Fucomannan-glucomannan of boletus fucosus, and composition and application of fucomannan-glucomannan of boletus fucosus

Abstract

The invention provides yellow-rose yellow-meat boletus fucomannan as well as a composition, a preparation method and application thereof, and belongs to the technical field of biological medicine mass health. The fucomannan 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 fucose glucomannan galactan of the boletus fucose is 10 kDa to 20 kDa, the polydispersity coefficient of the fucose glucomannan galactan is 1 to 2, and the molar ratio of glucose to mannose to fucose to galactose is 1.00 to 3.08 to 2.84 to 14.86. The boletus fucomannan and the composition containing the boletus fucomannan and the composition containing the boletus fucomannan and the composition containing the boletus fucomannan have the effects of promoting proliferation of beneficial bacteria, reducing the pH value of a microenvironment, increasing secretion of SCFAs and lactic acid, adjusting composition of intestinal microorganisms and the like, and have the function of improving the intestinal microecology. Therefore, the invention also discloses the fucomannan-glucomannan of the boletus roseus and the fucomannan-glucomannan of the boletus roseus and the fucomannan-glucomannan of the boletus roseus and the fucomannan-glucomannan of the boletus roseus and the fucomannan of the composition of the boletus roseus and the fucomannan of the composition of the boletus roseus and the fucomannan of the composition of the boletus roseus and the fucomannan of the composition of the composition of the composition of the composition of the composition of the composition of the composition of

Description

Technical Field

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

[0002] Rose-yellow Boletus ( Butyroboletus roseoflavus ) belongs to the genus *Boletus* of the family Boletaceae. Boletus Boletus edulis is an important type of edible fungus, distributed in Asia, Europe, and North America. In Yunnan Province, my country, it is widely exported to European countries as a specialty edible fungus, highly favored by consumers for its rich aroma, smooth and crisp texture, and medicinal properties. Modern pharmacological studies have shown that Boletus edulis possesses various biological activities, including anti-tumor, antioxidant, anti-inflammatory, anti-obesity, and anti-diabetic effects. However, current research mainly focuses on extracts and small-molecule active substances, while no reports have been made on polysaccharides, a key biomolecule and one of the fundamental substances for pharmacological efficacy, leaving the material basis for its therapeutic effects incompletely elucidated. Due to the large molecular weight, diverse types of sugar units and glycosidic bonds, and significant differences in the modification sites and degrees of sulfation / acetylation, polysaccharides exhibit complex structures. Since the activity of polysaccharides often depends on specific structural sequences, the study of the structure and function of fungal polysaccharides has become a critical technological challenge requiring breakthroughs in this field. To date, there are no publicly available reports on fucoglucomannan and its activity from Boletus edulis. Summary of the Invention

[0003] The purpose of this invention is to address the aforementioned shortcomings of the existing technology by providing a novel fucoidan from Boletus macrantha, its pharmaceutical composition, its preparation method, and its application in the preparation of prebiotics or drugs or foods that regulate intestinal flora.

[0004] Based on this, the present invention isolates and purifies a novel fucoidan from the higher fungus *Boletus macranthum*. The inventors have confirmed that the fucoidan can significantly improve intestinal microecology and regulate intestinal flora. The structure and function of the 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: This invention first provides a novel fucoidan from Boletus macranthae, abbreviated as BRP, which has the following chemical structure represented by the 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 *Boletus macrantha* fucose glucomannan described in this invention is fucose (Fuc), glucose (Glc), mannose (Man), and galactose (Gal).

[0007] The weight-average molecular weight of the *Boletus macrantha* fucoidan-glucomannan described in this invention is 10 kDa to 20 kDa, and the polysaccharide dispersion coefficient is 1 to 2.

[0008] The *Boletus macrantha* fucoidan of this invention is characterized in that the *Boletus macrantha* fucoidan is derived from the genus *Boletus* of the family Boletaceae. Boletus The fruiting bodies of the fungus were obtained through extraction and purification.

[0009] Furthermore, the fruiting body of the *Boletus rosaefolius* fungus is characterized by being selected from *Boletus rosaefolius*. Butyroboletus roseoflavus Fresh or dried fruiting bodies.

[0010] This invention provides a method for preparing the fucoidan from *Boletus macrantha*, comprising the following steps: The fruiting bodies of *Boletus macrantha* were extracted with ethanol, filtered or centrifuged to obtain residue; the residue was extracted with water to obtain a crude polysaccharide solution of *Boletus macrantha*; ethanol precipitation was used to obtain crude polysaccharide of *Boletus macrantha*; the fraction with a weight average molecular weight of 10 kDa to 20 kDa in the crude polysaccharide of *Boletus macrantha* was purified to obtain *Boletus macrantha* fucoidan-glucomannan.

[0011] The preparation method of *Boletus macrantha* fucoidan-glucomannan described in this invention further includes the following steps: After homogenizing the fruiting bodies of Boletus rosenbergii, defatting them with 95% ethanol or anhydrous ethanol, filtering or centrifuging to obtain the solid, then extracting with warm or hot water, filtering or centrifuging to remove the residue, precipitating the supernatant with ethanol to obtain Boletus rosenbergii polysaccharide, making its ethanol concentration reach 60%, and collecting the precipitate by centrifugation.

[0012] A further preferred step includes: defatting the powder of the *Boletus macrantha* fruiting body twice with 95% ethanol at a material-to-liquid ratio of 1:4 to 1:8. A preferred material-to-liquid ratio is 1:6. After filtration, the extract is discarded, and the residue is extracted twice with hot or warm water at a material-to-liquid ratio of 1:4 to 1:8. A preferred material-to-liquid ratio is 1:8.

[0013] Further, the solution obtained by hot or warm water extraction is centrifuged, and ethanol with a final concentration of 60% is added to the supernatant to precipitate the precipitate. The precipitate is then centrifuged and collected to obtain crude polysaccharide of Boletus macrantha.

[0014] In this invention, the method for refining the polysaccharide from the fruiting body of *Boletus macrantha* is selected from one or more of the following: quaternary ammonium salt precipitation, fractional alcohol precipitation, anion exchange chromatography, and gel size exclusion chromatography. Preferably, one or more of the following methods are used: quaternary ammonium salt precipitation, fractional alcohol precipitation, and anion exchange chromatography.

[0015] According to the preparation method provided by this invention, the monosaccharide composition of the *Boletus macrantha* fucoglucomannan is fucose (Fuc), glucose (Glc), mannose (Man), and galactose (Gal); its weight-average molecular weight is 10 kDa~20 kDa. The *Boletus macrantha* fucoglucomannan is a galactan with α(1→6) galactoside bonds as the main chain and side chains replaced by β-Glc, α-Fuc, α-Man, and α(1→4)Man.

[0016] Through in-depth experimental research, the inventors discovered that the novel *Boletus macrantha* fucoidan-glucomannan can significantly regulate intestinal flora and improve intestinal microecological homeostasis. This *Boletus macrantha* fucoidan-glucomannan exhibits properties such as regulating intestinal microbiome composition and improving intestinal microecology by improving the composition of intestinal flora, promoting the proliferation of beneficial bacteria, lowering the microenvironment pH, and increasing the secretion of SCFAs and lactic acid.

[0017] The present invention further provides a *Boletus macrantha* fucoidan-glucomannan composition, wherein the composition contains an effective amount of *Boletus macrantha* fucoidan-glucomannan.

[0018] Furthermore, the composition also includes an effective amount of *Boletus macrantha* fucoidan glucomannan and / or pharmaceutical and / or food excipients or flavoring agents.

[0019] The *Boletus rosaefolius* fucoidan and its compositions can be administered orally or parenterally. Specifically, the dosage form can be tablets, capsules, lyophilized powder, or oral liquid containing *Boletus rosaefolius* fucoidan as an active ingredient.

[0020] Furthermore, the present invention also discloses the application of *Boletus macrantha* fucoidan glucomannan or a combination thereof in the preparation of drugs or foods that regulate intestinal health and intestinal flora prebiotics.

[0021] In this invention, the dosage forms of the *Boletus macrantha* fucoidan glucomannan and its compositions include, but are not limited to, lyophilized powder, oral liquid, capsules, tablets, etc.

[0022] Compared with the prior art, the present invention has the following technical effects: The *Boletus macrantha* fucoidan-glucomannan provided in this invention is a first-time report. Through in-depth experimental research (see embodiments of this invention), the inventors have confirmed that the novel *Boletus macrantha* polysaccharide provided by this invention 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 macrantha* fucoidan-glucomannan, 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

[0023] Figure 1 The graph shows the molecular weight (A) and distribution curve (B) of BRP from Boletus macranthumiformis fucoidan.

[0024] Figure 2 HPLC chromatogram of BRP monosaccharide composition of Boletus rosifolia fucoidan.

[0025] Figure 3 Total ion chromatogram for BRP methylation of Boletus rosifolia fucoidan glucomannan.

[0026] Figure 4A -H is the BRP spectrum of *Boletus rosenbergii* fucoidan glucomannan, 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 C HSQC 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.

[0027] Figure 5 To illustrate the effect of *Boletus rosenbergii* fucoidan on gut microbiota composition, the figure shows: (A) Chao index; (B) Shannon index; (C) genus-level community composition; (D) species-level community composition; (E) relative abundance comparison analysis; and (F) LEfSe analysis to identify differentially abundant taxa.

[0028] Figure 6 The effects of *Boletus rosenbergii* fucoidan on SCFAs, lactic acid, and intestinal microenvironment pH are shown in the figure. (A) GC-MS spectrum of SCFAs; (B) Changes in microenvironment pH; (C) Total amount of SCFAs produced; (D) Total amount of lactic acid produced; (E) GC-MS spectrum of lactic acid; (F) Amount of acetic acid produced; (G) Amount of propionic acid produced; (H) Amount of butyric acid produced.

[0029] Figure 7 The effect of fucoidan from Boletus rosenbergii on probiotic proliferation is shown in the figure. (A) P. acidilactici The growth curve; (B) B. lactis The growth curve; (C) B. infantis The growth curve; (D) P. acidilactici SEM image of colon index; (E) B. lactis SEM image; (F) B. infantis SEM image.

[0030] Figure 8 Rheological properties and microstructure of *Boletus rosenbergii* fucoidan glucomannan. In the figure, (A) time-scan analysis of BRP; (B) steady-state shear flow curve of BRP; (C) frequency scan of BRP; (D) TEM image of BRP (2 μm); (E) TEM image of BRP (1 μm); (F) TEM image of BRP (500 nm).

[0031] Figure 9 The diagram shows the structure of BRP (bacteroidetes fucoidan glucomannan) from Boletus macranthum, where n represents the number of repeating sugar units and is a natural number. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] The *Boletus macrantha* fucoidan-glucomannan provided by this invention has a structure represented by the following general formula: In the formula: n represents the number of sugar repeating units, and n is a natural number.

[0035] The monosaccharide composition of the *Boletus macrantha* fucose glucomannan is fucose (Fuc), glucose (Glc), mannose (Man), and galactose (Gal).

[0036] The *Boletus macrantha* fucoidan-glucomannan is a galactan with α(1→6) galactoside bonds as the main chain and side chains replaced by β-Glc, α-Fuc, α-Man and α(1→4)Man.

[0037] The rose-yellow fleshy Boletus fucoidan is derived from Boletaceae and Boletus genus. Boletus The fruiting bodies were obtained through extraction and purification.

[0038] The fruiting body of the fungus *Boletus macranthum* is selected from *Boletus macranthum*. Butyroboletus roseoflavus Fresh or dried fruiting bodies.

[0039] Furthermore, the present invention provides a method for preparing fucoidan from Boletus macranthae, comprising the following steps: After homogenizing the fruiting bodies of *Boletus macrantha*, ethanol was extracted, and the solid was obtained by filtration or centrifugation. The solid was then extracted with water to obtain a crude polysaccharide solution of *Boletus macrantha*. Ethanol precipitation was used to obtain crude polysaccharide of *Boletus macrantha*. The fraction with a weight average molecular weight of 10 kDa to 20 kDa in the crude polysaccharide of *Boletus macrantha* was purified to obtain *Boletus macrantha* fucoidan-glucomannan.

[0040] In a specific example, the following steps are included: The fruiting bodies of *Boletus macrantha* can be fresh, dried, or powdered after homogenization of dried fruiting bodies. After homogenizing the *Boletus macrantha* fruiting bodies, extract with 95% ethanol, filter, and retain the solid residue.

[0041] Extract twice with warm or hot water, centrifuge to remove residue, and combine the supernatants. Then, add 95% ethanol to the supernatant to a final concentration of 60%, centrifuge (3500 g × 30 min) to obtain the alcohol precipitate, wash the precipitate three times with 95% ethanol, reconstitute with water, and freeze-dry.

[0042] The method for refining *Boletus rosaefolius* fucoidan-glucomannan is selected from one or more of the following methods: anion exchange column chromatography, gel size exclusion chromatography, dialysis, and ultrafiltration. Specifically, the crude *Boletus rosaefolius* fucoidan-glucomannan fraction can be reconstituted with deionized water, centrifuged to remove a small amount of insoluble matter, and then purified by fractional alcohol precipitation, gel size exclusion chromatography, dialysis, or ultrafiltration. The fraction containing polysaccharide, the retentate, or the permeate is collected and directly freeze-dried under vacuum or concentrated under reduced pressure followed by alcohol precipitation and vacuum drying to obtain the refined *Boletus rosaefolius* fucoidan-glucomannan, named BRP.

[0043] In a specific example, this invention uses DEAE-52 packing material. The column is packed, sample is loaded, and eluted sequentially with either salt-containing or salt-free elution solutions, with the eluent collected according to the actual performance of the packing material. The eluent may or may not be concentrated; it is then placed in a dialysis bag for dialysis or an ultrafiltration membrane for desalting. The desalted retentate is collected and then freeze-dried or vacuum-depressurized to obtain further purified *Boletus edulis* polysaccharide.

[0044] In a specific example, the present invention uses Sephadex G100 packing material, with water or 0.1 M NaCl as the eluent. The eluent is collected, analyzed by HPLC, and the eluents are combined. Dialysis or non-dialysis can be selected. Finally, the purified fucoidan of Boletus macranthum is obtained by vacuum freeze-drying or vacuum decompression drying.

[0045] This invention also provides a method for structural analysis of the above-mentioned *Boletus macrantha* fucoidan glucomannan, comprising the following steps: Molecular weight and distribution were determined using high-performance gel size exclusion chromatography with differential detector. After derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP), the monosaccharide composition of *Boletus rosenbergii* fucoidan was analyzed by high-performance liquid chromatography (HPLC) on an octadecylsilane-bonded silica gel column. The polysaccharide was methylated under alkaline conditions using halomethane reagents such as iodomethane, followed by hydrolysis under acidic conditions and reduction with sodium borodeuteride. The methylated sugar alcohols were acetylated with acetic anhydride and pyridine, and the extracted polysaccharide was analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the glycosidic bond configuration of *Boletus rosenbergii* fucoidan. The *Boletus rosenbergii* fucoidan sample was dissolved in heavy water and analyzed by nuclear magnetic resonance spectroscopy, mainly 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 1H- 13 C HSQC, 1 H- 13 C HMBC and HSQC-TOCSY correlation spectra.

[0046] Following the aforementioned structural analysis method, the structure of *Boletus rosenbergii* fucoglucomannan was analyzed. The *Boletus rosenbergii* fucoglucomannan exhibited only one well-symmetrical peak on HPGPC gel chromatography, with a weight-average molecular weight of 10 kDa–20 kDa and a polysaccharide dispersion index of 1.22. The monosaccharide composition of the *Boletus rosenbergii* fucoglucomannan consisted of fucose (Fuc), glucose (Glc), mannose (Man), and galactose (Gal). Methylation analysis revealed six different glycosidic bonds in this *Boletus rosenbergii* fucoglucomannan: Glc… p -(1→,Man p -(1→,Fuc p -(1→,→4)-Man p -(1→,→6)-Gal p -(1→,→2,6)-Gal p -(1→;Based on NMR results, the fucoidan of Boletus macranthum is a galactan with α(1→6) galactoside bonds as the main chain and the side chains replaced by β-Glc, α-Fuc, α-Man and α(1→4)Man.

[0047] Therefore, the *Boletus macrantha* fucoidan has the structural features shown in the following general formula: a galactan with α(1→6) galactoside bonds as the main chain and side chains replaced by β-Glc, α-Fuc, α-Man and α(1→4)Man.

[0048] In the formula, n represents the number of sugar repeating units, and n is a natural number.

[0049] Through in-depth experimental research, this invention has discovered that the *Boletus macrantha* fucoidan-glucomannan can improve the composition of intestinal flora, promote the proliferation of beneficial bacteria, lower the microenvironment pH, and increase the secretion of SCFAs and lactic acid, thus possessing functions such as regulating the composition of the intestinal microbiome and improving the intestinal microecology. Therefore, this invention also provides the application of the *Boletus macrantha* fucoidan-glucomannan in the preparation of prebiotic drugs and / or foods that improve intestinal microecology, regulate intestinal flora, and promote intestinal health.

[0050] The pharmaceutical composition of this invention includes the *Boletus rosenbergii* fucoidan glucomannan or its pharmaceutically acceptable salt or solvent compound, and pharmaceutically and / or food-acceptable excipients. Optionally, the excipients include pharmaceutical excipients, carriers and / or diluents and / or film-forming agents, etc.

[0051] In specific examples, the dosage form of the above-mentioned pharmaceutical composition is an injection, a solid preparation, or an oral liquid, such as a water injection and a lyophilized powder for injection, a capsule, an oral liquid, etc., and the excipients include excipients and / or flavoring agents, etc.

[0052] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but these specific embodiments do not constitute any limitation on the scope of the claims of the present invention.

[0053] Example 1 Extraction and purification of fucoidan from Boletus macranthum.

[0054] Extraction of crude polysaccharides from Boletus macrantha.

[0055] The fruiting bodies of *Boletus macrantha* were homogenized and extracted with 95% ethanol at a ratio of 1:6 (kg:L). The mixture was heated in a water bath at 60 °C for 4 h, cooled to room temperature, and extracted twice. The residue was filtered to obtain defatted samples and evaporated to dryness. The dried residue was placed in a reaction vessel, and deionized water (1:8 ratio) was added. The mixture was extracted at 60 °C for 6 h, twice, and centrifuged (3500 g × 30 min). The supernatant was collected. 95% ethanol was added to the supernatant until the final ethanol concentration was 60%, and the mixture was centrifuged (3500 g × 30 min) to obtain a 60% ethanol precipitate and a supernatant. The 60% ethanol precipitate was washed with 95% ethanol and anhydrous ethanol, respectively, and centrifuged to obtain crude polysaccharide. The crude polysaccharide was reconstituted with deionized water and then freeze-dried to obtain the crude polysaccharide sample.

[0056] Refined fucoidan from Boletus macranthae.

[0057] 1) Anion exchange column chromatography: After activation and packing of DEAE-52 column, elution was performed with deionized water and 0.1 NaCl, respectively, for 3 column volumes. The pH of the eluent was adjusted to 7, and the crude Boletus edulis polysaccharide was obtained by freeze drying.

[0058] 2) Gel column chromatography: Sephadex G100 gel was used. The ion-exchanged polysaccharide was weighed, dissolved in deionized water, centrifuged at 7000 g for 30 min, and the supernatant was filtered through a 0.22 μm filter and slowly and evenly injected into the top of the Sephadex G100 gel column. Elution was performed with 0.1 M NaCl solution at a flow rate of 0.1 mL / min, collecting 2 mL per tube. The polysaccharide content was detected using the phenol-sulfuric acid method. Elution curves of the polysaccharide components were plotted with absorbance as the ordinate and the number of tubes as the abscissa. Eluents belonging to the same elution peak were combined and desalted using a 5000 Da dialysis bag. After dialysis, the samples were lyophilized. The purified components obtained after Sephadex G100 purification were re-analyzed in the liquid chromatography phase, showing a single symmetrical peak, indicating the acquisition of pure polysaccharide from *Boletus macrantha*.

[0059] Example 2 Structural analysis of fucoidan glucomannan from Boletus macranthum.

[0060] Experimental methods 1.1. Molecular weight and its distribution The molecular weight and distribution of fucoidan prepared from Boletus macranthae in Example 1 were analyzed by high performance gel size exclusion chromatography-differential detector (HPGPC-RID).

[0061] Chromatographic instrument: Agilent Technologies 1260 series high-performance liquid chromatograph; Chromatographic conditions: Shodex OHpak SB-804 HQ (8 mm × 300 mm) column; column temperature: 40 °C; differential detector; mobile phase: 0.1 M NaCl; flow rate: 0.5 mL / min; Determination procedure: Accurately weigh the *Boletus macrantha* polysaccharide sample or a known molecular weight dextran reference standard, add deionized water to prepare a 1 mg / mL solution, filter through a 0.22 μm microporous membrane, and analyze the filtrate using the high-performance liquid chromatograph. Record the chromatogram. Data were processed using GPC software, a standard curve was plotted, and the molecular weight was calculated by substituting the data into the equation.

[0062] Monosaccharide composition analysis Take 1 mL each of the prepared sample solution, monosaccharide standard solution, and monosaccharide standard mixture (with deionized water as a blank control) and place them in their respective COD tubes. Add 1 mL of 4 M trifluoroacetic acid (TFA) and vortex to mix. Incubate at 110°C for 4 h for hydrolysis. After the reaction, evaporate to dryness using a rotary evaporator. Finally, add 500 μL of deionized water to dissolve the residue. Add 200 μL of 0.6 M NaOH solution and 400 μL of 0.5 M PMP-methanol solution to the COD tubes and derivatize at 70°C for 1 h. After the reaction, cool to room temperature and extract three times with chloroform. Filter the aqueous phase through a 0.22 μm filter membrane. Use the filtrate as the sample for liquid chromatography analysis. Chromatographic conditions: Agilent Technologies 1260 series high performance liquid chromatograph; column: Hadesil C18-Bio (250 mm × 4.6 mm, 5 µm); column temperature: 25 °C; mobile phase: 0.1 M phosphate buffer-acetonitrile (82:18); elution: flow rate 1 mL / min; injection volume 5 μL; time 90 min; detection: DAD detector, wavelength 250 nm.

[0063] Methylation analysis.

[0064] Take the sample and add 1 mL of NaOH / DMSO suspension, then add 1 mL of CH3I and react for 2 h. After the reaction is complete, add water to terminate the reaction, add 3 mL of chloroform for extraction, and extract three times. Combine the extracts. Back-extract three times with deionized water and evaporate to dryness. Dissolve the methylated sample in 1 mL of deionized water, then add 1 mL of 4 M TFA solution and hydrolyze at 120 °C for 2 h. After the reaction is complete, evaporate to dryness. Dissolve the acid-hydrolyzed sample in 1 mL of water, adjust the pH to alkaline with NaOH solution, add 15 mg of sodium borodeuteride, and reduce by stirring in a 50 °C water bath for 2 h. After the reaction is complete, evaporate to dryness. Add 1 mL of acetic anhydride and 1 mL of pyridine to the reduced sample, react at 100 °C for 1 h, add 1 mL of deionized water to terminate the reaction, add 3 mL of chloroform for extraction three times, back-extract the organic phase three times with deionized water, and evaporate to dryness. Dissolve in 1 mL of chromatographic grade dichloromethane, filter through a 0.22 μm filter membrane, and perform GC-MS analysis.

[0065] GC conditions: DB-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm); column temperature: initial temperature 80 °C, hold for 1 min, programmed temperature increase 5 °C / min to 250 °C, hold for 40 min; column flow rate 1.5 mL / min; injection port temperature 250 °C; split ratio 10:1; carrier gas was high-purity helium, injection volume 1 μL.

[0066] MS conditions: ionization mode EI; electron energy 70 eV; transfer line temperature 290 °C; ion source temperature 230 °C; quadrupole temperature 150 °C; mass range 50~600; comparison with the CCRC spectral database to determine the glycosidic bond type.

[0067] Nuclear magnetic resonance analysis.

[0068] Weigh 30 mg of *Boletus rosaefolius* polysaccharide sample and dissolve it in 0.5 mL of D2O. Freeze-dry the sample, exchanging the D2O three times. Then dissolve the freeze-dried sample in 0.5 mL of D2O (99.9% D, containing 0.05 wt. % internal standard 3-(trimethylsilyl)propionic-2,2,3,3- d In 4acid, sodium salt). Measured using a Bruker 800 MHz nuclear magnetic resonance spectrometer. 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.

[0069] Experimental results.

[0070] The properties of the *Boletus fucoidogalactan* fucoidan of the present invention are: white or off-white solid, tasteless, easily soluble in water, and hygroscopic.

[0071] The results of high performance gel size exclusion chromatography analysis showed that ( Figure 1 Fucoido-glucomannan extracted and purified from Boletus macrantha has only one symmetrical chromatographic peak, and its weight-average molecular weight ( ) Mw The content of the polysaccharide is 14.2 kDa, and the polysaccharide dispersion coefficient is 1.22.

[0072] The results of monosaccharide composition analysis showed that ( Figure 2 The HPLC chromatogram of the *Boletus macrantha* fucoidan-glucomannan after PMP pre-column derivatization showed four peaks: fucose, mannose, galactose, and glucose, indicating that the monosaccharide composition of the *Boletus macrantha* polysaccharide contains fucose, mannose, galactose, and glucose.

[0073] The results of methylation analysis are shown in Table 1 and Figure 3 The polysaccharide of *Boletus macrantha* contains six glycosidic bonds: Fuc p -(1→,Glc p -(1→,Man p -(1→,→4)-Man p -(1→,→6)-Gal p -(1→,→2,6)-Gal p -(1→).

[0074] Table 1. GC-MS signal assignment of polysaccharide methylation products of Boletus macrantha. The partial results of the nuclear magnetic resonance (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 5.07 ppm values ​​were attributed to t-α-d-Man. p and t-α-l-Fuc p The anomaly proton signal peak, δ H 5.00 ppm δ H 5.07ppm and δ H The 5.12 ppm concentration was assigned to α-d-1,6-Gal p α-d-1,2,6-Gal p and α-d-1,4-Man p The anomaly proton signal peak, δ H 4.54 ppm and δ H 4.53 ppm is attributed to t-β-d-Glc p and t-β-Me-d-Glc p The anomeric proton signal peak. The proton signals at other positions on the sugar ring (H-2~H-6) are shown in Figure 4.

[0075] exist 13 In the C NMR spectrum, the signal in the low field region δ C 104.38 ppm δ C 105.07 ppm δ C 105.19 and δ C105.57 ppm were attributed to t-α-l-Fuc p t-α-d-Man p α-d-1,4-Man p t-β-d-Glc p and t-β-Me-d-Glc p The anterior carbon signal peak, δ C The concentrations of 100.94 ppm and δC of 101.19 ppm were attributed to α-d-1,2,6-Gal. p and α-d-1,6-Gal p The anodic carbon signal peak. The carbon signal assignments at other positions on the sugar ring (C-2~C-6) are shown in Figure 4.

[0076] Based on the methylation results of BRP, the major structural fragment α-d-1,6-Gal p α-d-1,2,6-Gal p t-α-l-Fuc p t-α-d-Man p α-d-1,4-Man p t-β-d-Glc p and t-β-Me-d-Glc p They are named residues A, A', B, C, C', D, and D', respectively. For residues A-D'... 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 69.33, δ H 5.07 ppm), and the long-range correlation between C-6 of residue A' and H-1 of residue A ( δ C 69.37, δ H 5.00 ppm), indicating that the BRP backbone is also composed 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 C 80.92, δ H5.08 ppm), confirming that residue B is linked to C-2 of A'; the C-2 of residue A' is remotely correlated with the H-1 of residue C ( δ C 80.92, δ H 5.12 ppm), confirming that residue C is linked to C-2 of A'; the C-2 of residue A' is remotely correlated with the H-1 of residue C' ( δ C 80.92, δ H 5.12 ppm), confirming that residue C' is linked to C-2 of A'; C-4 of residue C' is remotely correlated with H-1 of residue C ( δ C 80.38, δ H 5.12 ppm), confirming that residue C is linked to C-4 of C'. The ROESY plot shows a long-range correlation between H-1 of residue D and H-2 of residue A'. δ H 4.54, δ H (3.85 ppm), indicating that residue D' is linked to residue A'. Based on the combined results of monosaccharide composition, methylation, and NMR analysis, the structure of BRP is inferred to be: α-d-1,6-Gal p and α-d-1,2,6-Gal p Alternating links form the main chain, with a side chain attached to the O-2 position of residue A'. This side chain contains t-α-l-Fuc. p t-α-d-Man p α-d-1,4-Man p ,t-β-d-Glc p and t-β-Me-d-Glc p Furthermore, t-α-d-Man is linked at position O-4 of residue C'. p .

[0077] In the formula, n represents the number of sugar repeating units, and n is a natural number.

[0078] In summary, its structural features are as follows: (1) The *Boletus rosenbergii* polysaccharide is a novel fucoidan extracted from the fruiting body of *Boletus rosenbergii*; (2) The connection mode of the *Boletus rosenbergii* fucoidan is: α-d-1,6-Gal p and α-d-1,2,6-Gal p Alternating glycosidic bonds form the main chain, and the side chains are linked by t-α-l-Fuc pt-α-d-Man p α-d-1,4-Man p t-β-d-Glc p and t-β-Me-d-Glc p A search of publicly available literature both domestically and internationally revealed that this arrangement and chemical structure of *Boletus macrantha* fucoidan glucomannan was not previously reported, making it a first-time discovery by the inventors.

[0079] Table 2. Rose-yellow Boletus fucoglucomannan 1 H and 13 Signal attribution of C NMR Example 3 The effects of Boletus macrantha polysaccharide on gut microbiota.

[0080] reagents MRS (glucose-free), Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; BRP (Boletus macranthae fucoidan), 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.

[0081] Experimental methods 2.1 Fermentation culture of gut microbiota.

[0082] 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), homogenized in PBS buffer, and centrifuged at 270 × g to remove large particulate impurities. The experiment used glucose-free MRS broth as the basal medium, with a medium-to-fecal slurry volume ratio of 9:1. The final concentration of the test compounds and the control (inulin) was 10 mg / mL. Anaerobic fermentation was carried out at 37°C for 24 hours using a Mitsubishi anaerobic gas-generating bag from Japan, with gentle shaking at 15 rpm throughout the fermentation process.

[0083] Metagenomic sequencing.

[0084] Genomic DNA was extracted using the EZNA™ Mag-Bind Soil DNA Kit (OMEGA, USA). After quality testing, the DNA was fragmented to an average size of approximately 150 bp using a Covaris S220 (Covaris, China). Metagenomic shotgun libraries were constructed using the Hieff NGS® Ultima Pro DNA Library Prep Kit for Illumina® (Yisheng, China). Paired-end sequencing was performed on a BGI DNBseq_T7 PE150 (BGI, China) from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Quality control of the sequence reads was achieved by using FASTP to trim adapters from paired-end reads and remove low-quality reads. Megahit was used to perform multi-sample hybrid assembly of clean reads. Then, bowtie2 was used to align the clean reads of each sample to the assembled contigs to obtain unmapped PE reads. SPAdes was then used to perform hybrid assembly of the unaligned reads. For the contigs generated by the two assembly processes, sequences smaller than 500bp were filtered out for statistical analysis and subsequent gene prediction and other downstream analyses.

[0085] Measurement of lactic acid and pH.

[0086] Determination of SCFAs and lactic acid: The intestinal flora fermentation broth was centrifuged at 13000 g for 30 min (temperature controlled at 4 °C). The supernatant was collected and 5 M HCl was added to lower the pH of the solution to 2. The acidified sample was extracted by adding anhydrous diethyl ether (1:1, v / v), vortexed, and incubated on ice for 5 min, followed by centrifugation at 10000 g for 5 min (temperature controlled at 4 °C). The diethyl ether layer containing SCFAs was transferred to a new centrifuge tube containing anhydrous Na2SO4 (for removing residual moisture). The remaining aqueous phase was extracted twice more with diethyl ether. All diethyl ether extracts were combined and mixed for subsequent derivatization.

[0087] Accurately measure the ether extract and transfer it to the glass inner tube of the GC vial. Add BSTFA and immediately tighten the cap, then vortex for 5 seconds. Leave the mixture in the GC vial and incubate at 70°C for 30 min to carry out the derivatization reaction. The derivatized sample is then ready for GC / MS analysis.

[0088] Analysis was performed using an Agilent 7890A gas chromatography-mass spectrometry (GC-MS) system equipped with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The temperatures of the injector, ion source, quadrupole, and interface were set to 260 °C, 230 °C, 150 °C, and 280 °C, respectively. Derivatized samples were injected in 1 μL split mode (10:1) with a solvent delay time of 3 min. Helium was used as the carrier gas at a flow rate of 1 mL / min. The column oven temperature program was as follows: initial temperature 40 °C held for 2 min, then increased to 150 °C at a rate of 15 °C / min and held for 1 min, and finally increased to 300 °C at a rate of 30 °C / min and held for 5 min.

[0089] The pH value of the fermentation broth was measured using a pH meter (PB-10, Sartorius).

[0090] Experimental results.

[0091] The effect of fucoidan from Boletus macranthum on gut microbiota composition.

[0092] To assess whether galactomannan possesses prebiotic functions, this invention analyzed its impact on the composition and structure of the gut microbiota to explore its prebiotic function. α-diversity analysis (Chao index) showed that the microbial community richness was essentially comparable between the Inulin group and the BRP group. Figure 5 (A), but Shannon index analysis showed significant differences in the composition and structure of the microbial community (A). Figure 5 (B). Notably, galactomannan selectivity increased the abundance of key beneficial bacteria genera, including Pediococcus ( Pediococcus ) and Bifidobacterium spp. Bifidobacterium The increase was the most significant ( ). Figure 5 (C). This selective enrichment effect was further confirmed at the species level (C). Figure 5 (D). Furthermore, the Kruskal-Wallis test, corrected for FDR, validated that, including A... naerostipes hadrus and Eubacterium eligens The abundance of several key butyrate-producing bacteria, including [list of bacteria], increased significantly. Figure 5 (E). Galactan intervention also promoted Lactiplantibacillus sp. and Lactobacillus acidophilus The inventors observed a significant increase in probiotics, including those rich in polysaccharide utilization sites (PULs). Bacteroides uniformisThe presence of Christensenellaceae R7 group indicates enhanced polysaccharide metabolism in the gut ecosystem. LEfSe analysis identified *Pediococcus* and *Bifidobacterium* as key genera significantly enriched in response to *Boletus macrantha* fucoglucomannan intervention. Figure 5 Both *Boletus flavonoids* (Category F) and *Boletus flavonoids* (Fucoidan) are recognized for their beneficial effects on gut microbiota and host immunity. Therefore, the *Boletus flavonoids* fucoidan / glucomannan described in this invention has potential for development into a prebiotic.

[0093] Effects of Boletus rosenbergii fucoidan on SCFAs, lactic acid and pH in micro-ecosystem.

[0094] 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 BRP group was 1.68 times higher than that inulin group ( ). Figure 6 In the middle (C), acetic acid, propionic acid, and butyric acid increased by 1.67 times, 1.86 times, and 1.61 times, respectively. Figure 6 (FH). As a key metabolic precursor of short-chain fatty acids, lactate levels were also significantly increased under the intervention of Boletus rosenbergii fucoidan glucomannan. Figure 6 The level in the middle DE group was 1.77 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, promoting beneficial bacteria (such as...) Pediococcus , Bifidobacterium and Limosilactobacillus proliferation () Figure 5 (C), and can also inhibit potential pathogens (such as C), Escherichia-Shigella coli and Fusobacterium mortiferum proliferation () Figure 5 (E). In summary, *Boletus macrantha* fucoidan-glucomannan can function as a microecological regulator to improve gut health by stimulating the production of lactic acid and short-chain fatty acids, lowering intestinal pH, and promoting structural changes in the gut microbiota.

[0095] Example 4 The effect of fucoidan from Boletus macranthae on probiotic proliferation.

[0096] 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.

[0097] reagents MRS (glucose-free), Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.; BRP (Boletus macranthum), prepared according to the method in 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.

[0098] Experimental methods 3.1 Effects on probiotic proliferation.

[0099] 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.

[0100] Microscopic observation of probiotics.

[0101] 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.

[0102] Experimental results.

[0103] Based on metagenomic annotation results, this study selected three important probiotic strains, including Pediococcus lactis (… P . acidilactici Bifidobacterium animalis subsp. lactis ( B . lactis ) and Bifidobacterium longum infantis subspecies ( B . infantisAn experiment was conducted to investigate the effect of BRP on the proliferation of probiotics. The results showed that BRP significantly promoted the proliferation of all three types of probiotics. Figure 7 This invention has surprisingly found that BRP outperforms common carbon source glucose and classic prebiotic inluin in promoting the proliferation of Pediococcus lactis, Bifidobacterium lactis, and Bifidobacterium infantis. This superior probiotic effect may be attributed to the unique molecular structure of BRP, particularly α-D-1,4-Man p The strains exhibited branching structure characteristics. Regarding proliferation kinetics, the growth curves of all strains in glucose, inluin, or BRP media all showed a typical S-shaped pattern, consistent with the basic laws of microbial population dynamics. Approximately 24 hours after inoculation, all strains entered a stationary phase, indicating a dynamic equilibrium between substrate utilization and metabolic byproduct accumulation. In summary, this invention demonstrates that Boletus galactan can directly stimulate probiotic proliferation, confirming its potential as a prebiotic for targeted regulation of the gut microbiota.

[0104] Example 5 Rheological properties and microstructure of fucoidan from Boletus macranthum.

[0105] Material The rose-yellow-fleshed Boletus fucoglucomannan was obtained by the method in Example 1.

[0106] method 2.1 Rheological properties Rheological testing was performed using an Anton Paar MCR 302e rheometer (Anton Paar GmbH, Graz, Austria) equipped with a 25 mm parallel plate clamp and a 1 mm test gap. A 5 mg / mL BRP polysaccharide aqueous solution was used as the test sample, with deionized water as the control. Steady-state flow curves were determined at 25 °C using flow sweep mode, with shear rates ranging from 0.01 to 1000 s⁻¹. Dynamic time-scan mode was used to characterize the time-dependent viscoelastic behavior of the samples under isothermal conditions (25 ± 0.1 °C) at 1% strain (within the linear viscoelastic range) and a fixed frequency of 1 Hz (6.28 rad / s) for 50 min. Frequency-dependent viscoelasticity was further evaluated under the same temperature and geometric conditions using dynamic frequency sweep: a fixed strain amplitude of 1% and an angular frequency sweep range of 0.1–300 rad / s.

[0107] microstructure The polysaccharide aqueous solution (1 mg / mL) was incubated at 85 °C for 6 hours to prepare the sample. The molecular morphology of the dried sample was observed using a transmission electron microscope (Talos L120C, Thermo Fisher Scientific) at an accelerating voltage of 80 kV.

[0108] result 3.1 Rheological properties In the time-scan test (1% strain, 1 Hz), BRP maintained stable G′ and G″ over 50 min. Figure 8 (A) confirmed its excellent structural stability. Under steady-state shear conditions, the polysaccharide solution (5 mg / mL, 25 °C) exhibited typical pseudoplastic behavior. Figure 8 BRP (B-type polymeric resin) exhibits a flow curve that follows a power-law model. It displays higher viscosity at high shear rates. The superior rheological properties of BRP are attributed to the mannose side chains in its molecule, which promote molecular chain entanglement and transient cross-linking, thus conferring higher viscoelasticity and time stability to the system. These properties make BRP more suitable for systems requiring thickening or structural stability. Frequency sweep tests (0.1–100 rad / s) further reveal its viscoelasticity (…). Figure 8 (C). BRP exhibits weak gel behavior, with G′ and G″ increasing with frequency. In the low-frequency region, G″>G′, reflecting viscosity dominance; in the high-frequency region, G′>G″, reflecting elastic dominance. Throughout the frequency range, BRP exhibits stronger intermolecular interactions and a denser entanglement network.

[0109] The mannose side chains in BRP enhance molecular chain entanglement and transient cross-linking, resulting in higher zero-shear viscosity, modulus, and structural stability, making it particularly suitable for structured systems such as emulsions and thickening sauces. In summary, the presence of mannose side chains in BRP significantly improves its functionality in structural design and stability regulation.

[0110] microstructure Transmission electron microscopy (TEM) characterization results (DF in Figure 4) revealed the microstructure of the polysaccharide at the nanoscale. BRP polysaccharide exhibited a complex three-dimensional network topology: obvious local thickening (chain diameter: 8–15 nm) could be observed at molecular connection points. These morphological features are consistent with the high branching density detected in the previous chemical structure analysis. The main chain with dense short side chain branches formed a multi-level interconnected network structure through inter-chain entanglement and cross-linking, which together confirmed its complex branching structure.

[0111] Example 6 Preparation of Rose Yellow Boletus Fucoido-Glucomannan Tablets

[0112] Material The same method as in Example 1 yields *Boletus fucoidogalactan* fucoidan, a food or pharmaceutical grade starch.

[0113] prescription 3. Preparation process The raw materials and excipients are homogenized and sieved for later use. The prescribed amount of PVPK30 is weighed and added to a 50% ethanol solution, while vigorous stirring to ensure thorough dispersion and dissolution, resulting in a clear, transparent solution. This solution is then sealed and stored for later use. The prescribed amounts of *Boletus macrantha* fucoidan, microcrystalline cellulose 101, lactose, and sodium carboxymethyl starch are weighed and dry-mixed for 15 minutes. The prepared binder is added, and the mixture is stirred for 3 minutes before granulation using an 18-mesh sieve. The granules are dried at 50-60 °C, then silica and magnesium stearate are added and mixed for 2 minutes before granulation again using a 16-mesh sieve. The semi-finished product content is determined, tablets are compressed, and film-coated to obtain the final product.

[0114] Example 7 Preparation of capsules containing *Boletus macrantha* fucoidan glucomannan.

[0115] Material The yellow-fleshed Boletus fucoido-glucomannan was obtained by the same method as in Example 1, and the starch was food or pharmaceutical grade.

[0116] prescription 3. Preparation process Weigh out the prescribed amounts of *Boletus macrantha* fucoidan and starch, and stir until fully mixed. Add an appropriate amount of talc, granulate using a wet ethanol method, sieve and dry, then fill into No. 2 capsule shells. Each capsule contains 80 mg of *Boletus macrantha* fucoidan, thus obtaining *Boletus macrantha* fucoidan capsules.

[0117] Example 8 Preparation of oral solution of *Boletus macrantha* fucoidan glucomannan.

[0118] Material The same method as in Example 1 yields *Boletus fucoidogalactan*, and the food or pharmaceutical grade flavoring agent is caramel flavoring.

[0119] prescription 3. Preparation process Weigh out the prescribed amounts of Boletus macrantha, fucoidan, glucomannan, sucrose, and caramel flavoring. Dissolve them completely in purified water, filter through a 0.2 μm microporous membrane, and fill each bottle with 4 mL of the filtrate using an oral liquid filling machine. After sealing, sterilize to obtain the final product.

[0120] Example 9 Rose-yellow Boletus fucoido-glucomannan freeze-dried powder for injection.

[0121] Materials: Rose-yellow Boletus fucoidan obtained by the same method as in Example 1, and water for injection.

[0122] prescription: Weigh the prescribed amount of *Boletus rosenbergii* fucoidan glucomannan, add water for injection, stir until completely dissolved, and sterilize by intermittent autoclaving. After the content is qualified, filter through a 0.22 μm microporous membrane; dispense into controlled vials, 0.5 mL per vial, partially stopper, place in a freeze-drying oven, freeze-dry according to the set freeze-drying curve, stopper, remove from the oven, cap, visually inspect for compliance, and package to obtain the finished product.

[0123] Freeze-drying process: Place the sample in the chamber, lower the partition temperature to -50 °C, and hold for 5 h; lower the cold trap to -50 °C and begin evacuation to 200 μbar. Sublimation begins: Heat to -15 °C at a constant rate for 2 h and hold for 3 h; heat to -5 °C at a constant rate for 3 h and hold for 6 h, maintaining a vacuum of 100 ~ 250 μbar; Drying continues: heat to 5 °C for 2 h and hold for 2 h, maintaining a vacuum of 150 ~ 200 μbar; heat to 15 °C for 1 h and hold for 2 h, maintaining a vacuum of 80 ~ 100 μbar; heat to 40 °C for 0.5 h and hold for 4 h, then evacuate to the lowest possible vacuum.

[0124] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0125] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should not be limited to the scope described in this application.

Claims

1. Rose-yellow-fleshed Boletus fucoidan, characterized in that, The *Boletus rosaefolius* fucoidan glucomannan 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 fucoidogalactan* according to claim 1, characterized in that, The weight-average molecular weight of the fucoidan-glucomannan from *Boletus macranthum* is 10 kDa to 20 kDa, the polysaccharide dispersion coefficient is 1 to 2, and the molar ratio of glucose:mannose:fucose:galactose is 1.00:3.08:2.84:14.

86.

3. The *Boletus fucoidogalactan* fucoidan according to claim 1, characterized in that, The rose-yellow-fleshed Boletus fucoidan glucomannan is derived from the genus Boletus in the family Boletaceae. Boletus The fruiting bodies were obtained through extraction and purification.

4. The *Boletus fucoidogalactan* according to claim 3, characterized in that, The fruiting bodies of the *Boletus macranthum* fungus are selected from *Boletus macranthum*. Butyroboletus roseoflavus Fresh fruiting bodies or dried bodies.

5. The method for preparing the fucoidan of Boletus macranthae according to any one of claims 1 to 4, characterized in that, The method includes the following steps: After homogenizing the fruiting bodies of *Boletus macrantha*, ethanol was extracted, and the residue was obtained by filtration or centrifugation. The residue was then extracted with water to obtain a crude polysaccharide solution of *Boletus macrantha*. Ethanol precipitation was used to obtain crude polysaccharide of *Boletus macrantha*. The fraction with a weight average molecular weight of 10 kDa to 20 kDa in the crude polysaccharide of *Boletus macrantha* was purified to obtain *Boletus macrantha* fucoidan-glucomannan.

6. The method for preparing the fucoidan from Boletus macranthae according to claim 5, characterized in that, After homogenizing the fruiting bodies of Boletus rosenbergii, 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 rosenbergii polysaccharide, making its ethanol concentration reach 60%, and collecting the precipitate by centrifugation.

7. The method for preparing *Boletus fucoidogalactan* according to claim 5, characterized in that, The steps for refining the *Boletus macrantha* fucoidan glucomannan are 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 macrantha* fucoidan according to any one of claims 1 to 4, characterized in that, The composition comprises an effective amount of *Boletus macrantha* fucoidan glucomannan and / or pharmaceutical excipients, excipients, or flavoring agents.

9. The use of the *Boletus macrantha* fucoidan-glucomannan as described in any one of claims 1 to 4 or the composition as described in claim 8 in the preparation of a drug or food for improving intestinal microecology.

10. The use of the *Boletus macrantha* fucoidan-glucomannan as described in any one of claims 1 to 4 or the composition as described in claim 8 in the preparation of prebiotic drugs or foods that regulate intestinal flora and promote intestinal health.

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