mesh copper mesh to remove tissue clumps. The filtrate was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 1 mL of sterile ddH2O was added. The suspension was thoroughly mixed by aeration for 20 seconds to lyse red blood cells. Serum-free 2× RPMI 1640 medium was then added and mixed. The cells were then washed twice with D-Hanks solution, resuspended in 4 mL of serum-free RPMI 1640 medium, filtered through a 200-mesh copper mesh to obtain a single-cell suspension, and counted using an automated cell counter.

[0103] Splenocytes were diluted to 5 × 10 6 / mL concentration, added into 96-well cell culture plates, 100 μL per well, and divided into experimental group A and experimental group B; and the wells with only RPMI 1640 culture medium were set as blank control groups, which were also divided into blank group A and blank group B.

[0104] LPS was diluted to a concentration of 20 μg / mL in RPMI 1640 medium supplemented with 20% (v / v) calf serum and added to each well of a 96-well plate containing experimental group A and blank group A at a rate of 100 μL. ConA solution was diluted to a concentration of 10 μg / mL in RPMI 1640 medium supplemented with 20% (v / v) calf serum and added to each well of a 96-well plate containing experimental group B and blank group B at a rate of 100 μL.

[0105] Each well was mixed and incubated in a 37°C, 5% CO2 incubator for 72 hours. At the 67th hour, 20 μL of MTT solution was added to each well. At the 72nd hour, 50 μL of 0.04 M HCl solution containing 20% ​​(w / v) SDS was added to each well. Finally, the cells were incubated in a 37°C, 5% CO2 incubator for 12 hours. The absorbance was measured at 570 nm.

[0106] The results are as follows Figure 8 As shown in Figure D, it can be seen that after the mice were injected with CTX, the proliferation activity of spleen lymphocytes in the mice was significantly reduced, and immunosuppression occurred. However, after the mice were gavaged with β-1,3-glucan, the proliferation activity of spleen lymphocytes in the mice was significantly improved, indicating that β-1,3-glucan can resist the immunosuppression caused by CTX and effectively play a role in enhancing immunity.

[0107] 5. Macrophage phagocytic index and phagocytic rate detection

[0108] Fluorescent microspheres were prepared with PBS solution containing 1% (w / v) BSA at a volume ratio of 1:1000, wrapped in tin foil, incubated in a dark water bath at 37°C for 30 minutes, and ultrasonically treated at 200W for 5 minutes to complete the pretreatment of the fluorescent microspheres.

[0109] In a clean bench, use sterile scissors and forceps to cut open the mouse skin. Use a 5mL syringe to draw up 5mL of sterile PBS and inject it into the mouse's peritoneal cavity. Gently pipette and aspirate the solution into a centrifuge tube placed on ice, repeating this three times. After all mouse peritoneal macrophages have been isolated, add 5% (v / v) fetal calf serum (FCS) and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, add 1mL of sterile ddH2O to each tube, mix thoroughly by pipetting for 20 seconds, and add sterile 2× DMEM high-glucose medium to lyse the red blood cells. Centrifuge again at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the solution in 4mL of sterile D-Hanks solution per tube. Repeat this process twice by centrifuging at 1500 rpm for 5 minutes. Discard the supernatant, add 4mL of DMEM high-glucose medium, resuspend thoroughly, filter through a 200-mesh copper mesh, and count the cells using a hemocytometer.

[0110] Macrophages were diluted to 4 × 10 cells / mL using DMEM high glucose medium containing 5% (v / v) fetal calf serum (FCS).5 2 mL of macrophage dilution solution was added to each well of a six-well plate, with triplicate wells per group. The cells were cultured in a 37°C, 5% CO2 cell culture incubator for at least 1.5 hours to ensure complete cell attachment. The culture medium from fully adhered peritoneal macrophages was carefully aspirated, 2 mL of D-Hanks solution was added, and the mixture was shaken and discarded. This was repeated twice. 2 mL of DMEM high-glucose medium containing 5% (v / v) fetal calf serum (FCS) was added, followed by 100 μL of pretreated fluorescent microspheres. The six-well plate was wrapped with tin foil and incubated in a 37°C, 5% CO2 cell culture incubator for 1.5 hours in the dark. The supernatant was discarded, and 3 mL of sterile PBS was added to each well to wash away unbound fluorescent microspheres. The microspheres were thoroughly scraped off with a cell scraper, dispersed with a pipette, and filtered through 200-mesh gauze into a 1.5 mL EP tube for flow cytometry analysis of the phagocytic capacity of peritoneal macrophages. The phagocytic capacity of peritoneal macrophages was determined by calculating the formulas "phagocytic index = number of fluorescent microspheres phagocytosed / total number of macrophages" and "phagocytic rate = number of cells phagocytosing fluorescent microspheres / total number of macrophages".

[0111] The results are as follows Figure 8 As shown in Figure E, it can be seen that after mice were injected with CTX, the phagocytic ability of peritoneal macrophages in mice was significantly reduced, and immunosuppression occurred. However, after mice were gavaged with β-1,3-glucan, the phagocytic ability of peritoneal macrophages in mice was significantly improved, indicating that β-1,3-glucan can resist the immunosuppression caused by CTX and effectively play a role in enhancing immunity.

[0112] VI. HE staining of spleen

[0113] (1) Dewaxing of sections: The spleen tissue was immersed in 4% (w / v) paraformaldehyde solution for 48 h, washed with dH2O, and dehydrated with 75% (v / v) ethanol for 2 h → 80% (v / v) ethanol for 2 h → 90% (v / v) ethanol for 1 h → 95% (v / v) ethanol for 0.5 h → anhydrous ethanol for 20 min → anhydrous ethanol for 20 min → anhydrous ethanol and xylene mixture (1:1, v / v) for 20 min, then transparentized with xylene for 20 min, and then the tissue was embedded in paraffin. Use a paraffin slicer to cut the embedded tissue block into slices with a thickness of 5 μm. Adhere the slices to a glass slide and soak them in xylene I for 10 min → xylene II for 10 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% (v / v) ethanol for 5 min → 90% (v / v) ethanol for 5 min → 80% (v / v) ethanol for 5 min → 70% (v / v) ethanol for 5 min → wash with distilled water.

[0114] (2) HE staining: After paraffin sections were stained with hematoxylin for 1 minute, they were rinsed with tap water, differentiated with 1% (v / v) hydrochloric acid alcohol for 10 seconds, rinsed with tap water again, and then blued with 1% (v / v) ammonia aqueous solution for 1 minute. They were rinsed with running water for 15 seconds, stained with eosin solution for 15 seconds, and rinsed with running water.

[0115] (3) Dehydration and sealing: Place the slices in 95% (v / v) ethanol I for 5 min → 95% (v / v) ethanol II for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 5 min → xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene and let them dry slightly, then seal them with neutral gum.

[0116] The images of the slices were collected by microscope and analyzed. Figure 9 As shown in Figure A, the spleen structure of mice in the control group was clear, with densely packed lymphocytes, regular red and white pulp structures, and clear white pulp margins. In the CTX group, the white pulp was blurred and reduced in size. In the CTX+β-1,3-glucan group, lymphocytes were densely packed, with densely packed white pulp and clear margins. This suggests that β-1,3-glucan can counteract the immunosuppression caused by CTX, effectively enhancing immunity.

[0117] 7. Splenic immune cell typing test

[0118] The number of spleen cells was detected and recorded using a cell counter, and then spleen cell suspension was prepared using RPMI1640 culture medium to ensure that each sample contained 5×10 5 Cells were collected and diluted to a volume of 1 mL with PBS containing 5% (v / v) fetal bovine serum. Centrifuge at 700g for 5 minutes and discard the supernatant. Resuspend the cells with PBS containing 5% (v / v) fetal bovine serum, add fluorescent flow cytometry antibodies PerCP CD45, FITC CD3 and APC / Cy7 CD19 according to the antibody instructions, and incubate on ice for 30 minutes. Centrifuge at 700g for 5 minutes and discard the supernatant. Add 1 mL of PBS solution containing 5% (v / v) fetal bovine serum to wash away the unspecifically bound dye. Centrifuge at 700g for 5 minutes and discard the supernatant. Resuspend the cells with 200 μL of PBS solution containing 5% (v / v) fetal bovine serum, filter with a 200-mesh filter, and detect immune cell surface markers by flow cytometry.

[0119] The results are as follows Figure 9 As shown in B-9C, after mice were injected with CTX, the total number of spleen cells and CD3 + T cells and CD19 +The number of B cells decreased significantly, and immunosuppression occurred. However, the spleen immune cell status of mice after oral administration of β-1,3-glucan was significantly improved, indicating that β-1,3-glucan can resist the immunosuppression caused by CTX and effectively play a role in enhancing immunity.

[0120] Example 3 Preparation of β-1,3-glucans of different molecular weights and comparison of their immune-enhancing activities

[0121] 1. Preparation methods of β-1,3-glucans with different molecular weights

[0122] S1. Primary water extraction: Weigh 600 g of pest-free Ganoderma lucidum fruiting bodies, wash, dry, slice, add to 15 L of water, soak for 12 h, then heat and extract at 100 ° C for 4 h. The resulting residue is filtered as residue 1, and the filtrate is extract 1;

[0123] S2. Secondary water extraction: Residue 1 was added to 15 L of water, heated at 100 ° C for 3 h, and the resulting residue was filtered to obtain residue 2, and the filtrate was obtained as extract 2;

[0124] S3. Primary alkaline extraction: Residue 2 was added to 12 L of 0.5 mol / L NaOH solution (so that the ratio of residue 2 to NaOH solution was 1 kg:20 L), and the mixture was heated at 80°C for 3 h. The resulting residue was filtered to obtain residue 3, and the filtrate was obtained as extract 3.

[0125] S4. Secondary alkaline extraction: Residue 3 was added to 12 L of 0.5 mol / L NaOH solution (so that the ratio of residue 3 to NaOH solution was 1 kg:20 L), and the extraction was heated at 80°C for 3 h. The resulting residue was filtered to obtain residue 4, and the filtrate was obtained as extract 4.

[0126] S5. Combined extracts: Neutralize extracts 3 and 4 with 37% (v / v) HCl (while adding hydrochloric acid and stirring thoroughly until the mixture is neutral), and then combine with extracts 1 and 2 to obtain a total extract;

[0127] S6. Filtration: Filter through four layers of 300-mesh gauze to remove insoluble matter. Then, filter through a ceramic membrane filtration system (0.2 μm) to remove impurities. Then, filter through a hollow fiber ultrafiltration system (MW: 3000 Da) to remove impurities and NaCl. Collect the liquid at the interception end.

[0128] S7. Alcohol precipitation to obtain β-1,3-glucan with a weight average molecular weight of >670 kDa: The liquid at the cut end was concentrated under reduced pressure at 60°C to a β-1,3-glucan concentration of 20 mg / mL, and then 95% (v / v) ethanol solution was added (to make the final ethanol concentration 50% (v / v)), and the mixture was allowed to stand at 4°C for 12 h. After centrifugation, the precipitate was collected and freeze-dried to obtain β-1,3-glucan with a weight average molecular weight of >670 kDa (determined by high performance gel exclusion chromatography, the results are as follows Figure 10 shown);

[0129] S8. Degrade to obtain β-1,3-glucan with a weight average molecular weight of 300KDa: Weigh 5g of Ganoderma lucidum β-1,3-glucan with a weight average molecular weight of >670KDa, dissolve the sample in 0.01M NaOH solution, stir until it is completely dissolved, and then add HCl to neutralize it, so that the final concentration of β-1,3-glucan is 1mg / mL. After ultrasonic degradation of β-1,3-glucan for 17h at a power of 1500W and a duty cycle of 4:4 (ultrasound for 4s, rest for 4s), centrifuge at 4500rpm for 20min to remove the precipitate, and pass the supernatant through a hollow fiber ultrafiltration system (MW: 3000Da) to remove salts and small molecules. The liquid at the interception end is collected, concentrated under reduced pressure at 60℃, and freeze-dried to obtain β-1,3-glucan with a weight average molecular weight of 300KDa (determined by high performance gel exclusion chromatography, the results are as follows Figure 10 shown).

[0130] S9. Degradation to obtain β-1,3-glucan with a weight average molecular weight of 150 KDa: 500 mg of β-1,3-glucan with a weight average molecular weight of 300 KDa was weighed and dissolved in 150 μL of distilled water. After degradation for 0.5 h using 2% H2O2 and 1.5 mM CuCl2, small molecules, H2O2 and CuCl2 were removed using a 7000 Da dialysis bag. The sample was concentrated to 20 mL using a rotary evaporator and freeze-dried to obtain a β-1,3-glucan with a weight average molecular weight of 150 KDa (determined by high performance gel exclusion chromatography, the results are as follows: Figure 10 β-1,3-glucan as shown).

[0131] S10. Degradation to obtain β-1,3-glucan with a weight-average molecular weight of 80 KDa: 500 mg of β-1,3-glucan with a weight-average molecular weight of 300 KDa was weighed and dissolved in 150 μL of distilled water. After degradation with 2% H2O2 and 1.5 mM CuCl2 for 1 h, small molecules, H2O2, and CuCl2 were removed using a 7000 Da dialysis bag. The sample was concentrated to 20 mL using a rotary evaporator and freeze-dried to obtain a β-1,3-glucan with a weight-average molecular weight of 80 KDa (determined by high-performance gel exclusion chromatography, the results are as follows: Figure 10 β-1,3-glucan as shown).

[0132] 2. Cell Culture

[0133] (1) Cell recovery: Take out the frozen RAW264.7 mononuclear macrophages from the liquid nitrogen tank, immediately place them in a 37°C water bath and shake gently. When the cells melt into an ice-water mixture, gently pour them into a centrifuge tube containing 5 mL of DMEM high-glucose medium containing 10% (v / v) fetal bovine serum. Centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend them in 10 mL of DMEM high-glucose medium containing 10% (v / v) fetal bovine serum, gently blow them apart, and transfer them to a 10 cm cell culture dish and culture them in a 37°C, 5% CO2 cell culture incubator.

[0134] (2) Cell passaging: When the cell density reaches 80%, the cell line is repeatedly dispersed into single cells using a pipette, centrifuged at 1000 rpm for 3 min, the supernatant discarded, 5 mL of DMEM high-glucose medium containing 10% (v / v) fetal bovine serum added, the cells dispersed, and 1 mL of the cell suspension was transferred to DMEM high-glucose medium containing 10% (v / v) fetal bovine serum. The cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0135] (3) Cell freezing: After the cells grow to the logarithmic phase, use a pipette to repeatedly disperse the cells into single cells, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 3 mL of FCS containing 5% DMSO, disperse the cells, and transfer 1 mL per tube to cryopreservation tubes. Place the tubes in a cryopreservation box, freeze at -80°C for 24 h, and then place in a liquid nitrogen tank for long-term storage.

[0136] 3. Determination of NO, TNF-α and IL-6

[0137] RAW264.7 mononuclear macrophage cells were cultured at a rate of 5 × 10 5 Cells were plated at a density of 100 cells / well in a 12-well plate. After 12 hours of attachment, the blank group was treated with DMEM high-glucose medium containing 10% (v / v) FCS, and the glucan-treated group was treated with 50, 100, and 200 μg / mL of 80 KDa, 150 KDa, 300 KDa, and >670 KDa β-1,3-glucans (β-1,3-glucan was dissolved in DMEM high-glucose medium containing 10% (v / v) FCS, and polymyxin B (PMB) was added to a final concentration of 25 μg / mL. The cells were incubated at 37°C for 24 hours before being added to the system. Both the blank and glucan-treated groups were incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours, and the cell culture supernatant was obtained.

[0138] 100 μL of cell culture supernatant was added to a 96-well plate, and 50 μL of 1% (w / v) sulfonamide and 50 μL of 0.1% (w / v) naphthylamine were added in sequence. After reacting at 25°C for 20 min, the absorbance at 545 nm was measured using a microplate reader to determine the NO content.

[0139] In addition, the levels of TNF-α and IL-6 in the cell culture supernatant were detected according to the instructions of the ELISA kit.

[0140] Figure 11 The results of the determination of NO, TNF-α and IL-6 contents show that compared with the control group, β-1,3-glucans with weight average molecular weights of 80KDa, 150KDa, 300KDa and >670KDa can promote macrophages to produce NO, TNF-α and IL-6, and β-1,3-glucan with a weight average molecular weight of 300KDa has the strongest activity in activating macrophages, indicating that it has the best effect in enhancing immunity.

[0141] Example 4 Activity Test of β-1,3-Glucan in Regulating Intestinal Flora Balance

[0142] 1. Animal husbandry

[0143] Six- to seven-week-old female C57BL / 6N mice were housed in a clean animal care facility. All animal husbandry and experimental procedures adhered to the guidelines in the Laboratory Animal Care and Use Manual. Mice were acclimated for 7 days, maintained at 25°C, and maintained on a 12-hour light / dark cycle. All mice received adequate maintenance feed and drinking water.

[0144] 2. Experimental Grouping and Drug Administration

[0145] The mice were randomly divided into 3 groups, with 10 mice in each group. The drug administration cycle was 28 days. The specific treatments were as follows:

[0146] (1) Control group: gavage with 200 μL of dH2O daily;

[0147] (2) CTX group: 200 μL of dH O was gavaged daily, and 4 mg / mL of cyclophosphamide (CTX) solution was intraperitoneally injected on days 23 and 24 (the dose of cyclophosphamide was 40 mg / kg mouse);

[0148] (3) CTX+GLP-D group: 200 mg / kg of β-1,3-glucan obtained in Example 1 was orally administered daily, and 4 mg / mL of cyclophosphamide (CTX) solution was intraperitoneally injected on days 23 and 24 (the cyclophosphamide dose was 40 mg / kg mouse).

[0149] The mice were killed by cervical dislocation on the 29th day after the first oral administration.

[0150] 3. Activity test for regulating intestinal flora balance

[0151] Mice were immersed in 75% (v / v) ethanol for 2 minutes and then dissected using sterilized scissors and forceps. The cecum was removed and the contents collected in 2 mL cryovials, quickly frozen in -196°C liquid nitrogen, and stored in a -80°C freezer. Beijing Novogene Technology Co., Ltd. was commissioned to extract bacterial genomes from the mouse cecal contents. The V4 region of the bacterial 16S rDNA gene was amplified by PCR using universal primers V4F (5′-GTGCCAGCMGCCGCGGTAA-3′) and V4R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplified product was purified and recovered using magnetic beads, and a sequencing library was prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit, which was then sequenced on a HiSeq instrument. Based on the valid data, ASVs (amplicon sequence variants) clustering and species classification analysis, calculation and other analyses were performed, and multiple sequence alignment of ASVs was continued to construct a phylogenetic tree, and the community structure differences of different samples and groups were further obtained.

[0152] The results are as follows Figure 12 As shown, β-1,3-glucan can effectively inhibit the growth of harmful intestinal bacteria (Desulfovibrio, Coriobacteriaceae_UCG-002, and Colidextribacter), and promote and / or do not inhibit the growth of beneficial intestinal bacteria (Clostridium sensu stricto 1, Bacteroides, Lactobacillus, and Faecalibacterium); among them, Faecalibacterium mainly includes Faecalibacterium prausnitzii, and Bacteroides mainly includes Bacteroides plebeius. This shows that β-1,3-glucan can effectively inhibit the growth of harmful intestinal bacteria and promote and / or do not inhibit the growth of beneficial intestinal bacteria, and is suitable for preparing products that regulate the balance of intestinal flora.

[0153] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of β-1,3-glucan in the preparation of a product for regulating the balance of intestinal flora, characterized in that: The regulating the balance of intestinal flora is to inhibit the growth of harmful intestinal bacteria and promote and / or not inhibit the growth of intestinal probiotics; wherein the harmful intestinal bacteria are one or more of Desulfovibrio, Coriobacteriaceae_UCG-002, and Colidextribacter, and the intestinal probiotics are one or more of Clostridium sensu stricto 1, Bacteroides, Lactobacillus, and Faecalibacterium.

2. The application according to claim 1, characterized in that The Bacteroides genus is Bacteroides plebeius.

3. The application according to claim 1, characterized in that The Faecalibacterium is Faecalibacterium prausnitzii.

4. Application of β-1,3-glucan in the preparation of products for enhancing immunity.

5. The use according to any one of claims 1 to 4, characterized in that: The β-1,3-glucan is extracted from Ganoderma lucidum.

6. The use according to any one of claims 1 to 4, characterized in that: The weight average molecular weight of the β-1,3-glucan is greater than 80 KDa.

7. The application according to claim 6, characterized in that The weight average molecular weight of the β-1,3-glucan is 300 KDa.

8. The use according to any one of claims 1 to 7, characterized in that: The preparation method of the β-1,3-glucan comprises the following steps: S1. Extracting Ganoderma lucidum at 95-100°C for 6-8 hours, and separating the residue and the extract; S2. extracting the residue obtained in S1 with alkali at 75-85°C for 5-7h, separating the residue and the extract, and neutralizing the extract to obtain a neutralized extract; S3. The extract obtained in S1 and the neutralized extract obtained in S2 are combined, and microfiltration, alcohol precipitation, and ultrasound are performed in sequence to obtain the β-1,3-glucan.

9. The application according to claim 8, characterized in that: The power of the ultrasound in S3 is 2000-4000W.

10. The use according to any one of claims 1 to 9, characterized in that: The product is one or more of food, medicine, and health care products.

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