Preparation method and application of polygonatum polysaccharide based on fermentation-ultrafiltration cascade

Abstract

The invention discloses a rhizoma polygonati polysaccharide preparation method and application based on fermentation-ultrafiltration cascade, and the method comprises the following steps: step 1, drying and crushing rhizoma polygonati tubers to obtain rhizoma polygonati powder, and mixing the rhizoma polygonati powder with a solvent to obtain a rhizoma polygonati suspension; 2, bacillus licheniformis is inoculated into the polygonatum sibiricum suspension in the step 1, and fermentation culture is carried out; and 3, filtering the fermented supernatant obtained in the step 2, and purifying the filtered supernatant by adopting an ultrafiltration membrane to obtain the required polygonatum polysaccharide. The polygonatum sibiricum polysaccharide obtained by the fermentation cascade ultrafiltration method is high in yield and purity, and the structure of the polysaccharide is not changed.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing technology, specifically to a method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade and its application. Background Technology

[0002] Polygonatum is a perennial herb belonging to the genus Asparagus in the family Liliaceae. It is widely distributed in Northeast, North, and Southwest China. As an important plant used for both food and medicine, it has been widely applied as an adjunct treatment for various chronic diseases, such as diabetes, atherosclerosis, immunodeficiency, and inflammatory diseases. The various active ingredients in Polygonatum provide a solid foundation for its application in modern functional foods and natural medicine development. Among them, polysaccharides are considered one of the main functional components of Polygonatum.

[0003] Currently, research on obtaining high-yield bioactive polysaccharides from natural sources through fermentation has received widespread attention. During fermentation, microorganisms secrete extracellular enzymes and other secondary metabolites, disrupting plant cell wall structures, promoting the release of active ingredients, and thus increasing polysaccharide yield. Furthermore, ultrafiltration technology, as a membrane separation technique based on molecular weight cutoff, has been widely used in recent years for the purification and fractionation of natural polysaccharides. Ultrafiltration can effectively remove impurities such as proteins, small sugars, organic acids, and inorganic salts, and operates at room temperature without phase change, thus offering advantages such as high efficiency, environmental friendliness, and no impact on polysaccharide structure. Summary of the Invention

[0004] This invention addresses the problems existing in the prior art by providing a method for preparing Polygonatum polysaccharides based on fermentation-ultrafiltration cascade and its application.

[0005] The technical solution adopted in this invention is: a method for preparing Polygonatum polysaccharides based on fermentation-ultrafiltration cascade, comprising the following steps: Step 1: Dry and pulverize the rhizome of Polygonatum to obtain Polygonatum powder. Mix the Polygonatum powder with a solvent to obtain a Polygonatum suspension. Step 2: Inoculate Bacillus licheniformis into the Polygonatum sibiricum suspension from Step 1 and ferment it. Step 3: After filtering the fermentation supernatant obtained in Step 2, the supernatant is purified by ultrafiltration membrane to obtain the desired Polygonatum polysaccharide.

[0006] Furthermore, in step 2, the mass concentration of the Polygonatum sibiricum suspension is 2–3 wt.%, and the volume ratio of Bacillus licheniformis solution to Polygonatum sibiricum suspension is 6–7:100; the concentration of the Bacillus licheniformis solution is 10. 8 CFU / mL.

[0007] Furthermore, the fermentation time in step 2 is 8–9 hours.

[0008] Furthermore, in step 3, the fermentation supernatant is filtered using a 0.45 μm filter membrane.

[0009] Furthermore, in step 3, the ultrafiltration membrane is a 10 k and a 1 k polyethersulfone membrane, and purification is performed using 10 k and 1 k ultrafiltration membranes, respectively.

[0010] Furthermore, in step 2, fermentation time, inoculum size, and substrate concentration are used as independent variables to design a single-factor experiment; the required fermentation time, inoculum size, and substrate concentration are predicted by a quadratic multinomial regression model using the polysaccharide dissolution rate as the response value.

[0011] Furthermore, in step 3, after ultrafiltration, an elution process is also performed.

[0012] Furthermore, the Bacillus licheniformis culture process is as follows: Bacillus licheniformis is inoculated into NB medium and cultured at 37 °C for 24 h, and the bacterial precipitate is obtained by centrifugation.

[0013] A polysaccharide of Polygonatum odoratum obtained by a fermentation-ultrafiltration cascade preparation method.

[0014] An application of Polygonatum polysaccharide, wherein the Polygonatum polysaccharide is used in the preparation of pharmaceuticals, foods or health products that regulate the structure of intestinal flora.

[0015] The beneficial effects of this invention are: Compared with the traditional alcohol precipitation method, this invention can improve the yield and purity of Polygonatum polysaccharide without changing the original structure of the polysaccharide. The preparation process is mild, green and efficient, and does not require organic reagents. Attached Figure Description

[0016] Figure 1 The results of polysaccharide dissolution rate under different fermentation parameters in the embodiments of the present invention are shown. a is the substrate concentration, b is the inoculum amount, and c is the fermentation time.

[0017] Figure 2 The results of Polygonatum polysaccharide obtained under different ultrafiltration conditions in the embodiments of the present invention are shown. a is the total sugar content, and b is the polysaccharide yield and polysaccharide purity.

[0018] Figure 3 The results of elution detection under different conditions in Example 2 of the present invention are shown in Figure a. a represents the results of different concentrations of NaCl with a tube count of 5 mL / tube, and b represents the results with a tube count of 4 mL / tube.

[0019] Figure 4 The images show the ultraviolet spectra of Polygonatum polysaccharide samples obtained in Example 2 and the comparative example of this invention.

[0020] Figure 5 The infrared spectra of Polygonatum polysaccharide samples obtained in Example 2 and the comparative example of the present invention are shown.

[0021] Figure 6 The images show SEM images of Polygonatum polysaccharide samples obtained in Example 2 and the comparative example of this invention.

[0022] Figure 7 The results of the monosaccharide composition test of the Polygonatum polysaccharide sample obtained in Example 2 of the present invention are shown in Figure a, which is the result of Example 1, and Figure b is the standard curve.

[0023] Figure 8 The NMR spectra of the Polygonatum polysaccharide sample obtained in Example 2 of this invention are shown below: a is the 1H NMR spectrum, b is the ¹³C NMR spectrum, c is the ¹H–¹H correlation spectrum, d is the ¹H–¹H full correlation spectrum, e is the ¹H–¹H nuclear Overhauser effect spectrum, and f is the ¹H–¹³C heteronuclear single quantum coherence spectrum.

[0024] Figure 9 This is a schematic diagram of the structure of the Polygonatum polysaccharide sample obtained in Example 1 of the present invention.

[0025] Figure 10 The animal experiment results using Polygonatum polysaccharide obtained in Example 1 of this invention are shown in the figures: a is the rate of change in mouse body weight, b is the DAI score, c is the organ index, and d is the colon length.

[0026] Figure 11 The results of staining mouse colon sections in an animal experiment using Polygonatum polysaccharide obtained in Example 1 of this invention.

[0027] Figure 12 The results of serum inflammatory factors in mice in animal experiments using Polygonatum polysaccharide obtained in Example 1 of this invention are shown. a is IL-6, b is IL-1β, c is TNF-α, and d is IL-10.

[0028] Figure 13 The positive area of ​​mouse colon slices in animal experiments using Polygonatum polysaccharide obtained in Example 1 of this invention is shown in Figure 1. a represents IL-6, b represents IL-1β, c represents TNF-α, and d represents IL-10.

[0029] Figure 14 The results of the intestinal microbiota analysis of mice in animal experiments using Polygonatum polysaccharide obtained in Example 1 of this invention are shown in the figures: a) principal coordinate analysis of intestinal microbiota at the ASV level; b) petal diagram of the number of unique and shared ASVs among groups; c) bar diagram of microbial community composition at the phylum and genus levels; and d) linear discriminant analysis of effect size (LEfSe). Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] A method for preparing Polygonatum polysaccharides based on fermentation-ultrafiltration cascade includes the following steps: Step 1: Dry and pulverize the rhizome of Polygonatum to obtain Polygonatum powder. Mix the Polygonatum powder with a solvent to obtain a Polygonatum suspension. Step 2: Inoculate Bacillus licheniformis into the Polygonatum sibiricum suspension from Step 1 and ferment; the mass concentration of the Polygonatum sibiricum suspension is 2-3 wt.%, and the volume ratio of Bacillus licheniformis solution to Polygonatum sibiricum suspension is 6-7:100; the concentration of the Bacillus licheniformis solution is 10. 8 CFU / mL. Fermentation time was 8–9 h. A single-factor experiment was designed with fermentation time, inoculum size, and substrate concentration as independent variables; the required fermentation time, inoculum size, and substrate concentration were predicted using a quadratic multinomial regression model with polysaccharide dissolution rate as the response value.

[0032] The culture process of Bacillus licheniformis is as follows: Bacillus licheniformis is inoculated into NB medium and cultured at 37 ℃ for 24 h. The bacterial cells are then centrifuged to obtain a precipitate.

[0033] Step 3: After filtering the fermentation supernatant obtained in Step 2, the supernatant was purified using an ultrafiltration membrane to obtain the desired Polygonatum polysaccharide. The fermentation supernatant was filtered using a 0.45 μm filter membrane, and the ultrafiltration membranes were 10 kJ and 1 kJ polyethersulfone membranes, respectively, for purification. Elution was also performed after ultrafiltration.

[0034] Example 1 A method for preparing Polygonatum polysaccharides based on fermentation-ultrafiltration cascade includes the following steps: Step 1: Wash and slice the Polygonatum rhizome, dry it to constant weight, pulverize it, and pass it through an 80-mesh sieve to obtain Polygonatum powder. Mix the Polygonatum powder with a solvent to obtain a Polygonatum suspension.

[0035] Step 2: Inoculate the existing preserved Bacillus licheniformis (from the Institute of Microbiology, Chinese Academy of Sciences, Beijing) into NB medium and incubate at 37 °C for 24 h. Centrifuge to obtain the bacterial pellet and resuspend it in 0.9% physiological saline to 10⁻⁶ oz. 8 CFU / mL, stored at 4 ℃ for subsequent fermentation.

[0036] The mass concentration of the Polygonatum sibiricum suspension was 3 wt.%, i.e., the substrate concentration was 3 wt.%; the volume ratio of Bacillus licheniformis solution to Polygonatum sibiricum suspension was 6:100, i.e., the inoculum size was 6 wt.%; the concentration of the Bacillus licheniformis solution was 10... 8 CFU / mL, fermentation time was 8 h.

[0037] A single-factor experiment was designed with fermentation time, inoculum size, and substrate concentration as independent variables. The required fermentation time, inoculum size, and substrate concentration were predicted using a quadratic multinomial regression model with polysaccharide dissolution rate as the response value. The process is as follows: The initial fermentation parameters were set as follows: substrate concentration 1%, inoculum size 6% (v / v), fermentation time 16 h, and fermentation temperature 37℃. In the single-factor optimization stage, the substrate concentration gradient was set to 1%, 3%, 5%, 7%, and 9%; the inoculum size gradient was set to 2%, 4%, 6%, 8%, and 10% (v / v); and the fermentation time gradient was set to 4, 8, 12, 16, and 20 h. The results are as follows: Figure 1 As shown.

[0038] Based on the results of the single-factor experiments, a Box-Behnken design (BBD) was conducted using Design-Expert 13 software, with polysaccharide dissolution rate (Y, %) as the response value and fermentation time (A, h), inoculum size (B, %), and substrate concentration (C, %) as independent variables. The level codes for each factor are shown in Table 1. 1, 0, and +1 correspond to the low, medium, and high levels, respectively. The experimental results are shown in Tables 2 and 3.

[0039] Table 1. Response Surface Design

[0040] Table 2. Results of Response Surface Experiment

[0041] Using the polysaccharide dissolution rate as the response value, a quadratic multinomial regression model was used to predict the required fermentation time, inoculum size, and substrate concentration. The results are shown in Table 3. The highest polysaccharide dissolution rate (66.5%) was achieved at a substrate concentration of 2.7%, an inoculum size of 6.5%, and a fermentation time of 9 h. Therefore, a substrate concentration of 2–3%, an inoculum size of 6–7%, and a fermentation time of 8–9 h were selected. In this example, a substrate concentration of 3 wt.%, an inoculum size of 6%, and a fermentation time of 8 h were selected. The results of the analysis of variance are shown in Table 3. Table 3. Analysis of Variance for Regression Models

[0042] Step 3: Remove macromolecular impurities from the fermentation supernatant obtained in Step 2 using a 0.45 μm filter membrane, and then purify the supernatant sequentially using 10 k and 1 k PES ultrafiltration membranes. Determine the total sugar content of the purified filtrate, collect the components that have passed through the 10 k and 1 k ultrafiltration membranes, concentrate and freeze-dry to obtain FPSP-2.

[0043] To illustrate the effectiveness of this invention, filtrates obtained from 100 k, 50 k, 10 k, and 1 k ultrafiltration membranes were collected, concentrated, and freeze-dried to obtain samples labeled FPS-100 k, FPS-50 k, FPS-10 k, and FPS-1 k, respectively. The concentrated and freeze-dried fermentation broth was labeled FPS-RAW.

[0044] The polysaccharide sample obtained in Example 1 was subjected to methylation analysis, and the procedure is as follows: The sample was dissolved in dimethyl sulfoxide (DMSO, 500 μL), and incubated with NaOH (1 mg) for 30 min. Iodomethane (250 μL) was then added for methylation for 1 h. After the reaction, a mixture of water and dichloromethane (1:2, v / v) was added, and the lower dichloromethane phase was collected. The resulting product was hydrolyzed with 2 M trifluoroacetic acid (TFA, 100 μL) under two different conditions: 60 °C for 1 h and 121 °C for 90 min. The hydrolysate was then reduced with 12 M ammonia (50 μL) and 1 M sodium borodeuteride (50 μL), and the reaction was terminated with glacial acetic acid. Acetic anhydride was then added, and acetylation was performed at 100 °C for 2.5 h. The target compound was obtained by dichloromethane extraction for subsequent analysis.

[0045] The obtained methylated products were detected using gas chromatography-mass spectrometry (GC-MS, Agilent 6890A-5977B, Agilent Technologies, USA), equipped with a BPX70 capillary column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The initial column temperature was set at 140 °C and held for 2 min, then increased to 230 °C at a rate of 3 °C / min and held at that temperature for 3 min.

[0046] The results are shown in Table 4. PSFP mainly has five glycosidic bonds, namely t-Fruf-(2→, t-Glcp-(1→, →1)-Fruf-(2→, →6)-Glcp-(1→ and →1,6)-Fruf-(2→, with fructose residues being the main components.

[0047] Table 4. Methylation Results

[0048] Example 2 The other steps in this embodiment are the same as in embodiment 1, except that the PSFP-2 obtained in embodiment 1 needs to undergo the following processing: FPSP-2 was reconstituted, proteins were removed using the Sevag method, the mixture was concentrated to half its volume under reduced pressure to remove organic matter, and then lyophilized.

[0049] FPSP-2 was first purified using a DEAE-agarose gel rapid flow column (1.6 cm × 20 cm). Elution was performed using gradients of different concentrations of NaCl solution (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L) at a flow rate of 2.5 mL / min. Five mL of eluent was collected from each tube, and the sugar content in the eluent was determined using the phenol-sulfuric acid method.

[0050] Subsequently, the FPSP-2-containing fraction was further purified using a Sephadex G-100 gel filtration column (1.6 cm × 60 cm) at a flow rate of 0.2 mL / min, with 4 mL of eluent collected per tube. The eluent was concentrated, dialyzed, and lyophilized, and designated as FPSP-2. The elution curve is shown below. Figure 3 As shown in the figure, there is only one elution peak, indicating that FPSP-2 is mainly composed of a homogeneous component.

[0051] To illustrate the effect of the above process, the sample FPSP-RAW obtained by eluting the concentrated and freeze-dried fermentation broth was denoted as FPSP-RAW.

[0052] To illustrate the effectiveness of the present invention, a comparative example is provided. Comparative Example 1 The other contents of this comparative example are the same as those of Example 1, except that 50 k and 10 k ultrafiltration membranes are used for purification in step 3, and the sample is designated as FPSP-1.

[0053] Comparative Example 2 The other contents of this comparative example are the same as those of Example 1, except that a 1 k ultrafiltration membrane is used for purification in step 3, and the sample is designated as FPSP-3.

[0054] Comparative Example 3 The other contents of this comparative example are the same as those of Example 1, except that in step 3, the fermentation supernatant is extracted with 4 times the volume of anhydrous ethanol for 12 h, the precipitate is collected, and after being dissolved and freeze-dried again, the sample is denoted as FPSP-AP.

[0055] Figure 2The graph shows the polysaccharide test results of samples obtained under different ultrafiltration conditions. It can be seen from the graph that the total sugar content decreased significantly after passing through a 1 k k ultrafiltration membrane, indicating that FPSPs are mainly concentrated between 1 and 10 k k. The yield and purity results of different components show that PSFP-1 has a purity of 64.62%, but its yield is only 4.81%, while PSFP-3 has a higher yield of 18.76%, but its purity is very low at only 42.37%. PSFP-2 has the highest yield and purity among the three ultrafiltration components. The traditional alcohol precipitation method yielded PSFP-AP with a yield of 20.62% and a purity of 44.52%, both lower than those obtained by ultrafiltration.

[0056] FPSP-RAW, FPSP-2, and FPSP-AP were scanned using a UV spectrophotometer in the range of 200-800 nm. The results are as follows: Figure 4 As shown in the figure, different treatment methods have a significant impact on the absorbance of the FPSP samples. Compared with FPSP-RAW, FPSP-2 has lower absorbance in the low-wavelength region, but its absorption curve trend is basically consistent with that of FPSP-RAW, indicating that ultrafiltration removes some small molecule impurities but does not significantly change the structure of the polysaccharide. In contrast, the absorption curve of FPSP-AP is completely different from that of FPSP-RAW, indicating that while alcohol precipitation removes more soluble substances, it also changes the composition or spectral characteristics of the sample.

[0057] Take an appropriate amount of potassium bromide, grind it, and press it into a thin sheet with a thickness of about 1 mm. Scan it on a Fourier transform infrared spectrometer in full-band mode, and use the obtained spectrum as a reference. Then, mix the polysaccharide sample with potassium bromide at a ratio of about 1 / 100 (m / m), grind it, and press it into a sheet. Also, scan it at 4000–400 cm⁻¹. -1 The Fourier transform infrared (FTIR) spectra of FPSP-2 and FPSP-AP were obtained by scanning in full-band mode within the wavenumber range. The results are as follows: Figure 5 As shown in the figure. It can be seen from the figure that the two are located between 1000 and 1200 cm. -1 The regions all exhibit distinct absorption peaks, characteristic of the C–O–C and C–O–H vibrations of polysaccharides, confirming that the obtained product is a polysaccharide. The difference lies in the absorption peak observed in FPSP-AP at 1745 cm⁻¹. -1 The presence of a characteristic absorption peak, typically attributed to the C=O stretching vibration of carboxyl or ester groups, suggests the possible presence of partially bound acidic polysaccharide components or esterified structures in FPSP-2. Meanwhile, PSFP-UF exhibits a peak at 1132 cm⁻¹. -1 and 927 cm -1 It exhibits a characteristic absorption peak at 1132 cm⁻¹. -1This structure is typically attributed to stretching vibrations of the C–O–C sugar ring, indicating its relative enrichment in FPSP-2; 927 cm⁻¹ -1 The nearby absorption peaks may be related to the vibrations of pyranose residues. These differences suggest that different separation methods may alter the composition and conformational characteristics of the polysaccharides. FPSP-AP is rich in acidic or esterified components, while FPSP-2 is unique in its glycosidic bond type and monosaccharide residue configuration.

[0058] The purified FPSP powder was immobilized on copper posts and covered with a thin layer of gold foil. SEM images were captured at 1500× magnification with an accelerating voltage of 15.0 kV. The results are as follows: Figure 6 As shown in the figure, FPSP-RAW exhibits an irregular sheet-like structure with obvious pores and rough texture, indicating a relatively loose internal structure. FPSP-AP has a denser surface morphology, with some sheets showing curling and breakage, suggesting that some molecules may aggregate or the structure may collapse during alcohol precipitation. FPSP-2, on the other hand, displays a regular sheet-like structure with a relatively smooth surface and fewer fragments, indicating a more uniform molecular arrangement.

[0059] The molecular weight of FPSP-2 obtained in Example 2 was determined using a gel permeation chromatography (GPC) system consisting of Ohpak SB-805 and SB-803 HQ columns. 100 μL of sample was injected, with a 0.1 M NaNO3 solution containing 0.02% NaN3 as the mobile phase at a flow rate of 0.6 mL / min. The purified FPSP was detected using a refractive index detector (RI, Optilab T-rEX, Wyatt Technology, USA) and a multi-angle laser light scattering detector (MALLS, DAWN HELEOS II, Wyatt Technology, USA). The results showed that the molecular weight of FPSP-2 was 5.4 kDa.

[0060] The monosaccharide composition of Polygonatum polysaccharide FPSP-2 obtained in Example 2 was determined as follows: High-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD) was used for determination. Given the instability of fructose (Fru) under high temperature and prolonged conditions, a milder hydrolysis method was employed for monosaccharide composition analysis. Specifically, polysaccharide samples were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 60 °C for 1 h, followed by drying under nitrogen; washing with methanol and drying again was then performed. This methanol washing was repeated 2–3 times, followed by dissolution in sterile water and transfer to sample vials for analysis.

[0061] The chromatographic analysis system employed a Thermo ICS 5000+ ion chromatography system (ICS5000+, Thermo Fisher Scientific, USA), using an electrochemical detector for the detection and analysis of monosaccharide components. This system was configured with a Dionex™ CarboPac™ PA20 column (150 × 3.0 mm, 10 μm) and an injection volume of 5 μL. Mobile phases A, B, and C were H₂O, 0.1 M NaOH, and 0.1 M NaOH + 0.2 M NaAc, respectively, with a flow rate of 0.5 mL / min and a column temperature of 30 °C. The gradient elution program was as follows: 0 min, A / B / C = 95:5:0 (V / V / V); 26 min, A / B / C = 85:5:10; 42 min, A / B / C = 85:5:10; 42.1 min, A / B / C = 60:0:40; 52 min, A / B / C = 60:40:0; 52.1 min, A / B / C = 95:5:0; 60 min, A / B / C = 95:5:0 (V / V / V).

[0062] Test results are as follows Figure 7 As shown in the figure, the monosaccharide composition of FPSP-2 can be seen. The main monosaccharide components are fructose (Fru) and glucose (Glc), with a ratio of Fru:Glc = 65.51:5.79.

[0063] The NMR analysis of the sample obtained in Example 2 was performed, and the results are as follows: Figure 8 As shown in Table 5, 50 mg of lyophilized sample powder was dissolved in 500 μL of D2O solution and detected on a Bruker 600 MHz nuclear magnetic resonance spectrometer (Avance II-600 MHz, Bruker, Switzerland). Figure 8 In the spectrum, a and b are one-dimensional nuclear magnetic resonance spectra, while c, d, e, and f are two-dimensional nuclear magnetic resonance spectra.

[0064] Table 5. FPSP-2 1 H and 13 C chemical shift

[0065] Based on the NMR and methylation results, the determined FPSP-2 structure is as follows: Figure 9 As shown.

[0066] To illustrate the effectiveness of the present invention, animal experiments were conducted using the sample obtained in Example 2, as follows: Thirty healthy male Kunming mice, aged four weeks, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. (Chengdu, China). The mice were housed at a constant temperature of 25 °C with a 12 h / 12 ​​h light / dark cycle and free access to fresh water and a basal diet. All animal experiments were conducted in accordance with relevant institutional and national guidelines for laboratory animal care and use, and were approved by the Ethics Committee of the Laboratory Animal Center of Sichuan University (Permit No.: SCU43-2025-02).

[0067] After one week of acclimatization, mice were randomly divided into five groups: a normal control group (Control), a colitis model group (DSS), a low-dose FPSP-2 group (FPL, 100 mg / kg), a medium-dose FPSP-2 group (FPM, 200 mg / kg), and a high-dose FPSP-2 group (FPH, 400 mg / kg). During the prevention period (one week), the Control and DSS groups were administered 0.2 mL of sterile saline by gavage, while the FPL, FPM, and FPH groups were administered 0.2 mL of a solution containing PSFP by gavage, at doses of 100, 200, and 400 mg / kg / day, respectively. All mice had free access to water.

[0068] During the modeling period (one week), the Control group continued to receive sterile saline and water; the DSS group received sterile saline and 3% DSS solution (m / V) freely available for drinking; the other groups were administered PSFP solution by gavage at the corresponding dose and freely available for drinking 3% DSS solution (m / V). Throughout the experiment, mouse weight was recorded every other day. At the end of the prevention and modeling periods, mouse feces were collected for gut microbiota analysis. On day 21, mice were euthanized and blood samples were collected. The liver and spleen were weighed, and the colon was removed and fixed in 4% paraformaldehyde solution for the following experiments.

[0069] The results of DAI scoring and organ index analysis are as follows: Figure 10 As shown in the figure, compared with the control group, the DSS group mice had a significant decrease in body weight. However, after treatment with low, medium, and high doses of FPSP-2, the trend of body weight loss was significantly improved, especially in the FPH group where body weight recovery was closest to normal. Disease Activity Index (DAI) scores showed a significant increase in the DSS group, while all doses of FP significantly reduced DAI scores, suggesting that FP can effectively alleviate clinical symptoms of colitis. Furthermore, DSS induction led to a significant increase in the spleen index, while FP intervention significantly reduced the spleen index, indicating its role in improving abnormalities in immune organs. Liver indices did not differ significantly among the groups.

[0070] The fixed colonic tissue was sequentially embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The tissue sections were scored in a blinded manner by two independent researchers without prior grouping information. The results of colonic length changes are as follows: Figure 11 As shown in the figure, the colon of mice in the DSS group was significantly shortened, while all doses of FP significantly prolonged the colon length, suggesting that FP has a certain tissue protective effect. Histopathological results further verified the above trend. The colon tissue structure of the control group was intact, and the glands were tightly arranged; the DSS group showed obvious epithelial shedding, crypt structure destruction, and inflammatory cell infiltration; while the degree of intestinal tissue damage in the FPL, FPM, and FPH groups was significantly reduced, the crypt structure was more intact, and the inflammatory cell infiltration was reduced, with the improvement becoming more significant with increasing dose.

[0071] Paraffin-embedded tissue sections underwent dewaxing, antigen retrieval (citrate buffer, pH 6.0), endogenous peroxidase blocking, and BSA blocking. Subsequently, sections were incubated overnight at 4 °C with primary antibodies (IL-1β, IL-6, TNF-α, and IL-10). After washing, sections were incubated at 37 °C with horseradish peroxidase (HRP)-labeled secondary antibody for 30 min. 3,3'-diaminobenzidine (DAB) staining was used, and cell nuclei were counterstained with hematoxylin. Finally, sections were dehydrated, cleared, and mounted. Quantitative analysis of DAB-positive staining was performed at 400× magnification using HALO image analysis software. Results are as follows: Figure 12 As shown.

[0072] The figure shows that the positive expression areas of IL-6, IL-1β, and TNF-α were significantly increased in the DSS group, while the positive expression area of ​​IL-10 was significantly decreased, indicating an active local inflammatory response. After PSFP intervention, the positive expression area of ​​inflammatory factors was significantly reduced, and IL-10 positive expression was restored, especially in the FPH group where the improvement was most significant.

[0073] Detection of inflammatory factors Following the instructions in the kit manual, the levels of cytokines in serum, including IL-1β, IL-6, IL-10, and TNF-α, were detected using an ELISA kit.

[0074] The results are as follows Figure 13As shown in the figure, the levels of inflammatory factors IL-6, IL-1β, and TNF-α in mouse serum significantly increased after DSS induction, while the levels of anti-inflammatory factor IL-10 significantly decreased, indicating that DSS successfully induced a systemic inflammatory response. After FP intervention, the levels of inflammatory factors decreased to varying degrees in all dose groups, with the FPH group showing the most significant decrease, indicating that PSFP has significant anti-inflammatory activity. Simultaneously, IL-10 levels were significantly higher in the FPL, FPM, and FPH groups than in the DSS group, suggesting that PSFP can promote the recovery of anti-inflammatory factors and improve immune balance.

[0075] 16S rRNA sequencing analysis PCR amplification of the V4 region of the bacterial 16S rRNA gene was performed using primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplification program was as follows: pre-denaturation at 98 °C for 1 min; followed by 30 cycles, each consisting of denaturation at 98 °C for 10 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 5 min. The PCR products were sequenced on a NovaSeq 6000 platform (Illumina, SD, USA). The amplification and sequencing of the 16S rRNA V4 region were performed by Beijing Novogene Bioinformatics Technology Co., Ltd.

[0076] The results are as follows Figure 14 As shown in the figure, plant extracts can alleviate colitis-related symptoms by regulating the gut microbiota. PCoA analysis revealed significant segregation of the gut microbiota composition among different groups. The control group and the DSS model group (DSS) showed significant differentiation on the PC1 and PC2 axes, suggesting that DSS induction led to significant changes in the gut microbiota structure. In contrast, the polysaccharide intervention groups (FPL, FPM, FPH), especially the medium-dose FPM group, were closer to the control group on the coordinate axes, indicating that polysaccharide intervention could restore the gut microbiota structure to a healthy state. The Venn diagram (Figure b) shows the distribution of common and unique OTUs among the different groups. The control group had the most unique OTUs, while the number of unique OTUs in FPL and FPH increased compared to the DSS group.

[0077] At the phylum level, the gut microbiota of mice in each group mainly consisted of Firmicutes, Bacteroides, Proteobacteria, and Actinomycetes. Compared with the control group, the Firmicutes group significantly decreased while the Proteobacteria group significantly increased, leading to a decrease in the Firmicutes / Bacteroides ratio, indicating an imbalance in the gut microbiota structure under colitis. After intervention with different doses of fermented Polygonatum polysaccharide, this imbalance was restored to some extent, with an increase in Firmicutes abundance and a significant decrease in Proteobacteria abundance, indicating that fermented Polygonatum polysaccharide has a good regulatory effect, with FPL and FPM showing better regulatory effects. At the genus level, Lactobacillus, unclassified_Muribaculaceae, Bacteroides, and Ligilactobacillus were the main dominant genera. After DSS treatment, beneficial bacteria such as Lactobacillus and Ligilactobacillus significantly decreased while harmful bacteria such as Turicibacter and Parabacteroides significantly increased, indicating that the inflammatory state promoted the proliferation of opportunistic pathogens and inhibited the growth of beneficial bacteria. After intervention with fermented Polygonatum polysaccharide, the above conditions were significantly improved, especially in the low and medium dose groups, suggesting that it can improve DSS-induced colitis by restoring the balance of intestinal microecology.

[0078] To further compare the differences in gut microbiota among the groups, the LEfSe method (LDA threshold > 3.0) was used to analyze the gut microbiota, and the results are shown in the figure. Significant differences were observed in multiple taxonomic units among the different treatment groups. In the Control group, *g__Rikenella*, *g__Butyricimonas*, and *s__Lactobacillus_intestinalis* were significantly enriched. The DSS model group was enriched mainly in *f_Sutterellaceae*, *f_Peptostreptococcaceae*, *g_Romboutsia*, and *s_unclassified_Turicimonas*, suggesting that these microbiota are characteristic imbalances in inflammatory states. *f_Sutterellaceae* is an opportunistic pathogen often associated with intestinal inflammation and mucosal damage. After intervention with fermented Polygonatum polysaccharide, the microbiota structure was significantly adjusted. The dominant bacteria enriched in the FPL group were mainly *o_Clostridiales* and *f_Clostridiaceae*, which are mostly butyrate-producing bacteria, helping to repair the intestinal barrier and inhibit inflammatory responses. FPM is enriched in the f_Enterococcaceae family, some members of which can regulate gut microbiota balance and inhibit the growth of harmful bacteria by producing lactic acid and antimicrobial substances. FPH is significantly enriched in the f_Prevotellaceae family, which is believed to promote polysaccharide metabolism and short-chain fatty acid production, thus having a positive effect on colon health.

[0079] Compared with existing alcohol precipitation methods, the preparation method of this invention can improve the yield and purity of Polygonatum polysaccharides without altering the original structure of the polysaccharides. Alcohol precipitation involves a phase transition, leading to polysaccharide aggregation, fragmentation, or structural changes. This invention uses ultrafiltration for purification, a gentle, green, and efficient process that does not require organic reagents and allows for continuous ultrafiltration.

Claims

1. A method for preparing Polygonatum polysaccharides based on fermentation-ultrafiltration cascade, characterized in that, Includes the following steps: Step 1: Dry and pulverize the rhizome of Polygonatum to obtain Polygonatum powder. Mix the Polygonatum powder with a solvent to obtain a Polygonatum suspension. Step 2: Inoculate Bacillus licheniformis into the Polygonatum sibiricum suspension from Step 1 and ferment it. Step 3: After filtering the fermentation supernatant obtained in Step 2, the supernatant is purified by ultrafiltration membrane to obtain the desired Polygonatum polysaccharide.

2. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 1, characterized in that, In step 2, the mass concentration of the Polygonatum sibiricum suspension is 2–3 wt.%, and the volume ratio of Bacillus licheniformis solution to Polygonatum sibiricum suspension is 6–7:100; the concentration of the Bacillus licheniformis solution is 10. 8 CFU / mL.

3. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 1, characterized in that, The fermentation time in step 2 is 8 to 9 hours.

4. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 1, characterized in that, In step 3, the fermentation supernatant is filtered using a 0.45 μm filter membrane.

5. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 1, characterized in that, In step 3, the ultrafiltration membranes are 10 k and 1 k polyethersulfone membranes, and purification is performed using 10 k and 1 k ultrafiltration membranes, respectively.

6. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 1, characterized in that, In step 2, fermentation time, inoculum size, and substrate concentration are used as independent variables to design a single-factor experiment; the required fermentation time, inoculum size, and substrate concentration are predicted by a quadratic multinomial regression model using polysaccharide dissolution rate as the response value.

7. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 1, characterized in that, In step 3, after ultrafiltration, an elution process is also performed.

8. The method for preparing Polygonatum polysaccharide based on fermentation-ultrafiltration cascade according to claim 2, characterized in that, The culture process of Bacillus licheniformis is as follows: Bacillus licheniformis is inoculated into NB medium and cultured at 37 ℃ for 24 h. The bacterial cells are then centrifuged to obtain a precipitate.

9. Polygonatum polysaccharide obtained by any one of the preparation methods described in claims 1 to 8.

10. The application of Polygonatum polysaccharide as described in claim 9, characterized in that, The Polygonatum polysaccharide is used in the preparation of medicines, foods, or health products that regulate the structure of intestinal flora.

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