Neoletus theabrown fucoidan mannogalactan as well as composition and application thereof

Abstract

The invention provides a new boletus theabrowns fucoidan mannogalactan, a composition thereof, a preparation method and application thereof, and belongs to the technical field of biological medicine mass health. The new boletus theabrownus fucoidan mannogalactan has a structure represented by a general formula in an attached drawing of the abstract, and n represents the number of sugar repeating units and is a natural number. The weight-average molecular weight of the new boletus theabrowns fucose mannogalactan is 10 kDa to 20 kDa, the polydispersity coefficient is 1 to 2, and the molar ratio of mannose to fucose to galactose is 1.00: 1.16: 3.85. The neo-boletus theabrown fucoidan mannan galactan and the composition containing the neo-boletus theabrown fucoidan mannan galactan have the effects of promoting proliferation of beneficial bacteria, reducing the pH value of a microenvironment, increasing secretion of SCFAs and lactic acid, regulating the composition of intestinal microorganisms and the like, and have the function of improving the intestinal microecology. Therefore, the invention also discloses the neo-boletus theabrownus fucoidan and a composition containing the neo-boletus theabrownus fucoidan, and application of the neo-boletus theabrownus fucoidan and the composition in preparation of prebiotic drugs and / or food for improving intestinal microecology, regulating intestinal flora and promoting intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and health technology, specifically relating to a novel *Boletus brownii* fucoidan, its composition, its preparation method, and its application. Background Technology

[0002] Boletus edulis is hailed as one of the world's four most prized edible fungi, widely distributed in Asia, Europe, and North America, and is also an important specialty biological resource of Yunnan Province, China. Its unique flavor, smooth and crisp texture, and traditionally recognized medicinal value make it highly sought after in the international market, especially in Europe where it enjoys stable export demand. *Boletus edulis* (also known as the brown-skinned Boletus) Neoboletus brunneissimus As a member of the genus *Boletus* in the family Boletaceae (family Boletaceae) Boletus Boletus edulis is an important type of edible fungus and enjoys a high reputation in the consumer market. Modern research shows that Boletus edulis is not only rich in nutrients but also possesses various biological activities such as anti-tumor, antioxidant, anti-inflammatory, and regulation of metabolic syndromes (such as obesity and diabetes). However, existing research mainly focuses on its small molecule active ingredients or crude extracts, while systematic research on polysaccharide macromolecules remains relatively weak. Due to the highly complex structure of polysaccharides, manifested in a wide molecular weight range, diverse glycosyl composition, varied glycosidic bond linkages, and uneven sites and degrees of modification of modifying groups (such as sulfate and acetyl groups), the fine structural analysis and structure-activity relationship studies have long faced technical bottlenecks, which directly restricts the in-depth elucidation of their pharmacodynamic material basis. Therefore, the study of the structure and function of fungal polysaccharides has become a key scientific task that urgently needs to be broken through in this field. To date, there are no publicly reported studies on fucomanganese galactan from Boletus brownii and its activities. Summary of the Invention

[0003] The purpose of this invention is to address the aforementioned shortcomings of the existing technology by providing a novel *Boletus fumonis* galactomannan, its pharmaceutical composition, its preparation method, and its application in the preparation of drugs or foods that improve intestinal microecology, as well as its application in the preparation of prebiotic drugs or foods that regulate intestinal flora and promote intestinal health.

[0004] Based on this, the present invention isolates and purifies a novel fucoidan from the higher fungus *Boletus brownii*. The inventors have experimentally confirmed that the fucoidan can significantly improve the intestinal microecology and regulate the intestinal flora. The structure and function of the *Boletus brownii* polysaccharide are reported for the first time.

[0005] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution: The present application provides a novel structure of tea-brown Boletus rock fucoidan mannose galactose, which is abbreviated as NBP, and has a chemical structure represented by the following general formula: In the formula, n represents the number of sugar repeating units, and n is a natural number.

[0006] The monosaccharide composition of the tea-brown Boletus rock fucoidan mannose galactose is fucose (Fucose, Fuc), mannose (Mannose, Man) and galactose (Galactose, Gal).

[0007] The weight average molecular weight of the tea-brown Boletus rock fucoidan mannose galactose is 10 kDa-20 kDa, and the polysaccharide dispersion coefficient is 1-2.

[0008] The tea-brown Boletus rock fucoidan mannose galactose is characterized in that it is obtained from Boletus of Boletaceae Boletus The fruiting body of the fungus is obtained by extraction and purification.

[0009] Further, the tea-brown Boletus fungus fruiting body is optionally selected from the tea-brown Boletus Neoboletus brunneissimus Fresh fruiting body or dried body.

[0010] The present application provides a preparation method of the tea-brown Boletus rock fucoidan mannose galactose, comprising the following steps: The fresh fruiting body of the tea-brown Boletus is hot extracted with ethanol, filtered or centrifuged to obtain a residue; the residue is extracted with water to obtain a tea-brown Boletus crude polysaccharide solution; the tea-brown Boletus crude polysaccharide is obtained by ethanol precipitation; and the component with a weight average molecular weight of 10 kDa-20 kDa in the refined tea-brown Boletus crude polysaccharide is obtained to obtain the tea-brown Boletus rock fucoidan mannose galactose.

[0011] The preparation method of the tea-brown Boletus rock fucoidan mannose galactose further comprises the following steps: The fresh fruiting body of the tea-brown Boletus is homogenized, then defatted with 95% ethanol or anhydrous ethanol, filtered or centrifuged to obtain a solid, then extracted with warm water or hot water, filtered or centrifuged to remove the residue, and then the supernatant is precipitated with ethanol to obtain tea-brown Boletus polysaccharide, so that the alcohol concentration reaches 60%, and the precipitate is collected by centrifugation.

[0012] Further preferred steps include: defatting the tea-brown Boletus fruiting body twice with 95% ethanol at a solid-liquid ratio of 1:4-1:8. The preferred solid-liquid ratio is 1:6. After filtration, the extract is discarded, and the residue is extracted twice with hot water or warm water at a solid-liquid ratio of 1:4-1:8. The preferred solid-liquid ratio is 1:8.

[0013] Further, the hot water or warm water extracted solution is centrifuged, and 60% ethanol is added to the supernatant to precipitate the polysaccharide, which is then centrifuged and collected to obtain the crude polysaccharide of X. ferruginea.

[0014] In the present application, the method for refining the polysaccharide of X. ferruginea is selected from one or more of quaternary ammonium salt precipitation, fractional alcohol precipitation, anion exchange chromatography, and gel exclusion chromatography. Preferably, the method is selected from one or more of quaternary ammonium salt precipitation, fractional alcohol precipitation, and anion exchange chromatography.

[0015] According to the preparation method provided in the present application, the monosaccharide composition of the fucoidan mannogalactan of X. ferruginea is fucose (Fucose, Fuc), mannose (Mannose, Man), and galactose (Galactose, Gal), and the weight average molecular weight thereof is 10 kDa-20 kDa. The fucoidan mannogalactan of X. ferruginea is a galactose with a main chain of →6)-α-d-Gal p -(1→ and side chains of t-α-l-Fuc p , t-α-d-Man p , and →3)-α-l-Fuc p -(1→.

[0016] The inventors have found through in-depth experimental research that the fucoidan mannogalactan of X. ferruginea with the novel structure can significantly regulate intestinal flora and improve the homeostasis of intestinal microecology. The fucoidan mannogalactan of X. ferruginea can improve the composition of intestinal flora, promote the proliferation of beneficial bacteria, reduce the microenvironment pH, and increase the secretion of SCFAs and lactic acid, thereby exhibiting the characteristics of regulating the composition of intestinal microbiome and improving the intestinal microecology.

[0017] The present application further provides a fucoidan mannogalactan composition of X. ferruginea, which contains an effective amount of the fucoidan mannogalactan of X. ferruginea.

[0018] Further, the composition further comprises an effective amount of the fucoidan mannogalactan of X. ferruginea and / or a pharmaceutical and / or food excipient or flavoring agent.

[0019] The fucoidan mannogalactan of X. ferruginea and the composition thereof can be administered in the form of oral or parenteral administration. Specifically, the dosage form optionally comprises a tablet, a capsule, a lyophilized powder, or an oral solution, which contains the fucoidan mannogalactan of X. ferruginea as an active ingredient.

[0020] This invention discloses the application of the aforementioned *Boletus brownii* fucoidan or pharmaceutical composition in pharmaceuticals or foods that improve intestinal microecology.

[0021] This invention also discloses the use of *Boletus brownii* fucoidan or a combination thereof in the preparation of drugs or foods that regulate intestinal health and intestinal flora prebiotics.

[0022] In this invention, the dosage forms of the *Boletus brownii* fucoidan and its compositions include, but are not limited to, lyophilized powder, oral liquid, capsules, tablets, etc.

[0023] Compared with the prior art, the present invention has the following technical effects: The *Boletus fumonis* galactomannan polysaccharide provided in this invention is reported for the first time. Through in-depth experimental research (see embodiments of this invention), the inventors have confirmed that this novel *Boletus fumonis* polysaccharide can promote the proliferation of beneficial bacteria, lower the pH of the microenvironment, increase the secretion of SCFAs and lactic acid, and regulate the composition of intestinal microbiota, thus improving the intestinal microecology. Therefore, this invention provides a novel *Boletus fumonis* galactomannan polysaccharide, its preparation method, and its composition, which can be used to prepare prebiotic drugs and / or foods that improve intestinal microecology, regulate intestinal flora, and promote intestinal health. Attached Figure Description

[0024] Figure 1 The graph shows the molecular weight (A) and distribution (B) of fucoidan NBP from *Neocorhizoma brownii*.

[0025] Figure 2 HPLC chromatogram of NBP monosaccharide composition of fucoidan from *Neocorhizoma brownii*.

[0026] Figure 3 Total ion chromatogram for NBP methylation of *Neocorhizoma brownii* fucomanganese NBP.

[0027] Figure 4A -H represents the NBP NMR spectrum of *Neocorhizoma Chamoiformis* fucomannan galactan, where Figure 4(A) shows the one-dimensional NMR spectrum. 1 H spectrum; Figure 4 (B) one-dimensional NMR. 13 C-spectrum; Figure 4 (C) 1 H- 1 H COSY spectrum; Figure 4 (D) 1 H- 1 H TOCSY spectrum; Figure 4 (E) 1 H- 13 CHSQC spectrum; Figure 4(F) 1 H- 13 C HMBC spectrum; Figure 4 (G) HSQC-TOCSY spectrum; Figure 4 (H)1 H- 1 H ROESY spectrum.

[0028] Figure 5 Effects of tea-brown bolete fucoidan on the composition of intestinal flora, (A) Chao index; (B) Shannon index; (C) community composition at the genus level; (D) community composition at the species level; (E) relative abundance comparison analysis; (F) LEfSe analysis to identify different abundance groups.

[0029] Figure 6 Effects of tea-brown bolete fucoidan on SCFAs, lactic acid and intestinal pH, (A) GC-MS spectrum of SCFAs; (B) change of microenvironment pH; (C) total production of SCFAs; (D) total production of lactic acid; (E) GC-MS spectrum of lactic acid; (F) acetic acid production; (G) propionic acid production; (H) butyric acid production.

[0030] Figure 7 Effects of tea-brown bolete fucoidan on the proliferation of probiotics, (A) P. acidilactici growth curve of Lactobacillus acidophilus; (B) B. lactis growth curve of Bifidobacterium longum; (C) B. infantis growth curve of Bifidobacterium animalis lactis; (D) P. acidilactici SEM image of colon index of Lactobacillus acidophilus; (E) B. lactis SEM image of Lactobacillus acidophilus; (F) B. infantis SEM image of Bifidobacterium longum.

[0031] Figure 8 Rheological properties and microstructure of tea-brown bolete fucoidan, (A) time sweep analysis of NBP; (B) steady shear flow curve of NBP; (C) frequency sweep of NBP; (D) TEM image of NBP (2 μm); (E) TEM image of NBP (1 μm); (F) TEM image of NBP (500 nm).

[0032] Figure 9 Structure diagram of tea-brown bolete fucoidan NBP, wherein: n represents the number of sugar repeating units, and n is a natural number. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present application, the present application will be described more fully below, and preferred embodiments of the present application will be given. However, the present application can be realized in many different forms, and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. The use of the terms "and / or" includes a combination of one or more of the associated listed items.

[0035] The tea-brown bolete fucoid manno-galactan provided by the present application has the following general structure: In the formula, n represents the number of sugar repeating units, and n is a natural number.

[0036] The monosaccharide composition of the tea-brown bolete fucoid manno-galactan is fucose (Fucose, Fuc), mannose (Mannose, Man), and galactose (Galactose, Gal).

[0037] The tea-brown bolete fucoid manno-galactan is a galactan with a main chain of →6)-α-d-Gal p -(1→, and side chains of t-α-l-Fuc p , t-α-d-Man p , and →3)-α-l-Fuc p -(1→.

[0038] The tea-brown bolete fucoid manno-galactan is obtained by extraction and purification from the fruiting bodies of the boletaceae family and boletus Boletus .

[0039] The tea-brown bolete fucoid manno-galactan is obtained by extraction and purification from the fruiting bodies of the boletaceae family and boletus Neoboletus brunneissimus fresh fruiting bodies or dried bodies.

[0040] Further, the present application provides a preparation method of tea-brown bolete fucoid manno-galactan, comprising the following steps: After homogenizing the fresh fruiting bodies of tea-brown bolete, ethanol extraction, filtration or centrifugation, a residue is obtained; water extraction of the above residue to obtain a tea-brown bolete crude polysaccharide solution; ethanol precipitation to obtain tea-brown bolete crude polysaccharide; refining the component with a weight average molecular weight of 10 kDa-20 kDa in the tea-brown bolete crude polysaccharide to obtain tea-brown bolete fucoid manno-galactan.

[0041] In one specific example, the following steps are included: the fruiting bodies of tea-brown bolete can be fresh fruiting bodies or dried bodies. After homogenizing the fresh fruiting bodies of tea-brown bolete, 95% ethanol extraction, and filtration to retain the solid residue.

[0042] Extract twice with warm or hot water, combine the supernatant after centrifugation to remove the residue. Then, add 95% ethanol to the supernatant to a final concentration of 60%, centrifuge (3500 g x 30 min), obtain the alcohol precipitate, wash the alcohol precipitate with 95% ethanol 3 times, resuspend with water, and freeze-dry.

[0043] The method for purifying the tea-brown boletus fucoidan mannan is selected from one or more of anion exchange column chromatography, gel exclusion chromatography column method, dialysis method and ultrafiltration method. Specifically, the crude tea-brown boletus fucoidan mannan component is resuspended with deionized water, a small amount of insoluble matter is removed by centrifugation, and the purified tea-brown boletus fucoidan mannan is obtained by optional fractionation, alcohol precipitation, gel exclusion chromatography, dialysis or ultrafiltration method, etc. The purified tea-brown boletus fucoidan mannan is named NBP.

[0044] In a specific example, the present application uses DEAE-52 filler, and the filler is columned, loaded and eluted with salt-containing or salt-free elution solution according to the actual performance of the filler, and the eluate is collected. The eluate can be concentrated or not, and is desalted by dialysis or ultrafiltration, and the desalted eluate is collected, and vacuum freeze-drying or vacuum reduced pressure drying is performed, to obtain further purified tea-brown boletus polysaccharide.

[0045] In a specific example, the present application uses Sephadex G100 filler, and water or 0.1 M NaCl is used as eluent, the eluate is collected, HPLC analysis is performed, the eluate is combined, dialysis is selected or not, and finally vacuum freeze-drying or vacuum reduced pressure drying is performed, to obtain purified tea-brown boletus fucoidan mannan.

[0046] The present application also provides a structural analysis method of the above-mentioned tea-brown boletus fucoidan mannan, comprising the following steps: The molecular weight and its distribution are determined by high-performance gel exclusion chromatography-differential detector. The monosaccharide composition analysis is completed by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization combined with high-performance liquid chromatography technology of octadecylsilane bonded silica gel packing column. To further analyze the glycosidic bond connection mode, the polysaccharide is methylated under alkaline conditions, and after acid hydrolysis, sodium borodeuteride reduction and acetylation derivatization, it is analyzed by gas chromatography-mass spectrometry (GC-MS). Finally, nuclear magnetic resonance spectroscopy technology is used, including one-dimensional 1 H、 13 C spectrum and two-dimensional 1 H- 1 H COSY、 1 H- 1 H TOCSY,1 H- 1 HROESY, 1 H- 13 C HSQC, 1 H- 13 C HMBC and HSQC-TOCSY, high-level structure resolution was completed in deuterated aqueous solvent system.

[0047] According to the above structure resolution method, the structure of the fucosylated mannosyl galactan of X. theae was analyzed. The fucosylated mannosyl galactan of X. theae only showed one symmetrical chromatographic peak on HPGPC gel chromatography, and the weight average molecular weight thereof was 10 kDa~20 kDa, and the polysaccharide dispersion coefficient thereof was 1.17; the monosaccharide composition of the fucosylated mannosyl galactan of X. theae was fucose (Fucose, Fuc), mannose (Mannose, Man) and galactose (Galactose, Gal). Methylation analysis showed that the fucosylated mannosyl galactan of X. theae existed 5 different glycosidic bonds: terminal Fuc p , terminal Man p ,→3)-Fuc p -(1→,→6)-Gal p -(1→,→2,6)-Gal p -(1→; combined with the NMR results, the fucosylated mannosyl galactan of X. theae was a galactan with→6)-α-d-Gal p -(1→ as the main chain, and the side chain was substituted by t-α-l-Fuc p , t-α-d-Man p , and→3)-α-l-Fuc p -(1→.

[0048] Therefore, the fucosylated mannosyl galactan of X. theae has the structural characteristics shown in the general formula:→6)-α-d-Gal p -(1→ as the main chain, and the side chain was substituted by t-α-l-Fuc p , t-α-d-Man p , and→3)-α-l-Fuc p -(1→.

[0049] In the formula, n represents the number of sugar repeating units, and n is a natural number.

[0050] The present application further provides application of the tea-brown boletus fucoidan in preparing a prebiotic drug and / or food for improving intestinal microecology, regulating intestinal flora and promoting intestinal health.

[0051] The pharmaceutical composition of the present application comprises the tea-brown boletus fucoidan or a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, and a pharmaceutically and / or food acceptable excipient. Optionally, the excipient comprises a pharmaceutical excipient, a carrier and / or a diluent and / or a film-forming agent, etc.

[0052] In specific examples, the dosage form of the above-mentioned pharmaceutical composition is an injection or a solid preparation or an oral liquid, such as an aqueous injection and a freeze-dried injection for injection, a capsule, an oral liquid, etc., and the excipient comprises an excipient and / or a flavoring agent, etc.

[0053] The present application is described in detail below with specific examples in conjunction with the accompanying drawings, but these specific examples do not constitute any limitation on the scope of the claims of the present application.

[0054] Example 1 Extraction and purification of tea-brown boletus fucoidan.

[0055] Extraction of tea-brown boletus crude polysaccharide

[0056] After homogenization treatment of fresh fruiting bodies of tea-brown boletus, 95% ethanol was added at a solid-liquid ratio of 1:6 (kg:L), and reflux extraction was performed at 60 °C for 4 h. After cooling, the filtrate was filtered, and the extraction was repeated twice. The filtrate was combined, and the residue was dried. After defatting, the dried residue was added with deionized water at a solid-liquid ratio of 1:8 (kg:L), and was placed in a reaction kettle for extraction at 60 °C for 6 h, repeated twice. The extraction liquid was combined, and centrifuged at 3500 g for 30 min, and the supernatant was collected. 95% ethanol was slowly added to the supernatant, and the final ethanol concentration was adjusted to 60%. After standing and precipitating, the precipitate was centrifuged under the same conditions, and the precipitate component was collected. The obtained precipitate was washed with 95% ethanol and anhydrous ethanol, and centrifuged to obtain the crude polysaccharide product. Finally, the crude polysaccharide was redissolved with deionized water, and freeze-dried to obtain the tea-brown boletus crude polysaccharide sample.

[0057] Purification of tea-brown boletus fucoidan

[0058] 1) Anion exchange column chromatography: The crude polysaccharide sample of X. badius was subjected to preliminary separation and purification by DEAE-52 cellulose column. The column was eluted with deionized water and 0.1 M NaCl solution in a stepwise manner, and each eluent was used in an amount of 3 column volumes. The elution fractions at different concentrations were collected, desalted by dialysis in a 5000 Da dialysis bag after adjusting the pH to neutral (pH 7.0), and then freeze-dried to obtain the crude polysaccharide components separated based on charge differences.

[0059] 2) Gel column chromatography: For further fine purification, Sephadex G-100 gel column was used for molecular sieve chromatography. The polysaccharide sample purified by ion exchange was dissolved in deionized water, centrifuged at 8000 g for 30 min to remove insoluble substances, and then filtered through a 0.22 μm microporous filter. The filtrate was slowly loaded onto the top of the gel column, eluted with 0.1 M NaCl solution at a constant flow rate of 0.1 mL / min, and collected in 2 mL per tube.

[0060] The polysaccharide content of each tube of eluate collected was determined by the phenol-sulfuric acid method, and the elution curve was plotted with the number of tubes as the abscissa and the absorbance at 490 nm as the ordinate. According to the elution profile, the components belonging to the same elution peak were combined and subjected to desalting treatment by dialysis in a 5000 Da dialysis bag. The desalted sample was freeze-dried to obtain the refined polysaccharide of X. badius. Finally, high performance liquid chromatography (HPLC) analysis of the component showed a single, symmetrical chromatographic peak, confirming that a pure polysaccharide with uniform structure was obtained.

[0061] Example 2 Structural analysis of fucoidan from X. badius.

[0062] Experimental methods 1.1 Molecular weight and its distribution The molecular weight and its distribution of the fucosylated mannan prepared from the example 1 were determined by high performance gel permeation chromatography with refractive index detection (HPGPC-RID). The analysis was performed on an Agilent 1260 series high performance liquid chromatography system, with a Shodex OHpak SB-804 HQ column (8 mm × 300 mm) for chromatographic separation, a column temperature of 40 °C, 0.1 M NaCl aqueous solution as the mobile phase, a flow rate of 0.5 mL / min, and a refractive index detector for signal acquisition. The polysaccharide sample and dextran standards of different molecular weights were accurately weighed and dissolved in deionized water to prepare 1 mg / mL solutions, which were filtered through a 0.22 μm microporous membrane and then injected for analysis. The chromatographic data were processed using GPC software, and the molecular weight-retention time standard curve was drawn based on the retention time of the dextran standards. The sample data were substituted into the fitting equation to calculate the molecular weight and distribution parameters.

[0063] Monosaccharide composition analysis 1 mL of the sample solution (1 mg / mL), 1 mL of the monosaccharide standard solution (1 mg / mL), and 1 mL of the monosaccharide mixed standard solution (1 mg / mL) were placed in COD test tubes with deionized water as a blank control, 1 mL of 4 M TFA was added, and the mixture was vortexed and then hydrolyzed in an oven at 110 °C for 4 h. After the reaction was completed, the hydrolyzate was evaporated using a rotary evaporator, and the residue was dissolved in 500 μL of deionized water. Then, 200 μL of 0.6 M NaOH solution and 400 μL of 0.5 M PMP-methanol solution were added successively, and the derivatization reaction was carried out at 70 °C for 1 h. After cooling to room temperature, the mixture was extracted with chloroform three times, and the aqueous phase was filtered through a 0.22 μm membrane. The obtained filtrate was used as the sample for subsequent liquid chromatography analysis. The chromatographic analysis was performed on an Agilent 1260 series high performance liquid chromatography system, using a Hadesil C18-Bio column (250 mm × 4.6 mm, 5 μm) with a column temperature of 25 °C, a mobile phase of 0.1 M phosphate buffer-acetonitrile (82:18, v / v) for isocratic elution, a flow rate of 1 mL / min, an injection volume of 5 μL, a run time of 90 min, and a DAD detector with a detection wavelength of 250 nm.

[0064] Methylation analysis The sample was added to 1 mL of NaOH / DMSO suspension, and methylation derivation, chloroform extraction and water phase washing were sequentially performed. The organic phase was collected and concentrated and dried. The methylation product was dissolved in 4 M TFA solution, and hydrolysis was performed at 120 °C for 2 h, and the solvent was evaporated and dried again. After the hydrolysis product was redissolved in water and adjusted to an alkaline environment, sodium borodeuteride was added, and reduction was performed at 50 °C water bath for 2 h. After the reaction was completed, the solvent was evaporated and dried. Subsequently, acetylation was performed with acetic anhydride-pyridine system at 100 °C for 1 h. After the reaction was terminated, chloroform extraction, water washing and purification were performed, the organic phase was concentrated, dissolved in chromatographic grade dichloromethane, filtered through a 0.22 μm filter membrane, and analyzed by GC-MS.

[0065] GC-MS analysis was performed using a DB-5MS capillary column (30 m x 0.25 mm x 0.25 μm), with an initial column temperature of 80 °C (maintained for 1 min), increased to 250 °C at a rate of 5 °C / min (maintained for 40 min), a carrier gas of high-purity helium at a flow rate of 1.5 mL / min, an injection port temperature of 250 °C, a split ratio of 10:1, and an injection volume of 1 μL. MS settings included an EI ionization source (70 eV), an ion source temperature of 230 °C, a transfer line temperature of 290 °C, a quadrupole rod temperature of 150 °C, and a mass scan range of m / z 50-600. The glycosidic bond types were finally resolved by comparing the CCRC standard mass spectral database.

[0066] Nuclear magnetic resonance analysis Tea-brown boletus polysaccharide sample 30 mg was weighed and dissolved in 0.5 mL D2O, freeze-dried, and exchanged with D2O three times. The freeze-dried sample was dissolved in 0.5 mL D2O (99.9 atom % D, containing an internal standard 0.05 wt. % 3-(trimethylsilyl) propionic-2,2,3,3- d acid, sodium salt). A Bruker 800 MHz nuclear magnetic resonance spectrometer was used to determine 1 H / 13 C NMR spectrum and two-dimensional spectrum 1 H- 1 H COSY, 1 H- 1 H TOCSY, 1 H- 1 H ROESY, 1 H- 13 C HMBC, 1 H- 13 C HSQC and HSQC-TOCSY were analyzed and processed using MestReNova software.

[0067] Experimental results The properties of the *Neocorhizium anisopliae* fucoidan of the present invention are: white or off-white solid, tasteless, easily soluble in water, and hygroscopic.

[0068] Results of high performance gel size exclusion chromatography (GSC) Figure 1 Fucomanganese purified from *Boletus brownii* has only one symmetrical chromatographic peak, and its weight-average molecular weight ( ) Mw The content of the polysaccharide is 16.1 kDa, and the polysaccharide dispersion index is 1.17.

[0069] The results of monosaccharide composition analysis showed that ( Figure 2 The HPLC chromatogram of the *Boletus brownii* fucoidan-galactan after PMP pre-column derivatization showed three peaks: fucose, mannose, and galactose, indicating that the monosaccharide composition of the *Boletus brownii* polysaccharide contains fucose, mannose, and galactose.

[0070] The results of methylation analysis are shown in Table 1 and Figure 3 The polysaccharide of *Boletus edulis* contains five glycosidic bonds: terminalFuc p Terminal Man p ,→3)-Fuc p -(1→,→6)-Gal p -(1→,→2,6)-Gal p -(1→).

[0071] Table 1. GC-MS signal assignment of polysaccharide methylation products of *Boletus brownii*. The partial results of NMR detection and analysis are shown in Figure 4. 1 Signal in the low field region of the H NMR spectrum δ H 5.12 ppm and δ H The 5.13 ppm values ​​were attributed to t-α-d-Man. p and t-α-l-Fuc p The anomaly proton signal peak, δ H 5.01 ppm and δ H 5.07 ppm is attributed to →6)-α-d-Gal p -(1→ (A), →2,6)-α-d-Gal p -(1→ (A') and →3)-α-l-Fuc p The anomeric proton signal peak is shown in Figure 4. The proton signals at other positions on the sugar ring (H-2~H-6) are shown in Figure 4.

[0072] exist 13 In the C NMR spectrum, the signal in the low field region δ C 104.41 ppm δ C 105.25 ppm and δ C 105.26 ppm were attributed to t-α-l-Fuc p t-α-d-Man p and →3)-α-l-Fuc p -(1→ anomaly carbon signal peak, δ C The values ​​of 100.74 ppm and δC of 101.07 ppm are attributed to →2,6)-α-d-Gal p -(1→and→6)-α-d-Gal p -(1→ anodic carbon signal peak. The carbon signal assignments at other positions on the sugar ring (C-2~C-6) are shown in Figure 4.

[0073] Based on the methylation results of NBP, the main structural fragment →6)-α-d-Gal p -(1→, →2,6)-α-d-Gal p -(1→, t-α-l-Fuc p , →3)-α-l-Fuc p -(1→, and t-α-d-Man p They are named residues A, A', B, B', and C, respectively. For residue AC... 1 H and the corresponding 13 C signal attribution was determined using COSY, HSQC, and HSQC-TOCSY maps. The glycosidic bond sequence was determined using HMBC and ROESY maps: the HMBC map showed a long-range correlation between C-6 of residue A and H-1 of residue A'. δ C 69.34, δ H 5.07 ppm), and the long-range correlation between C-6 of residue A' and H-1 of residue A ( δ C 69.84, δ H 5.01 ppm), indicating that the NBP backbone consists of α-D-1,2,6-Gal p and α-D-1,6-Gal p Alternating linkages are present. The HMBC map further reveals a long-range correlation between C-2 of residue A' and H-1 of residue B. δ C80.38, δ H 5.13 ppm), confirming that residue B is linked to C-2 of A'; C-2 of residue A' is remotely related to H-1 of residue C ( δ C 80.38, δ H 5.12 ppm), confirming that residue C is linked to C-2 of A'; C-2 of residue A' is remotely related to H-1 of residue B' ( δ C 80.38, δ H 5.07 ppm), confirming that residue B' is linked to C-2 of A'; C-3 of residue B' is remotely related to H-1 of residue C ( δ C 80.38, δ H 5.12 ppm), confirming that residue C is linked to C-3 of C'; C-3 of residue B' is remotely related to H-1 of residue B ( δ C 80.38, δ H 5.13 ppm), confirming that residue B is linked to C-3 of B'. Based on the monosaccharide composition, methylation and NMR analysis results, the structure of NBP is deduced as: →6)-α-d-Gal p -(1→ is the main chain, the side chain is connected at the O -2 position of residue A', which contains α-l-Fuc p , t-α-d-Man p and →3)-α-l-Fuc p -(1→, and the side chain is connected at the O -3 position of residue C', which contains α-l-Fuc p and t-α-d-Man p .

[0074] In the formula, n represents the number of sugar repeating units, and n is a natural number.

[0075] In summary, the structural characteristics are: (1) the tea-brown new boletus polysaccharide is a novel fucoidan mannose galactose polysaccharide extracted from the fruiting body of tea-brown new boletus; (2) the connection mode of the tea-brown new boletus fucoidan mannose galactose polysaccharide is: →6)-α-d-Gal p -(1→ is the main chain, and the side chain contains t-α-l-Fuc p , t-α-d-Man p , and →3)-α-l-Fuc p-(1→ Substituted galactan. A search of publicly available literature both domestically and internationally revealed no previously reported arrangement or chemical structure of the *Neocorhizoma Chaemochromatoma* fucomanganese, making this a first-time discovery by the inventors.

[0076] Table 2. Fucomanganese from *Neocorhizoma Chamomicae* 1 H and 13 Signal attribution of C NMR Example 3 The effects of *Boletus brownii* polysaccharide on gut microbiota.

[0077] reagents MRS (glucose-free), Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; *Neoboletus fucomanganese*, abbreviated as NBP, prepared according to the method of Example 1 of this invention. Inluin, Beijing Mairuida Technology Co., Ltd.; Anaerobic gas-generating bags and anaerobic culture bags were purchased from Mitsubishi Gas Chemical Co., Ltd., Japan.

[0078] Experimental methods 2.1 Fermentation culture of gut microbiota The use and collection of fecal samples were approved by the ethics committee (approval number: PA20250816001). Fresh fecal samples were collected from six healthy volunteers (3 males and 3 females). After resuspending the samples in PBS buffer, large particles were removed by centrifugation at 270 g to obtain a gut microbiota suspension. The fecal microbiota suspension was inoculated into MRS medium without glucose at a 9:1 volume ratio, and test Boletus edulis polysaccharide and positive control inulin were added to achieve a final concentration of 10 mg / mL for both. Anaerobic culture was performed using a Mitsubishi anaerobic gas-generating bag to create a strictly anaerobic environment. The system was subjected to anaerobic fermentation at 37°C and constant shaking at 15 rpm for 24 h.

[0079] Metagenomic sequencing Genomic DNA was extracted using E.Z.N.A™ Mag-Bind Soil DNA Kit extraction kit (OMEGA, USA). After quality testing, the DNA was fragmented to an average size of about 150 bp using Covaris S220 (Covaris, China). The metagenomic shotgun library was constructed by Hieff NGS® Ultima Pro DNA Library Prep Kit for Illumina® kit (Yixing, China). Double-end sequencing was performed on Sangon Biotech (Shanghai) Co., Ltd. (China) Huada DNBseq_T7 PE150 (Huada, China). The quality control of sequence reads was achieved by trimming the adapters of double-end reads and removing low-quality reads through fastp. The clean reads were mixed and assembled using megahit, and then the clean reads of each sample were aligned to the assembled contigs using bowtie2 to obtain unmapped PE reads; the unmapped reads were further mixed and assembled using SPAdes; for the contigs generated by the two times of assembly, sequences less than 500 bp were filtered, and downstream analysis such as statistics and subsequent gene prediction was performed.

[0080] Determination of lactic acid and pH The intestinal flora fermentation broth was centrifuged at 13000 g for 30 min at 4 °C to collect the supernatant, and then the supernatant was acidified to pH 2.0 using 5 M HC1 for the determination of short-chain fatty acids (SCFAs) and lactic acid. The supernatant was collected and acidified to pH 2.0 using 5 M HC1. Then liquid-liquid extraction was performed using anhydrous ether at a ratio of 1:1 (v / v), vortexed, and then incubated in an ice bath for 5 min, and then centrifuged at 10000 g for 5 min at 4 °C. The ether phase was collected and transferred to a centrifuge tube containing anhydrous Na2SO4 to remove residual water, and the water phase was extracted twice. The total ether extract was mixed and used. In the derivatization process, the ether extract was accurately measured and placed into a GC sample bottle with a syringe, and then BSTFA reagent was added, sealed, vortexed for 5 s, and then reacted at 70 °C for 30 min to complete the derivatization. GC-MS analysis was performed using an Agilent 7890A system with a DB-5MS capillary column (30 m x 0.25 mm x 0.25 μm), and the temperatures of the inlet, ion source, quadrupole and transfer line were 260 °C, 230 °C, 150 °C and 280 °C, respectively, with helium as the carrier gas (flow rate 1 mL / min). The split injection mode was used (split ratio 10:1, injection volume 1 μL), and the solvent delay was 3 min. The column temperature program was as follows: initial 40 °C for 2 min, increased to 150 °C at 15 °C / min, held for 1 min, and then increased to 300 °C at 30 °C / min, held for 5 min. The pH value of the fermentation broth was determined using a Sartorius PB-10 pH meter.

[0081] Experimental results 3.1 The effect of tea-brown bolete fucoidan mannan on the composition of intestinal flora.

[0082] To evaluate whether the galactan has a prebiotic function, the present application analyzes its effect on the composition and structure of intestinal microbiota to explore its prebiotic potential. Alpha diversity analysis (Chao1 index) shows that the microbial community richness of the inulin group and the NBP group is basically the same (Fig. 2A), but the Shannon index analysis shows that there are significant differences in the composition and structure of the microbial community (Fig. 2B). Notably, the galactan selectively increases the abundance of key beneficial bacteria, among which the increase of Pediococcus (Fig. 2C) and Bifidobacterium (Fig. 2D) is the most significant. This selective enrichment effect is further confirmed at the species level (Fig. 2E). In addition, the FDR-corrected Kruskal-Wallis test verifies that the relative abundance of Pediococcus and Bifidobacterium is significantly higher in the galactan group than in the control group (Fig. 2F). Figure 5 Figure 5 Pediococcus Bifidobacterium Figure 5 Figure 5 Faecalibacterium prausnitzii Eubacterium eligens ​​​​​​​The abundance of key butyrate-producing bacteria, including those mentioned above, increased significantly. Figure 5 (E). Galactan intervention also promoted Ligilactobacills sp. and Lactobacillus acidophilus The proliferation of probiotics was observed. Simultaneously, this invention observed a significant increase in bacteria rich in polysaccharide utilization sites (PULs), including... Bacteroides uniformis and Christensenellaceae The R7 group indicates enhanced polysaccharide metabolism in the gut ecosystem. LEfSe analysis identified *Pediococcus* and *Bifidobacterium* as key genera significantly enriched in response to galactan intervention. Figure 5 Both *N. Brugia* (F) and *N. Brugia* genera are recognized for their beneficial effects on gut microbiota and host immunity. Overall, *N. Brugia* has great potential for development into prebiotics.

[0083] Effects of *Boletus brownii* fucoidan on SCFAs, lactic acid, and pH in a micro-ecosystem.

[0084] This invention has found that, compared with inulin, galactan supplementation can significantly increase the total amount of short-chain fatty acids and the levels of each component. Figure 6 (A). Specifically, the total short-chain fatty acid content in the NBP group was 1.12 times higher than that in the inulin group ( ). Figure 6 In the middle (C), acetic acid, propionic acid, and butyric acid increased by 1.11 times, 1.46 times, and 1.23 times, respectively. Figure 6 (Middle FH). As a key metabolic precursor of short-chain fatty acids, lactate levels were also significantly increased under galactan intervention (6D-E in the figure), reaching 1.32 times that of the Inulin group. This increase is related to Pediococcus A significant increase in abundance was associated with ( Figure 5 (E. spp.), this genus is recognized as a high-lactic-acid-producing bacterium. Increased concentrations of short-chain fatty acids and lactic acid together lead to a significant decrease in the pH of the fermentation broth. Figure 6 (B). This acidification is crucial for shaping the microbial community structure and can promote beneficial bacteria (such as...). Pediococcus , Bifidobacterium and Limosilactobacillus proliferation () Figure 5 (C). In summary, Boletus polysaccharides demonstrate potential as a microecological regulator for improving gut health by stimulating the production of lactic acid and short-chain fatty acids, lowering intestinal pH, and promoting compositional changes in the gut microbiota.

[0085] Example 4 The effect of fucoidan from *Boletus brownii* on probiotic proliferation.

[0086] experimental strains Pediococcus acidilactici ( P. acidilactici ), Bifidobacterium animalis subsp. Lactis ( B . lactis )and Bifidobacterium longum subsp. Infantis ( B . infantis Purchased from Guangdong Provincial Center for Microbial Culture Collection.

[0087] reagents MRS medium (glucose-free), Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; *Neoboletus fumonisin*, abbreviated as NBP, prepared according to the method of Example 1 of this invention. Inluin, Beijing Mairuida Technology Co., Ltd.; glucose, Sinopharm Chemical Reagent Co., Ltd.; anaerobic gas-generating bags and anaerobic culture bags were purchased from Mitsubishi Gas Chemical Co., Ltd., Japan.

[0088] Experimental methods 3.1 Effects on the proliferation of probiotics The strain was inoculated into MRS medium and anaerobically cultured at 37 °C until mid-log growth (OD600 = 0.6–0.8). Then, 4% (v / v) of the bacterial suspension was transferred to fresh MRS medium containing 0.5% (w / v) carbon sources (glucose, inluin, and BRP) and cultured for another 72 hours. OD600 values ​​were measured at 0, 8, 16, 24, 32, 40, 48, 56, 64, and 72 h.

[0089] Microstructure observation of probiotics Bacterial scanning electron microscope (SEM) samples were prepared using a classic chemical fixation and critical point drying method: First, bacterial cells were collected by centrifugation, then initially fixed with 2.5% glutaraldehyde and subsequently fixed with 1% osmium tetroxide to maintain their morphology; then, they were dehydrated sequentially with ethanol of varying concentrations, and finally dried non-destructively by replacing the ethanol with liquid CO2 using a critical point dryer; finally, the samples were adhered to the sample stage and ion-sputter coated to make their surface conductive for electron microscopy observation.

[0090] Experimental results Based on metagenomic annotation results, this invention selected three key probiotic strains: Pediococcus lactis ( P . acidilactici Bifidobacterium animalis subsp. lactis ( B . lactis ) and Bifidobacterium longum infantis subspecies ( B . infantis Fermentation experiments showed that NBP significantly promoted the proliferation of all three probiotic strains. Figure 7). In terms of proliferation kinetics, all strains showed typical S-shaped growth curves in glucose, Inluin or NBP medium, which was consistent with the basic law of microbial population dynamics. All strains entered the stationary phase after about 24 h of inoculation, indicating that a dynamic balance between substrate utilization and metabolic byproduct accumulation was reached. In summary, this study confirmed that fucosylated mannan from X. badius could directly stimulate the proliferation of probiotics, indicating that it could be used as a prebiotic for targeted regulation of intestinal flora.

[0091] Example 5 Rheological properties and microstructure of fucosylated mannan from X. badius.

[0092] Materials Fucosylated mannan from X. badius was obtained by the method of Example 1.

[0093] Methods 2.1 Rheological properties Rheological tests were performed using an Anton Paar MCR 302e rheometer (Anton Paar GmbH, Graz, Austria) equipped with a 25 mm parallel plate fixture, with a test gap set to 1 mm. The NBP polysaccharide aqueous solution at 5 mg / mL was used as the test sample, and deionized water was used as the control. The steady-state flow curve was determined by flow sweep mode at 25 °C, with a shear rate range of 0.01–1000 s⁻¹. The time-dependent viscoelastic behavior of the sample was characterized by dynamic time sweep mode at isothermal conditions (25 ± 0.1 °C) with a 1% strain (within the linear viscoelastic range) and a fixed frequency of 1 Hz (6.28 rad / s) for a 50 min test. The frequency-dependent viscoelasticity was further evaluated by dynamic frequency sweep under the same temperature and geometry conditions: the fixed strain amplitude was 1%, and the angular frequency sweep range was 0.1–300 rad / s.

[0094] Microstructure The polysaccharide aqueous solution (1 mg / mL) was incubated at 85 °C for 6 h to prepare the sample, and the molecular morphology of the dried sample was observed using a transmission electron microscope (Talos L120C, Thermo Fisher Scientific) at an acceleration voltage of 80 kV.

[0095] Results 3.1 Rheological properties NBP showed rheological characteristics centered on transient network and weak gel behavior. In the time stability test (Figure 2A), the viscosity of the sample decreased rapidly within 10 min, and then gradually stabilized. The viscosity of the control sample remained stable throughout the test. The results showed that the NBP polysaccharide solution had a weak gel-like structure, which was not stable in time. Figure 8Figure 2A, which shows an initial modulus rise followed by a gradual relaxation, indicating that it forms a dynamic and unstable transient network structure. Steady shear tests confirm that the NBP has pseudoplastic (shear-thinning) fluid characteristics (Figure 2B), although it has a slightly higher zero-shear viscosity, its flexible molecular backbone leads to a viscosity reduction under high shear conditions. In addition, frequency sweep results show that G' and G" increase with increasing frequency (Figure 2C), presenting a weak gel feature with low-frequency dominated viscosity and high-frequency dominated elasticity, and a low overall modulus, meaning that its intermolecular interactions are weak. This flexible backbone and highly reversible network structure endow the NBP with excellent flowability, making it very suitable for application in flowable formulation systems such as beverages and soft gels. Figure 8 Figure 2A, which shows an initial modulus rise followed by a gradual relaxation, indicating that it forms a dynamic and unstable transient network structure. Steady shear tests confirm that the NBP has pseudoplastic (shear-thinning) fluid characteristics (Figure 2B), although it has a slightly higher zero-shear viscosity, its flexible molecular backbone leads to a viscosity reduction under high shear conditions. In addition, frequency sweep results show that G' and G" increase with increasing frequency (Figure 2C), presenting a weak gel feature with low-frequency dominated viscosity and high-frequency dominated elasticity, and a low overall modulus, meaning that its intermolecular interactions are weak. This flexible backbone and highly reversible network structure endow the NBP with excellent flowability, making it very suitable for application in flowable formulation systems such as beverages and soft gels. Figure 8 Figure 2A, which shows an initial modulus rise followed by a gradual relaxation, indicating that it forms a dynamic and unstable transient network structure. Steady shear tests confirm that the NBP has pseudoplastic (shear-thinning) fluid characteristics (Figure 2B), although it has a slightly higher zero-shear viscosity, its flexible molecular backbone leads to a viscosity reduction under high shear conditions. In addition, frequency sweep results show that G' and G" increase with increasing frequency (Figure 2C), presenting a weak gel feature with low-frequency dominated viscosity and high-frequency dominated elasticity, and a low overall modulus, meaning that its intermolecular interactions are weak. This flexible backbone and highly reversible network structure endow the NBP with excellent flowability, making it very suitable for application in flowable formulation systems such as beverages and soft gels.

[0096] Microstructure Transmission electron microscopy (TEM) characterization results (Figure 3A) reveal the conformation of polysaccharides at the nanoscale. The NBP mainly exhibits linear chain structures with sparse short branches, presenting a uniform dispersion and limited entanglement (chain diameter: 5-10 nm). Figure 7 Figure 3A, which shows an initial modulus rise followed by a gradual relaxation, indicating that it forms a dynamic and unstable transient network structure. Steady shear tests confirm that the NBP has pseudoplastic (shear-thinning) fluid characteristics (Figure 2B), although it has a slightly higher zero-shear viscosity, its flexible molecular backbone leads to a viscosity reduction under high shear conditions. In addition, frequency sweep results show that G' and G" increase with increasing frequency (Figure 2C), presenting a weak gel feature with low-frequency dominated viscosity and high-frequency dominated elasticity, and a low overall modulus, meaning that its intermolecular interactions are weak. This flexible backbone and highly reversible network structure endow the NBP with excellent flowability, making it very suitable for application in flowable formulation systems such as beverages and soft gels.

[0097] Example 6 Preparation of tablet of Thelephora terrestris fucoidan.

[0098] Materials The Thelephora terrestris fucoidan obtained by the method of Example 1, food or pharmaceutical grade starch.

[0099] Prescription 3. Preparation process The raw and auxiliary materials were sieved and prepared for use; the PVP K30 was weighed according to the prescription amount, added to a 50% ethanol solution, and simultaneously stirred to disperse and dissolve it thoroughly, forming a transparent and clear solution, which was sealed for later use. The Thelephora terrestris fucoidan, microcrystalline cellulose 101, lactose, and carboxymethyl starch sodium were weighed according to the prescription amount, dry-mixed for 15 min, and then the prepared binder was added. After stirring for 3 min, the granules were sieved with a 18-mesh sieve. The granules were dried at 50-60 °C, and then the silicon dioxide and magnesium stearate were added and mixed for 2 min before sieving with a 16-mesh sieve. The content of the semi-finished product was measured, the tablets were pressed, and the film coating was applied, resulting in the final product.

[0100] Example 7 Preparation of capsule of Thelephora terrestris fucoidan.

[0101] Materials The Thelephora terrestris fucoidan obtained by the method of Example 1, food or pharmaceutical grade starch.

[0102] Prescription 3. Preparation process Weigh the prescription amount of tea-brown bolete fucoidan and starch, stirring to mix completely. Add the appropriate amount of talc, wet granulation with ethanol, sieve and dry, then load into No. 2 capsule shells, each capsule body filled with 80 mg of tea-brown bolete fucoidan, to prepare tea-brown bolete fucoidan capsules.

[0103] Example 8 Preparation of tea-brown bolete fucoidan oral liquid.

[0104] Materials The tea-brown bolete fucoidan obtained by the method of Example 1, and the food or drug grade flavoring agent are caramel flavor.

[0105] Prescription 3. Preparation process Weigh the prescription amount of tea-brown bolete fucoidan, sucrose and caramel flavor, add purified water to completely dissolve, then filter through a 0.2 μm microporous filter, and then fill into bottles by an oral liquid filling machine, seal and sterilize to obtain the product.

[0106] Example 9 Tea-brown bolete fucoidan lyophilized powder injection.

[0107] Materials: The tea-brown bolete fucoidan obtained by the method of Example 1, and water for injection.

[0108] Prescription Weigh the prescription amount of tea-brown bolete fucoidan and water for injection, stir to completely dissolve, and then sterilize by intermittent heat compression. After the content is qualified, filter through a 0.22 μm microporous filter, and then divide and fill into regulated vials, each vial containing 0.5 mL, half-tightened, placed in a freeze-drying box, and then freeze-dried according to the set freeze-drying curve, tight, taken out of the box, crimped, and then inspected, packaged to obtain the finished product.

[0109] Freeze-drying process: Put the sample into the box, and reduce the temperature of the baffle to -50 °C for 5 h; reduce the temperature of the cold trap to -50 °C, and start vacuumizing to 200 μbar. Start sublimation: 2 h of uniform temperature rise to -15 °C for 3 h; 3 h of uniform temperature rise to -5 °C for 6 h, and vacuum keeping at 100 ~ 250 μbar; further drying: 2 h of temperature rise to 5 °C for 2 h, vacuum keeping at 150 ~ 200 μbar; 1 h of temperature rise to 15 °C for 2 h, vacuum keeping at 80 ~ 100 μbar; 0.5 h of temperature rise to 40 °C for 4 h, and vacuum to the lowest.

[0110] The technical features of the above-described embodiments can be combined in any manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered as falling within the scope of the present disclosure.

[0111] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that, for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should not be limited by the scope described in the present application.

Claims

1. A type of fucoidan from *Neocorhizoma Chamomicae*, characterized in that... The *Boletus brownii* fucoidan-galactan has a structure represented by the following general formula: In the chemical formula: n represents the number of repeating units of the sugar, and n is a natural number.

2. The *Boletus fumonis* galactomannan according to claim 1, characterized in that, The weight-average molecular weight of the fucoidan-galactan from *Boletus brownii* is 10 kDa to 20 kDa, the polysaccharide dispersion coefficient is 1 to 2, and the molar ratio of mannose:fucose:galactose is 1.00:1.16:3.

85.

3. The *Boletus fumonis* galactomannan according to claim 1, characterized in that, The brown-brown Boletus fumonis galactomannan is derived from the genus Boletus in the family Boletaceae. Boletus The fruiting bodies were obtained through extraction and purification.

4. The *Boletus fumonis* galactomannan according to claim 3, characterized in that, The fruiting body of *Boletus brownii* mentioned above is selected from *Boletus brownii*. Neoboletus brunneissimus Fresh fruiting bodies or dried bodies.

5. The method for preparing *Boletus fumonis* galactomannan according to any one of claims 1 to 4, characterized in that, The method includes the following steps: After homogenizing fresh fruiting bodies of *Boletus brownii*, the mixture was extracted with ethanol by heat, filtered, or centrifuged to obtain residue. The residue was then extracted with water to obtain a crude polysaccharide solution of *Boletus brownii*. The crude polysaccharide was obtained by ethanol precipitation. The fraction with a weight average molecular weight of 10 kDa to 20 kDa in the crude polysaccharide of *Boletus brownii* was refined to obtain *Boletus brownii* fucoidan-galactan.

6. The method for preparing *Boletus fumonis* galactomannan according to claim 5, characterized in that, After homogenizing the fresh fruiting bodies of *Boletus brownii*, defatting them with ethanol, extracting them with warm or hot water, filtering or centrifuging to remove residue, and precipitating the supernatant with ethanol to obtain *Boletus brownii* polysaccharide, making its ethanol concentration reach 60%, and collecting the precipitate by centrifugation.

7. The method for preparing *Boletus fumonis* galactomannan according to claim 5, characterized in that, The steps for refining *Boletus brownii* fucoidan galactomannan include one or more of the following: fractional alcohol precipitation, anion exchange column chromatography, gel size exclusion chromatography, dialysis, and ultrafiltration.

8. A composition containing the *Boletus fumonis* galactomannan as described in any one of claims 1 to 4, characterized in that, The composition comprises an effective amount of *Neocorhizoma Chamoiformis* fucoidan and / or pharmaceutical excipients, excipients, or flavoring agents.

9. The use of the *Boletus fumonis* fucoidan-galactan according to any one of claims 1 to 4 or the composition according to claim 8 in the preparation of a medicine or food for improving intestinal microecology.

10. The use of the *Boletus fumonis* fucoidan galactomannan according to any one of claims 1 to 4 or the composition according to claim 8 in the preparation of prebiotic drugs or foods that regulate intestinal flora and promote intestinal health.

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