Grifola frondosa polysaccharide GFP22, a preparation method thereof and application thereof in preparing prebiotic products and auxiliary antibacterial drugs

Abstract

The present application discloses a grifola frondosa polysaccharide GFP22, a preparation method thereof and application thereof in preparing prebiotic products and auxiliary antibacterial drugs. The grifola frondosa polysaccharide GFP22 is a mannan glucan formed by polymerization of mannose and glucose, the ratio of mannose and glucose is 2.88:5.24, has the following structure, and the weight average molecular weight ranges from 4.9 kDa. The grifola frondosa polysaccharide GFP22 can promote synthesis of intestinal short-chain fatty acids and regulate intestinal flora homeostasis, promote proliferation of bifidobacterium, inhibit proliferation of klebsiella, and is expected to be developed into a kind of prebiotic product and a sugar drug for auxiliary anti-klebsiella drug.R:β-D-Glcp-(1→6)-β-D-Manp-(1→.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a Grifola frondosa polysaccharide GFP22, its preparation method, and its application in the preparation of prebiotic products and adjuvant antibacterial drugs. Background Technology

[0002] The gut microbiota refers to the microorganisms residing in the intestines. There are approximately 10 trillion microorganisms in the human gut, playing crucial roles in regulating nutrient absorption, maintaining epithelial cell development, and modulating innate immunity. The homeostasis and diversity of the gut microbiota are closely related to host cell proliferation and neural signaling pathways. An imbalance in the gut microbiota, with excessive proliferation of harmful bacteria and a decrease in beneficial bacteria, can lead to a series of diseases related to immunodeficiency and metabolic disorders.

[0003] Klebsiella pneumoniae (KP) belongs to the family Enterobacteriaceae and the genus Klebsiella. It is a Gram-negative facultative anaerobic bacterium that is commonly found in the environment, such as water, soil, and vegetation. It can colonize the respiratory or intestinal tract of humans or animals and is a common zoonotic opportunistic pathogen. Klebsiella pneumoniae (KP) is one of the most prevalent opportunistic pathogens in the world, highly pathogenic to humans and animals, causing pneumonia, metritis, mastitis, and other purulent inflammations, and even sepsis.

[0004] Edible fungal polysaccharides are carbohydrate polymers derived from edible fungi. They are resistant to endogenous digestive enzymes and are neither hydrolyzed nor absorbed in the small intestine. Because edible fungal polysaccharides cannot be digested and degraded by the human body, and because the gut microbiota colonizing the intestinal lumen contains abundant polysaccharide-hydrolyzing enzymes, edible fungal polysaccharides are primarily metabolized and degraded by the gut microbiota, thus affecting the gut microbiota and regulating host health. These beneficial polysaccharides or dietary fibers are often referred to as prebiotics, which can promote the proliferation of corresponding beneficial bacteria. Consuming a certain amount can provide beneficial microorganisms to the host.

[0005] Grifola frondosa, a fungus belonging to the genus Grifola in the family Polyporaceae, is one of the rare edible and medicinal fungi that has been developed and utilized in recent years. Grifola frondosa contains polysaccharides, sterols, and polyphenols, and possesses anti-tumor, immunomodulatory, and hypoglycemic effects. The Grifola frondosa strain HMGIM-W151021 is a high-polysaccharide-producing strain successfully domesticated from the wild by the Institute of Microbiology, Guangdong Academy of Sciences. The strain's preservation number is GDMCC No: 61165, and it has been published in Chinese Patent ZL 202210673582.4. However, its polysaccharide structure and its regulatory effects on the intestinal flora have not yet been reported. Summary of the Invention

[0006] This invention presents a simple and effective process and method for extracting and purifying polysaccharides from edible fungi. Using the proprietary strain *Grifola frondosa* HMGIM-W151021 as raw material, a mixed polysaccharide extract was obtained, which was further purified to obtain *Grifola frondosa* polysaccharide GFP22 with uniform charge and molecular weight. In vitro experiments demonstrated that this polysaccharide can regulate the balance of intestinal flora, inhibit pathogenic *Klebsiella pneumoniae*, and promote beneficial bacteria such as *Bifidobacterium*. Furthermore, after anaerobic fermentation in the intestine, this polysaccharide can produce short-chain fatty acids, which can regulate the body's immune and metabolic homeostasis, and hold promise for development into a novel prebiotic or a saccharide-based drug to assist in the treatment of pneumonia and related diseases caused by *Klebsiella pneumoniae*.

[0007] This invention provides a Grifola frondosa polysaccharide GFP22, which is a mannoglucan with a weight-average molecular weight of 4.90 kDa and has the following structure:

[0008]

[0009] R:β-D-Glcp-(1→6)-β-D-Manp-(1→.

[0010] The aforementioned Grifola frondosa polysaccharide GFP22 is a polysaccharide polymerized from mannose and glucose, with a molar ratio of mannose to glucose of 2.88:5.24.

[0011] This invention also provides a method for preparing the above-mentioned Grifola frondosa polysaccharide GFP22, comprising the following steps:

[0012] (1) Polysaccharide extraction: Boiling water was used to extract the fruiting bodies of Grifola frondosa. The extract was collected, concentrated, and centrifuged. The supernatant was then dialyzed, precipitated with alcohol, and centrifuged to collect the precipitate, yielding crude polysaccharide GFP.

[0013] (2) Polysaccharide purification: Crude polysaccharide GFP was purified by DEAE Sepharose. TMThe secondary crude polysaccharide GFP2 was obtained by separation using a Fast Flow anion exchange column; the secondary crude polysaccharide was further purified by a Sephacryl S-200HR gel column to obtain the Grifola frondosa polysaccharide GFP22.

[0014] Preferably, in step (1), the ethanol used for precipitation is 95% ethanol by volume, and the volume of the ethanol used for precipitation is 3 to 5 times that of the concentrate.

[0015] Preferably, in step (1), before ethanol precipitation, the concentrate is centrifuged and then the supernatant is dialyzed to remove water-soluble impurities and small molecule components.

[0016] Preferably, in step (2), when performing the separation on the anion exchange column, deionized water, 0.1M and 0.2M NaCl solutions are used sequentially for gradient elution, and the elution peak components eluted by 0.1M NaCl solution are collected to obtain the secondary crude polysaccharide GFP2.

[0017] Preferably, in step (2), during further purification on a Sephacryl S-200HR gel column, elution is performed using a 0.15M NaCl solution (containing 0.05M phosphate buffer, pH=6.8), and the collected eluted fraction is Grifola frondosa polysaccharide GFP22.

[0018] The identification of the obtained Grifola frondosa polysaccharide GFP22 in this invention includes determination of molecular weight and monosaccharide composition, followed by analysis of its structural characteristics using methylation combined with GC-MS and nuclear magnetic resonance.

[0019] The present invention also provides the use of the above-mentioned Grifola frondosa polysaccharide GFP22 in the preparation of prebiotic products or adjuvant antibacterial drugs.

[0020] Preferably, the prebiotic product can promote the production of short-chain fatty acids in the intestine and regulate the homeostasis of the intestinal flora.

[0021] Preferably, the short-chain fatty acids include acetic acid, propionic acid, and butyric acid; the regulation of intestinal flora homeostasis includes promoting the proliferation of Bifidobacteria and inhibiting the proliferation of Klebsiella pneumoniae.

[0022] Preferably, the target bacteria of the adjuvant antibacterial drug include Klebsiella pneumoniae.

[0023] The present invention also provides a prebiotic product or an adjuvant antibacterial drug, which contains the above-mentioned Grifola frondosa polysaccharide GFP22 as an active ingredient.

[0024] The present invention has the following beneficial effects:

[0025] This invention utilizes Grifola frondosa polysaccharide GFP22 to prepare intestinal prebiotics, promoting the enrichment of Bifidobacteria in the gut, inhibiting Klebsiella pneumoniae, and promoting the production of short-chain fatty acids by intestinal microorganisms. Grifola frondosa polysaccharide GFP22 does not directly inhibit harmful bacteria, but after fermentation, it significantly inhibits Klebsiella pneumoniae and other pathogenic microorganisms. Currently used antibiotics, while significantly inhibiting pathogenic bacteria, also suppress beneficial bacteria, causing intestinal flora imbalance. Exogenous probiotic supplementation may not achieve the desired colonization of specific probiotics, and some probiotics require low-temperature storage, resulting in high transportation and storage costs. The intestinal prebiotics of this invention have significant application value in the biomedical field. Attached Figure Description

[0026] Figure 1 The purity chromatogram of the Grifola frondosa polysaccharide GFP22 obtained in Example 1 is shown by high-performance gel permeation chromatography.

[0027] Figure 2 This is an ion chromatogram of the monosaccharide composition in the Grifola frondosa polysaccharide GFP22 obtained in Example 1.

[0028] Figure 3 This is the GC-MS spectrum of the glycosidic bonds in the Grifola frondosa polysaccharide GFP22 obtained in Example 1.

[0029] Figure 4 The Grifola frondosa polysaccharide GFP22 obtained in Example 1 1 H NMR and 13 C NMR spectrum.

[0030] Figure 5 The effect of fermenting Grifola frondosa polysaccharide GFP22 obtained in Example 1 for 24 h on the composition and structure of intestinal microorganisms.

[0031] Figure 6 The content of SCFA in the Grifola frondosa polysaccharide GFP22 obtained in Example 1 after 24 hours of in vitro fecal microbial fermentation. (A) Total carbohydrate concentration; (B) pH value; (C) Acetic acid concentration; (D) Propionic acid concentration; (E) Butyric acid concentration; Compared with group Con, the contents of acetic acid, propionic acid, and butyric acid showed statistically significant differences. ** P<0.01 *** P<0.001.

[0032] Figure 7 The inhibitory effects of Grifola frondosa polysaccharide GFP22 and fermentation broth prepared in Example 1 on Klebsiella pneumoniae (A) and the promoting effects of Grifola frondosa polysaccharide GFP22 on Bifidobacterium longum and Bifidobacterium pseudodocatenulatum (B, C) are shown. Detailed Implementation

[0033] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0034] Example 1: Extraction, isolation, purification, and structural characterization of Grifola frondosa polysaccharide GFP22

[0035] 1. Extraction and separation of polysaccharides

[0036] The fruiting bodies of *Grifola frondosa* strain HMGIM-W151021 were extracted multiple times using boiling water extraction. The sugar content of the extract was determined by the sulfuric acid-phenol method until the sugar reaction was no longer obvious. The extracts were combined, concentrated under reduced pressure, and centrifuged to remove the precipitate. The supernatant was dialyzed against running water for two days. The dialysate was precipitated with 95% ethanol at a concentration-to-ethanol ratio of 1:4 (v / v) and allowed to stand overnight. The supernatant was discarded, the precipitate was collected by centrifugation, and dried to obtain crude *Grifola frondosa* polysaccharide GFP (4.2%).

[0037] 2. Purification of polysaccharides

[0038] (1) Each time, take 4.0g of crude polysaccharide GFP and dissolve it in 45mL of deionized water, stir overnight, centrifuge and take the supernatant to load onto DEAE Sepharose. TM A Fast Flow anion exchange column was used for gradient elution with deionized water and different concentrations of NaCl solution (0.1M, 0.2M) at a flow rate of 3 mL / min, collected automatically. 50 μL of each tube was treated with the sulfuric acid-phenol method for color development, and the absorbance was measured at 490 nm using a microplate reader. Elution curves were plotted using absorbance and elution volume. Based on the elution curves, the separated polysaccharides were collected, concentrated under reduced pressure, dialyzed, and freeze-dried to obtain the eluted fraction with 0.1M NaCl. After drying, the secondary crude polysaccharide GFP2 (1.65 g) was obtained.

[0039] (2) Each time, 50 mg of the secondary crude polysaccharide GFP2 was dissolved in 3 mL of 0.15 M NaCl in pH 6.8 phosphate buffer, centrifuged (12,000 r / min), and the supernatant was loaded onto a Sephacryl S-200HR gel column. The column was eluted with 0.15 M NaCl solution (containing 0.05 M phosphate buffer, pH = 6.8) at a flow rate of 0.5 mL / min using an automated collector. After colorimetric analysis using the sulfuric acid-phenol method, absorbance was measured using an ELISA reader, and elution curves were plotted. The desired fraction was collected, concentrated, dialyzed, and freeze-dried to obtain homogeneous Grifola frondosa polysaccharide GFP22 (21.2 mg, 42.4%).

[0040] 3. Structural identification of polysaccharides

[0041] (1) The characteristic spectra of Grifola frondosa polysaccharide GFP22 on tandem TSKgel G5000SWXL and G3000SWXL analytical gel columns are as follows: Figure 1 As shown, the chromatographic conditions were: mobile phase: 0.1M NaNO3 solution; flow rate: 0.5 mL / min; column temperature: 30℃; Agilent 1260 HPLC system; detectors: differential detector and UV detector. The weight-average molecular weight of the sample was calculated based on the polysaccharide standard curve: Dextran series standards with known molecular weights (2 kDa, 5 kDa, 12 kDa, 20 kDa, 50 kDa, 120 kDa, 200 kDa, 500 kDa, 670 kDa) were dissolved in the mobile phase to prepare solutions with a concentration of 5 mg / mL. After centrifugation, the supernatant was collected and automatically injected for analysis. A standard curve was plotted using GPC software, and the weight-average relative molecular mass of Grifola frondosa polysaccharide GFP22 was determined to be 4.90 kDa. Gel permeation chromatography analysis of the homogeneity and weight-average molecular weight of Grifola frondosa polysaccharide GFP22 is shown below. Figure 1 As shown.

[0042] (2) Take 5 mg of Grifola frondosa polysaccharide GFP22, add 2 mL of 2M TFA, and hydrolyze at 120℃ for 3 h. Accurately pipette the acid-hydrolyzed solution into a tube and dry it with nitrogen evaporation apparatus. Add 5 mL of water and vortex to mix well. Pipe 50 μL and add 950 μL of deionized water, centrifuge at 12000 rpm for 5 min. Take the supernatant for ion chromatography analysis. Column: Dionex Carbopac TM PA20 (3*150mm); Mobile phase: A: H2O; B: 15mM NaOH-100mM NaOAC; Flow rate: 0.3mL / min; Injection volume: 5μL; Column temperature: 30℃; Detector: Electrochemical detector. Ion chromatography of monosaccharide composition in Grifola frondosa polysaccharide GFP22 is as follows: Figure 2 As shown.

[0043] (3) Take 10 mg of Grifola frondosa polysaccharide GFP22, place it in a glass reaction flask, add 1 mL of anhydrous DMSO, quickly add methylation reagent A, seal, dissolve under sonication, and then add methylation reagent B. React in a magnetically stirred water bath at 30℃ for 60 min. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction. Take the methylated polysaccharide, add 1 mL of 2M trifluoroacetic acid (TFA) for hydrolysis for 90 min, and evaporate to dryness using a rotary evaporator. Add 2 mL of double-distilled water to the residues, reduce with 60 mg of sodium borohydride for 8 hours, add glacial acetic acid for neutralization, rotary evaporate, dry in a 100℃ oven, then add 1 mL of acetic anhydride for acetylation, react at 100℃ for 1 h, and cool. Then add 3 mL of toluene, concentrate under reduced pressure and evaporate to dryness, repeat 4-5 times to remove excess acetic anhydride. Dissolve the acetylated product in 3 mL of CH2Cl2 and transfer to a separatory funnel, add a small amount of distilled water and shake well, remove the upper aqueous solution, and repeat this process 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, and the volume was adjusted to 10 mL, which was then placed in a liquid chromatography vial. Analysis was performed using a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometry system to determine the acetylated product samples. GC-MS conditions: RXI-5 SILMS column 30 m × 0.25 mm × 0.25 μm; temperature program: initial temperature 120 °C, increased to 250 °C / min at 3 °C / min; hold for 5 min; injection port temperature 250 °C, detector temperature 250 °C / min, helium carrier gas, flow rate 1 mL / min. The GC-MS chromatogram of the glycosidic bonds in Grifola frondosa polysaccharide GFP22 is shown below. Figure 3 As shown.

[0044] (4) The physicochemical constants of Grifola frondosa polysaccharide GFP22 are as follows:

[0045] GFP22: White powder; dissolve 30 mg of Grifola frondosa polysaccharide GFP22 in 0.5 mL of D2O, and add 2.5 μL of acetone as an internal standard (δ). H =2.29ppm, δ C =31.5ppm), and one-dimensional nuclear magnetic resonance spectra were measured at 25℃ on a Bruker AVANCE III 600M NMR spectrometer. 1 H NMR and 13 C NMR spectra as follows Figure 4 As shown, 13 C and 1 H NMR data can be found

[0046] Table 1. The specific structure of GFP22 polysaccharide is as follows:

[0047]

[0048] R:β-D-Glcp-(1→6)-β-D-Manp-(1→

[0049] This is a novel polysaccharide structure not previously reported in the literature.

[0050] Table 1: Carbon and hydrogen spectrum data of GFP22 (in H2O, 1 H for 600MHz, 13 (C for 150MHz) Table 3 1 H and 13 C NMR assignment of GFP22(in D2O)

[0051]

[0052] Example 2: The role of Grifola frondosa polysaccharide GFP22 in regulating gut microbiota homeostasis.

[0053] Fresh feces were collected from three volunteers. The uncontaminated middle portion was placed in a 15 mL sterile centrifuge tube and immediately added to sterile phosphate buffer solution (0.1 mol / L, pH = 7.2) to obtain a fecal solid-liquid mixture (10%, w / v). The mixture was homogenized and centrifuged, and the supernatant was collected. 2 g of peptone, 2 g of yeast extract, 2 mg of NaHCO3, 0.5 g of bile salts, 0.5 g of cysteine ​​hydrochloride, 0.1 g of NaCl, 40 mg of K2HPO4, 20 mg of heme, 10 mg of MgSO4, 10 mg of CaCl2, 80 1 mL of Tween, 1 mg of resazurin, and 10 μL of vitamin K1 were added to ultrapure water to a volume of 1 L. The pH was adjusted to 7.0 to prepare a basic nutrient growth medium. The prepared medium was sterilized (121℃, 20 min) and placed in a clean bench for later use. Con group: 0.5 mL fecal microbial culture + 4.5 mL culture medium + blank; GFP22 group: 0.5 mL fecal microbial culture + 4.5 mL culture medium + GFP22. The total fermentation volume was 5 mL, filled into vials, sealed, and tripled for each group. Anaerobic fermentation was carried out in a 37℃ constant temperature anaerobic incubator. Fermentation products were collected at 0 and 24 h. The fermentation products were centrifuged, and the supernatant was used to detect polysaccharide content, acidity, and short-chain fatty acids. The precipitate was used for 16S rRNA sequencing analysis (sequencing commissioned to Shanghai Meiji Biotechnology Co., Ltd.).

[0054] The results showed that after 24 hours of fermentation, the polysaccharide content in the GFP22 group was significantly reduced, indicating that the polysaccharides were utilized by gut microbiota during fermentation. The pH of the culture decreased significantly after fermentation, while the content of short-chain fatty acids, including acetic acid, propionic acid, and butyric acid, increased significantly. Figure 6The Ace, Shannon, and Simpson indices indicate that the polysaccharide GFP22 group enhanced the richness and diversity of the gut microbiota. At the phylum level, after 24 hours of fermentation, compared with Con, polysaccharide GFP22 significantly inhibited Proteobacteria and increased the abundance of Actinobacteria and Firmicutes. At the genus level, polysaccharide GFP22 significantly inhibited Klebsiella and increased the abundance of Catenibacterium and Bifidobacterium. Figure 5 ).

[0055] Example 3: Inhibitory effect of Grifola frondosa polysaccharide GFP22 and its fermentation broth on Klebsiella pneumoniae

[0056] Strain: Klebsiella pneumoniae (ATCC BAA-1902, purchased from Guangdong Provincial Microbial Culture Collection Center).

[0057] The supernatant of the GFP22 polysaccharide fermentation was obtained by collecting the fermentation product after 24 hours of fermentation according to the method in Example 2, centrifuging the fermentation product, and collecting the supernatant.

[0058] Klebsiella pneumoniae was cultured on BL agar medium. Single colonies from the plates were inoculated into 50 mL of BL liquid medium and cultured for 3-4 hours. 10 μL of bacterial culture, 90 μL of BL medium, and 100 μL of polysaccharide GFP22 or polysaccharide GFP22 fermentation supernatant were added to each well. OD was automatically measured every 30 minutes using a microplate reader. 600 Gentamicin was added as a positive control, and fermentation supernatant without polysaccharide GFP22 or with polysaccharide GFP22 was added as a blank control.

[0059] The results showed that polysaccharide GFP22 does not directly exert antibacterial activity. However, the fermentation supernatant of polysaccharide GFP22 after fermentation with intestinal flora exhibited antibacterial activity. This may be because polysaccharide GFP22 produces some antibacterial metabolites after fermentation, thereby playing an indirect antibacterial role. Figure 7 A).

[0060] Example 4: Growth-promoting effect of Grifola frondosa polysaccharide GFP22 on Bifidobacterium longum and B. pseudodocatenulatum

[0061] Single colonies from the plates were inoculated into MRS broth medium and cultured at 37°C with a shaking incubator at 150 rpm for 48 hours. The basal culture medium was prepared (the preparation method was the same as the basic nutrient growth medium in Example 2). After sterilization, the cultured bacterial suspensions were inoculated into different groups at a 10% (v / v) inoculation rate. The Con group consisted of 0.5 mL of Bifidobacterium suspension + 4.5 mL of basal culture medium + blank, and the GFP22 group consisted of 0.5 mL of Bifidobacterium suspension + 4.5 mL of basal culture medium + GFP22. Both groups were anaerobic cultured at 37°C, and the OD value of the fermentation broth was measured at 600 nm using a microplate reader. 600 The values ​​were used to plot growth curves. The results showed that different concentrations (2.5 mg / mL, 5 mg / mL) of the polysaccharide GFP22 promoted the growth of both *Bifidobacterium longum* and *Bifidobacterium pseudodocatenulatum*. Figure 7 B, C).

[0062] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Grifola frondosa polysaccharide GFP22, characterized in that, Includes the following steps: (1) Polysaccharide extraction: Boiling water was used to extract the fruiting bodies of Grifola frondosa. The extract was collected, concentrated, and centrifuged. The supernatant was then dialyzed, precipitated with alcohol, and centrifuged to collect the precipitate, yielding crude polysaccharide GFP. (2) Polysaccharide purification: Crude polysaccharide GFP was purified by DEAE Sepharose. TM The secondary crude polysaccharide GFP2 was separated using a Fast Flow anion exchange column and eluted sequentially with deionized water, 0.1 M, and 0.2 M NaCl solutions. The elution peak fraction eluted with 0.1 M NaCl solution was collected to obtain the secondary crude polysaccharide GFP2. The secondary crude polysaccharide GFP2 was then further purified using a Sephacryl S-200HR gel column and eluted with a 0.15 M NaCl solution at pH 6.8 containing 0.05 M phosphate buffer. The elution fraction was collected to obtain the Grifola frondosa polysaccharide GFP22.

2. The preparation method according to claim 1, characterized in that, In step (1), the ethanol used for precipitation is 95% ethanol by volume, and the volume of ethanol used for precipitation is 3 to 5 times that of the concentrate.

3. The Grifola frondosa polysaccharide GFP22 prepared by the preparation method according to claim 1.

4. The Grifola frondosa polysaccharide GFP22 according to claim 3, characterized in that, The aforementioned Grifola frondosa polysaccharide GFP22 is a mannoglucan polymerized from mannose and glucose, and has the following structure: R: β-D-Glcp-(1→6)-β-D-Manp-(1→.

5. The Grifola frondosa polysaccharide GFP22 according to claim 4, characterized in that, The weight-average molecular weight of the Grifola frondosa polysaccharide GFP22 is 4.90 kDa, and the ratio of mannose to glucose is 2.88:5.

24.

6. Use of the Grifola frondosa polysaccharide GFP22 according to any one of claims 3-5 in the preparation of prebiotic products or adjuvant antibacterial drugs.

7. The use according to claim 6, characterized in that, The prebiotic products described above can promote the production of short-chain fatty acids in the gut and regulate gut microbiota homeostasis.

8. The use according to claim 7, characterized in that, The short-chain fatty acids include acetic acid, propionic acid, and butyric acid; the regulation of intestinal flora homeostasis includes promoting the proliferation of Bifidobacteria and inhibiting the proliferation of Klebsiella pneumoniae.

9. The use according to claim 6, characterized in that, The target bacteria of the aforementioned adjuvant antibacterial drug include Klebsiella pneumoniae.

10. A prebiotic product or adjuvant antibacterial drug, characterized in that, It contains the Grifola frondosa polysaccharide GFP22 as described in any one of claims 3-5 as an active ingredient.

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