A low molecular weight rhizoma polygonati fructan of probiotic fermentation origin, a preparation method and purposes thereof

Abstract

The application discloses a low-molecular-weight Polygonatum sibiricum fruit polysaccharide fermented by probiotics, a preparation method and application thereof. The low-molecular-weight Polygonatum sibiricum fruit polysaccharide is obtained by directional anaerobic fermentation of Polygonatum sibiricum powder by Bifidobacterium adolescentis, combined with fractional alcohol precipitation and column chromatography purification. The weight average molecular weight of the fruit polysaccharide is 4.850*10 3 Da, the number average molecular weight is 3.570*10 3 Da, and the fructose content is 94.32%. Animal experiments show that the Polygonatum sibiricum fruit polysaccharide can significantly improve the species richness and diversity of intestinal flora, regulate the flora community structure, promote the proliferation of beneficial bacteria such as Bifidobacterium and Muribaculum, and has a significant prebiotic function. The application provides a new technical scheme for deep processing and utilization of Polygonatum sibiricum resources and regulation of intestinal flora.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, specifically to a low molecular weight Polygonatum fructan prepared by probiotic fermentation and its application in regulating intestinal flora and improving intestinal microecological balance. Background Technology

[0002] Polygonatum sibiricum is a traditional Chinese medicinal and edible plant resource, rich in polysaccharides, saponins, flavonoids, and other active ingredients. Among them, Polygonatum polysaccharides, as one of the main active substances, have been extensively studied in recent years. Existing research shows that Polygonatum polysaccharides possess various biological activities, including antioxidant, immunomodulatory, metabolic regulation, and improvement of gut microbiota. With the development of research on "gut microbiota-host health," naturally derived polysaccharides, as important dietary carbon sources, can influence the composition of the gut microbiota and the production of metabolites through selective utilization by gut microbes. Therefore, utilizing plant-derived polysaccharides to regulate gut microbiota has become an important research direction in the fields of functional foods, biomedicine, and nutritional intervention.

[0003] Currently, the main methods for preparing Polygonatum polysaccharides include water extraction and alcohol precipitation, ultrasound-assisted extraction, and enzyme-assisted extraction. Polysaccharides obtained by traditional extraction methods usually have high molecular weight and complex structures, which limits their solubility, diffusivity, and utilization efficiency by intestinal flora.

[0004] In recent years, microbial fermentation technology has been explored for structural modification and functional enhancement of plant polysaccharides due to its advantages such as mild reaction conditions, high selectivity, and environmental friendliness. However, existing technologies lack solutions for how to utilize probiotics with specific polysaccharide utilization capabilities to perform targeted fermentation of Polygonatum sibiricum raw materials, obtain low molecular weight Polygonatum sibiricum polysaccharides with specific molecular weight ranges and well-defined structural characteristics, and systematically evaluate their specific regulatory effects on the gut microbiota.

[0005] Therefore, developing a low molecular weight Polygonatum polysaccharide with specific structural characteristics and intestinal flora regulation function prepared by probiotic fermentation is of great significance for improving the utilization value of Polygonatum resources and developing new functional foods. Summary of the Invention

[0006] In view of this, the purpose of this application is to provide a low molecular weight Polygonatum fructan derived from probiotic fermentation, its preparation method, and its uses. This application obtained a strain of Bifidobacterium adolescentis with high efficiency in utilizing Polygonatum fructans through screening, and carried out directed anaerobic fermentation using Polygonatum powder as the sole carbon source. Combined with fractional alcohol precipitation and column chromatography purification processes, a weight-average molecular weight of 4.850 × 10⁻⁶ was obtained. 3 Da, with a number-average molecular weight of 3.570 × 10⁻⁶. 3This application presents a low molecular weight Polygonatum fructan with a fructose content as high as 94.32%. This low molecular weight Polygonatum fructan can significantly improve the species richness and diversity of intestinal flora, regulate the community structure, and promote the proliferation of specific beneficial bacteria, thereby effectively improving intestinal microecological imbalance. This application achieves targeted regulation of Polygonatum saccharide structure, providing a new technical solution for the development of functional prebiotic products.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, this application provides a method for preparing fermentation-derived low molecular weight Polygonatum fructans, comprising the following steps:

[0009] The probiotic Bifidobacterium adolescentis was inoculated into a fermentation medium with Polygonatum sibiricum powder as the sole carbon source for anaerobic fermentation to obtain Polygonatum sibiricum fermentation broth.

[0010] The fermentation broth of Polygonatum was subjected to solid-liquid separation, and the supernatant was collected;

[0011] The supernatant was subjected to fractional alcohol precipitation to collect the polysaccharide precipitate, which was then dried to obtain crude Polygonatum polysaccharide.

[0012] The crude polysaccharide of Polygonatum was purified by ion exchange chromatography and gel filtration chromatography in sequence. The fraction corresponding to the target polysaccharide peak was collected and dried to obtain low molecular weight Polygonatum fructan.

[0013] In some embodiments, the anaerobic fermentation conditions are: inoculum size 5% (v / v), culture temperature 37 °C, initial pH 7.0, strictly anaerobic culture, and fermentation time 12 h.

[0014] In some embodiments, the fractional alcohol precipitation includes: concentrating the fermentation supernatant, adding anhydrous ethanol to a final concentration of 40% (v / v), allowing it to stand at 4 °C, centrifuging to remove the precipitate, adding anhydrous ethanol to the supernatant to a final concentration of 80% (v / v), allowing it to stand at 4 °C, centrifuging to collect the polysaccharide precipitate.

[0015] In some embodiments, the ion exchange chromatography is DEAE-52 ion exchange column chromatography, specifically including: dissolving the crude polysaccharide of Polygonatum odoratum and loading it onto a DEAE-52 ion exchange column, and sequentially eluting with deionized water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L and 0.5 mol / L NaCl solutions, detecting the polysaccharide content using the phenol-sulfuric acid method, and collecting the fraction corresponding to the main peak according to the elution curve.

[0016] In some embodiments, the gradient elution flow rate is 10 mL / min.

[0017] In some embodiments, the gel filtration chromatography is Sephadex G-75 gel column chromatography, specifically including: dissolving the polysaccharide component purified by ion exchange chromatography and loading it onto a Sephadex G-75 gel column, performing isocratic elution with deionized water as the mobile phase at a flow rate of 1 mL / min, detecting the polysaccharide content using the phenol-sulfuric acid method, and collecting the fraction corresponding to the main peak according to the elution curve.

[0018] Secondly, this application provides a low molecular weight Polygonatum fructan, which is prepared by the preparation method described in the first aspect.

[0019] In some embodiments, the low molecular weight fructan has a weight-average molecular weight of 4.850 × 10⁻⁶. 3 Da, with a number-average molecular weight of 3.570 × 10⁻⁶. 3 Da has a polydispersity index of 1.358; and its monosaccharide composition contains 94.32% fructose, 2.88% glucose, and 2.80% mannose.

[0020] Thirdly, this application provides the application of the low molecular weight Polygonatum fructans described in the second aspect in the preparation of drugs or functional foods that regulate intestinal flora.

[0021] In some embodiments, the regulation of gut microbiota includes at least one of the following effects:

[0022] (a) Enhance the species richness and community diversity of the gut microbiota, including increasing the Chao1 index, ObservedOTUs index and Shannon index;

[0023] (b) Regulate the gut microbiota community structure to shift the microbiota structure in the disease state toward the normal direction;

[0024] (c) Increase the relative abundance of Actinobacteria and Bacteroidetes at the phylum level, and decrease the relative abundance of Firmicutes;

[0025] (d) Increase the relative abundance of Bifidobacteriaceae and Muribacaceae at the family level;

[0026] (e) Increase the relative abundance of Bifidobacterium and Muribacula at the genus level, and decrease the relative abundance of Broutella.

[0027] Fourthly, this application provides a functional product for regulating gut microbiota, comprising the low molecular weight Polygonatum fructans described in the second aspect and a food- or pharmaceutically acceptable carrier or excipient.

[0028] Compared with the prior art, this application has at least the following beneficial effects:

[0029] This application utilizes the probiotic *Bifidobacterium adolescentis* to conduct directed anaerobic fermentation of *Polygonatum sibiricum* powder. The reaction conditions are mild, requiring no strong acid or alkali treatment, and the fermentation cycle is short. Combined with fractional alcohol precipitation and column chromatography purification processes, a weight-average molecular weight of 4.850 × 10⁻⁶ was obtained. 3 Da, with a number-average molecular weight of 3.570 × 10⁻⁶. 3 This low-molecular-weight Polygonatum fructan, with a polydispersity index of 1.358 and a fructose content as high as 94.32%, overcomes the technical shortcomings of traditional Polygonatum polysaccharides, which are characterized by high molecular weight, wide distribution, and unclear structure. Animal experiments have shown that this low-molecular-weight Polygonatum fructan can significantly improve the species richness and diversity of the intestinal flora in mice with kidney injury, shifting the flora structure in the disease state towards the normal direction. It selectively regulates the flora composition at the phylum, family, and genus levels, significantly increasing the relative abundance of beneficial bacteria such as Actinobacteria, Bacteroidetes, Bifidobacteriaceae, Bifidobacterium, and Bacillus murikinae, exhibiting significant prebiotic function. This provides a new technical solution for the deep processing and utilization of Polygonatum resources and the development of functional products for regulating intestinal flora. Attached Figure Description

[0030] Figure 1 This application provides examples of the growth and sugar conversion of different strains fermenting Polygonatum at different time points; wherein... Figure 1 A represents the growth curves (OD) of each strain. 600 (1B shows the change of pH value of each strain's culture system with culture time, 1C shows the change of total sugar content of each strain's culture system with culture time, and 1D shows the change of reducing sugar content of each strain's culture system with culture time.)

[0031] Figure 2 The column chromatography elution curves for crude Polygonatum polysaccharide provided in the embodiments of this application are shown; wherein 2A is the elution curve of DEAE-52 cellulose ion exchange column chromatography, and 2B is the elution curve of Sephadex G-75 gel filtration column chromatography.

[0032] Figure 3 The results of homogeneity and molecular weight determination of Polygonatum polysaccharides provided in the embodiments of this application are shown.

[0033] Figure 4 The results of monosaccharide composition analysis of low molecular weight Polygonatum fructans provided in the embodiments of this application; wherein 4A is the standard curve for detecting fructose content in PSP by the Seliwanoff method. Figure 4B shows the HPLC chromatograms of PMP derivatives from the mixed monosaccharide standards and PSP samples. The monosaccharide standards, in peak order, are: Man (mannose), Rha (rhamnose), GlcA (glucuronic acid), GalA (galacturonic acid), Glc (glucose), Gal (galactose), Ara (arabinose), and Fuc (fucose). Combining the two assay methods, the main monosaccharide components in PSP were determined to be fructose, glucose, and mannose.

[0034] Figure 5 This document presents the results of the regulatory effect of low molecular weight Polygonatum fructans (PSP) provided in the embodiments of this application on the gut microbiota of a chronic kidney disease model mouse; where 5A is the Chao1 index, used to assess the species richness of the gut microbiota in each group; 5B is the Good's coverage index, used to assess the coverage of the sample microbial community by the sequencing depth in each group; and 5C is the Observed... The OTUs index is used to assess the number of OTUs actually detected in each group of gut microbiota; 5D is the Shannon index, used to comprehensively assess the species diversity and evenness of each group of gut microbiota; 5E is principal component analysis (PCA), used to show the spatial distribution differences of the gut microbiota community structure in each group of mice; 5F is principal coordinate analysis based on Jaccard distance (PCoA), used to show the similarity or difference of the gut microbiota community structure in each group of mice; 5G is a bar chart of the relative abundance of gut microbiota at the phylum level, used to show the distribution of the top-abundant phyla in each group; 5H is a bar chart of the relative abundance of gut microbiota at the family level, used to show the distribution of the top-abundant families in each group; 5I is a heatmap of gut microbiota changes at the genus level, showing the key genera with significant differences between groups and their relative abundance changes. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0036] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0037] In this application, unless the context clearly indicates otherwise, the terms “including,” “comprising,” “containing,” “having,” etc., shall be understood as open-ended and mean “including but not limited to.”

[0038] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may be adjusted according to the desired performance. At a minimum, each numerical parameter should be interpreted based on the reported significant figures and by applying common rounding techniques.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains.

[0040] Example 1: Screening of fermentation strains with the ability to utilize Polygonatum sibiricum

[0041] This embodiment is used to screen for superior strains suitable for fermentation preparation of low molecular weight Polygonatum fructans.

[0042] 1.1 Preparation of Polygonatum odoratum powder

[0043] The dried Polygonatum sibiricum material (Liaoning, Hubei Zexi Traditional Chinese Medicine Technology Co., Ltd.) was pulverized and passed through a 60-mesh sieve to obtain Polygonatum sibiricum powder for later use.

[0044] 1.2 Preparation of screening culture medium

[0045] A screening medium was constructed using Polygonatum sibiricum powder as the sole exogenous carbon source. The screening medium (1 L) consisted of: Polygonatum sibiricum powder (2.5 g), peptone (10.0 g), liver extract powder (5.0 g), beef extract powder (3.0 g), yeast extract powder (5.0 g), tryptone (8.0 g), sodium chloride (1.0 g), dipotassium hydrogen phosphate (1.0 g), potassium dihydrogen phosphate (1.0 g), FeSO4·7H2O (0.01 g), MnSO4 (0.005 g), and L-cysteine ​​(0.5 g), with a pH of 7.2 ± 0.1. No additional exogenous carbon sources such as glucose or lactose were added to ensure that the strain's growth primarily relied on carbohydrates from Polygonatum sibiricum. The above components were accurately weighed, dissolved in 1 L of distilled water, and autoclaved at 116 ℃ for 30 min, then cooled for later use.

[0046] 1.3 Candidate Strains

[0047] Different strains were selected as screening strains, including *Bifidobacterium longum* (CGMCC 1.15639, China General Microbiological Culture Collection Center), *Bifidobacterium bifidum* (CGMCC 1.5091, China General Microbiological Culture Collection Center), *Bifidobacterium adolescentis* (CGMCC 1.2190, China General Microbiological Culture Collection Center), and *Bifidobacterium animalis* (CGMCC 1.15623, China General Microbiological Culture Collection Center).

[0048] 1.4 Screening, Cultivation, and Sample Collection

[0049] The activated bacterial cultures of each candidate strain were inoculated into the above-mentioned Polygonatum odoratum single carbon source screening medium at the same inoculation ratio (5%, v / v) and cultured under anaerobic conditions at 37 ℃. Fermentation broth samples were collected at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 36 h, 48 h and 72 h for subsequent detection of various indicators.

[0050] 1.5 Indicator Testing

[0051] OD of culture medium was detected using a multi-functional microplate reader. 600 The pH values ​​were used to evaluate the growth ability of the strain in the Polygonatum culture system; pH changes in the culture medium were detected using a pH meter; changes in total sugar content were determined using the phenol-sulfuric acid method; and changes in reducing sugar content were determined using the DNS method.

[0052] 1.6 Screening Results

[0053] The results are as follows Figure 1 As shown. By Figure 1 As can be seen from A, different strains exhibit varying growth abilities in the Polygonatum culture system, with Bifidobacterium adolescentis showing better growth ability and OD... 600 The value was significantly higher than that of other strains during the culture period. For example... Figure 1 As shown in Figure B, the pH of the culture system for each strain gradually decreased with prolonged culture time, indicating that the strains could utilize the sugars derived from Polygonatum sibiricum to produce acid through metabolism. Figure 1 C and Figure 1 As shown in D, different strains exhibit varying abilities to utilize carbohydrates derived from Polygonatum. The total sugar content in the Bifidobacterium adolescentis culture system decreased most significantly, while the reducing sugar content showed a typical polysaccharide utilization characteristic of first increasing and then decreasing, indicating that this strain has a strong ability to utilize and convert carbohydrates derived from Polygonatum.

[0054] Overall bacterial growth capacity (OD) 600 Based on the characteristics of pH changes, total sugar consumption, and reducing sugar conversion, *Bifidobacterium adolescentis* was screened as the dominant strain for the fermentation preparation of low molecular weight fructan in this application. The preservation number of this *Bifidobacterium adolescentis* strain is CGMCC 1.2190.

[0055] Example 2: Preparation of Polygonatum sibiricum fermentation broth by fermentation of Bifidobacterium adolescentis

[0056] This embodiment is used to obtain the fermentation broth of Polygonatum sibiricum after fermentation with Bifidobacterium adolescentis, which is used as a raw material for subsequent extraction and purification.

[0057] 2.1 Preparation of fermentation culture medium

[0058] The dried Polygonatum sibiricum raw material was pulverized and passed through a 60-mesh sieve to obtain Polygonatum sibiricum powder. A fermentation medium was prepared using the Polygonatum sibiricum powder as the sole carbon source. Except for adjusting the amount of Polygonatum sibiricum powder added from 2.5 g / L to 20 g / L, the other components and contents were the same as in Example 1.2. The culture medium was sterilized by autoclaving at 116 ℃ for 30 min before use.

[0059] 2.2 Strain activation and inoculation

[0060] The Bifidobacterium adolescentis obtained in Example 1 was activated and cultured (activation conditions were the same as in Example 1.3). The activated Bifidobacterium adolescentis culture was inoculated into the above fermentation medium at an inoculation rate of 5% (v / v).

[0061] 2.3 Anaerobic fermentation culture

[0062] The inoculated culture system was subjected to anaerobic fermentation under the following conditions:

[0063] The culture temperature was 37 ℃; the initial pH was adjusted to approximately 7.0; the culture conditions were strictly anaerobic; the fermentation time was 12 h; and the stirring speed was 150 rpm.

[0064] 2.4 Monitoring of the fermentation process

[0065] During fermentation, the OD of the culture system was periodically monitored according to the method in Example 1. 600 Changes in pH value, total sugar content, and reducing sugar content.

[0066] 2.5 Determination of fermentation endpoint

[0067] The trends of various indicators during fermentation were consistent with the culture characteristics of *Bifidobacterium adolescentis* in Example 1. Specifically: within 0-12 h after inoculation, OD... 600The rapid increase in pH indicates that *Bifidobacterium adolescentis* has adapted to the nutrient environment derived from *Polygonatum sibiricum* and entered an active growth phase. Simultaneously, the total sugar content continues to decrease, indicating that the strain can effectively utilize the sugars from *Polygonatum sibiricum* and promote the transformation of sugar components. With further extension of fermentation time (after 12 hours), although the total sugar content continues to decrease, cell growth gradually stabilizes, and the pH continues to decrease (acidity continues to increase), indicating that microbial metabolic activity is enhanced during long-term culture and may further consume or transform the already formed low-molecular-weight sugar components.

[0068] The purpose of this application is to utilize the metabolic activity of *Bifidobacterium adolescentis* to directionally convert carbohydrates derived from *Polygonatum sibiricum* into low-molecular-weight *Polygonatum sibiricum* fructans with specific molecular weight characteristics, rather than to minimize the residual sugar content in the culture system. Therefore, considering the bacterial growth status, carbohydrate utilization efficiency, and structural stability of the target product, 12 h was selected as the preferred fermentation time for this application.

[0069] After fermentation, the Polygonatum sibiricum fermentation broth was obtained and used for subsequent polysaccharide extraction and purification.

[0070] Example 3: Extraction and purification of low molecular weight Polygonatum fructans

[0071] This embodiment is used to extract and purify low molecular weight Polygonatum fructan from the Polygonatum fermentation broth obtained in Example 2.

[0072] 3.1 Solid-liquid separation

[0073] The Polygonatum fermentation broth obtained in Example 2 was centrifuged at 8000 rpm for 10 min to remove the bacterial cells and unused Polygonatum solid residue, and the supernatant was collected.

[0074] 3.2 Concentration of supernatant

[0075] The resulting supernatant was concentrated under reduced pressure (rotary evaporation) to 1 / 5 to 1 / 10 of its original volume (the specific concentration factor depends on the initial volume, in order to facilitate subsequent alcohol precipitation).

[0076] 3.3 Fractional alcohol precipitation

[0077] (1) 40% ethanol precipitation: Transfer the above concentrate to a suitable container, and add anhydrous ethanol dropwise under slow stirring until the final ethanol concentration of the system reaches 40% (v / v). Place the mixture at 4 ℃ and let it stand for 12 h. Then centrifuge at 8000 rpm for 5 min to remove the polysaccharide precipitate with larger molecular weight and collect the supernatant.

[0078] (2) Precipitation with 80% ethanol: Anhydrous ethanol was added dropwise to the supernatant under slow stirring until the final ethanol concentration reached 80% (v / v). The mixture was allowed to stand at 4 °C for 12 h. Then, it was centrifuged at 8000 rpm for 5 min to collect the crude polysaccharide precipitate.

[0079] 3.4 Obtaining crude polysaccharides

[0080] The precipitate obtained from the above 80% ethanol precipitation was redissolved with an appropriate amount of deionized water, and the residual ethanol was removed by rotary evaporation to obtain a crude polysaccharide solution. The crude polysaccharide solution was then freeze-dried (pre-freezing temperature -80 ℃, vacuum degree 20.0 Pa, freeze-drying time 12 h) to obtain crude Polygonatum polysaccharide.

[0081] 3.5 DEAE-52 ion exchange column chromatography purification

[0082] (1) Sample preparation: The crude polysaccharide of Polygonatum obtained in 3.4 was dissolved in an appropriate amount of deionized water, centrifuged (10000 rpm, 10 min) and filtered (0.22 μm) for later use.

[0083] (2) Column equilibration: The DEAE-52 cellulose ion exchange column (200 mL) was fully equilibrated with deionized water beforehand.

[0084] (3) Sample loading and elution: The above polysaccharide solution was loaded onto a DEAE-52 ion exchange column. After adsorption, the sample was eluted sequentially with deionized water and NaCl solutions of different concentrations. The elution program is shown in Table 1 below:

[0085] Table 1 Elution Procedure

[0086]

[0087] The elution flow rate was controlled at 10 mL / min. 10 mL of eluent was collected from each tube during the elution process.

[0088] (4) Elution curve plotting and target fraction collection: The polysaccharide content of each eluent tube was determined using the phenol-sulfuric acid method (specific method as in Example 1.7). Elution curves were plotted with the tube number as the x-axis and the absorbance value detected by the phenol-sulfuric acid method as the y-axis. The results are as follows: Figure 2 As shown in Figure A. Based on the elution curve, the fraction corresponding to the main peak with a clear polysaccharide response signal (i.e., [the fraction]) was collected. Figure 2 (10-20 tubes in section A). Combine the target fractions, concentrate under reduced pressure, and freeze-dry to obtain the DEAE-52 purified polysaccharide fraction.

[0089] 3.6 Sephadex G-75 gel filtration chromatography purification

[0090] (1) Sample preparation: Dissolve the DEAE-52 purified polysaccharide component obtained in 3.5 in an appropriate amount of deionized water, and then filter it by centrifugation for later use.

[0091] (2) Column equilibration: Sephadex G-75 gel columns were pre-equilibrated with deionized water.

[0092] (3) Loading and elution: The above polysaccharide solution was loaded onto a Sephadex G-75 gel column and eluted isocratically with deionized water as the mobile phase at a flow rate of 1 mL / min. 3 mL of eluent was collected from each tube during the elution process.

[0093] (4) Elution curve plotting and target fraction collection: The polysaccharide content of each eluent tube was determined using the phenol-sulfuric acid method (specific method as in Example 1.7). Elution curves were plotted with the tube number as the x-axis and the absorbance value detected by the phenol-sulfuric acid method as the y-axis. The results are as follows: Figure 2 As shown in B. Based on the elution curve, collect the fraction corresponding to the main peak (i.e., ...). Figure 2 Tubes B (40-70, specific tube number range subject to actual test results) were combined, concentrated under reduced pressure, and freeze-dried to obtain fermentation-derived low molecular weight Polygonatum fructan (hereinafter referred to as "PSP").

[0094] Example 4: Structural Characterization of Low Molecular Weight Polygonatum Fructus

[0095] This embodiment characterizes the structure of the low molecular weight Polygonatum fructan obtained in Example 3.

[0096] Size exclusion chromatography-multi-angle laser light scattering (SEC-MALS) was used to analyze the obtained Polygonatum fructans. The sample was dissolved in ultrapure water, filtered through a membrane, and then analyzed. The molecular weight distribution of the sample was comprehensively analyzed using a differential refractive index detector (dRI), a multi-angle laser light scattering detector (LS), and a viscosity detection signal (DP). The results are as follows. Figure 3 As shown, the dRI, LS, and DP signals all exhibit symmetrical peaks, and the retention times of the three detection signals are consistent, indicating that the obtained low molecular weight Polygonatum fructans have good purity and molecular weight uniformity. The calculated weight-average molecular weight (Mw) is 4.850 × 10⁻⁶. 3 Da; the number-average molecular weight (Mn) is 3.570 × 10⁻⁶. 3 Da; the polydispersity index is 1.358.

[0097] The monosaccharide composition of the obtained low molecular weight Polygonatum fructans was analyzed using a combination of PMP pre-column derivatization and Seliwanoff assay. The specific method is as follows: 5 mg of sample was dissolved in 3 mL of trifluoroacetic acid (2 mol / L TFA) and hydrolyzed at 120 °C for 2 h. Methanol was repeatedly added and residual TFA was removed under reduced pressure. Subsequently, NaOH solution (0.3 mol / L) and PMP-methanol solution (0.5 mol / L) were added for derivatization, and the reaction was carried out in a water bath at 70 °C for 30 min. After the reaction, the mixture was neutralized with hydrochloric acid, and excess PMP reagent was removed by chloroform extraction. The aqueous phase was collected for high-performance liquid chromatography (HPLC) analysis. HPLC analysis was performed using a C18 column (4.6 mm × 250 mm, 5 μm) with gradient elution of acetonitrile and 0.02 mol / L ammonium acetate solution as the mobile phase. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 254 nm. Simultaneously, the fructose content in the sample was determined using the Seliwanoff assay. The sample solution (250 μg / mL) was mixed with Seliwanoff reagent and reacted in a water bath at 100 °C for 10 min. The absorbance was measured at 484 nm, and a standard curve was established using fructose standards to calculate the fructose content.

[0098] The results are as follows Figure 4 As shown. Figure 4 A is the standard curve for detecting fructose content in PSP using the Seliwanoff method. Substituting the sample absorbance into this standard curve, the fructose concentration in the PSP sample was found to be 235.8 μg / mL, which, after conversion, yielded a fructose content of 94.32% in the PSP. Figure 4 B shows the HPLC chromatograms of the mixed monosaccharide standard and the PMP derivative of the PSP sample. Due to the poor detection of fructose after PMP derivatization, the chromatogram mainly showed two peaks: glucose and mannose. Combining the two methods, the main monosaccharide components in PSP were determined to be fructose, glucose, and mannose; the relative content of fructose was 94.32%, glucose was 2.88%, and mannose was 2.80%, indicating that this product is a fructan derived from Polygonatum sibiricum with fructose residues as the main component.

[0099] Example 5: The regulatory effect of low molecular weight Polygonatum fructan on the intestinal flora of a mouse model of chronic kidney disease.

[0100] This example is used to evaluate the regulatory effect of low molecular weight Polygonatum fructans (PSP) obtained in Example 3 on the intestinal flora of a chronic kidney disease model mouse.

[0101] Male C57BL / 6 mice aged 6-8 weeks were randomly divided into a normal control group (Ctrl), an adenine model group (Adenine), and a low molecular weight Polygonatum fructan intervention group (AdePSP), with 12 mice in each group. Except for the normal control group, the mice in the other groups were fed a diet containing 0.2% adenine to establish a kidney disease model. During the modeling period, the AdePSP group was given low molecular weight Polygonatum fructan prepared in Example 3 by gavage daily at a dose of 200 mg / kg / day, while the Ctrl and Adenine groups were given an equal volume of distilled water. The intervention was continued for 3 weeks.

[0102] After the intervention, fecal samples were collected from mice in each group, and total microbial DNA was extracted. High-throughput sequencing of the 16S rRNA gene was performed. After quality control, the sequencing data underwent sequence processing, species classification, and community diversity analysis. The gut microbiota composition of different treatment groups was also compared.

[0103] Experimental results are as follows Figure 5 As shown. Alpha diversity analysis results showed that compared with the Adenine group, the Chao1 index and Observed OTUs in the AdePSP group were significantly increased (P<0.05), and the Shannon index was also significantly increased (P<0.05), indicating that low molecular weight Polygonatum fructan can improve the species richness and community diversity of the intestinal flora in adenine-induced nephropathy model mice; the Good's coverage of each group was close to 1, indicating that the sequencing depth could cover the main microbial communities in the samples well ( Figure 5 A-5D).

[0104] PCA and PCoA based on Jaccard distance showed that the gut microbiota community structure of the Adenine group and the Ctrl group was significantly separated, indicating that adenine treatment caused changes in the gut microbiota structure; the AdePSP group was significantly separated from the Adenine group and shifted towards the Ctrl group, indicating that low molecular weight Polygonatum fructans can regulate the adenine-induced abnormal gut microbiota structure. Figure 5 E, 5F).

[0105] Regarding the microbial community composition, compared with the Adenine group, the AdePSP group showed the following changes at the phylum level: the relative abundance of Actinobacteriota increased from 0.75% to 1.75% (P<0.05), Bacteroidetes increased from 11.00% to 26.07% (P<0.05), Firmicutes decreased from 53.44% to 41.02% (P<0.05), and the Firmicutes / Bacteroidota ratio decreased from 4.86 to 1.57. Figure 5 G).

[0106] At the family level, after AdePSP intervention, the relative abundance of Bifidobacteriaceae significantly increased from 0.27% to 1.04% (P<0.05), Muribaculaceae significantly increased from 7.09% to 18.70% (P<0.05), and Lachnospiraceae significantly decreased from 35.44% to 20.14% (P<0.05). Figure 5 H).

[0107] At the genus level, after AdePSP intervention, the relative abundance of *Bifidobacterium* significantly increased from 0.39% to 1.42% (P<0.05), and *Muribaculaceae* significantly increased from 7.31% to 18.35% (P<0.05). Simultaneously, *Parabacteroides*, *Akkermansia*, and *Anaerostipes* showed an upward trend, while *Blautia* decreased from 24.48% to 4.80% (P<0.05), and the relative abundance of *Desulfovibrio* decreased. Figure 5 I).

[0108] In summary, the low molecular weight Polygonatum fructan prepared in this application can improve the species richness and community diversity of the intestinal flora in adenine-induced nephropathy model mice, and can change the community structure of the intestinal flora, regulating the relative abundance of various bacterial groups at the phylum, family and genus levels. Among them, it can significantly increase the relative abundance of Bifidobacterium, Bifidobacterium and Muribacillus genus-related bacterial groups, indicating that the low molecular weight Polygonatum fructan has the effect of regulating the structure and composition of the intestinal flora under renal injury.

[0109] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for preparing low molecular weight Polygonatum fructans from fermentation, characterized in that, Includes the following steps: The probiotic Bifidobacterium adolescentis was inoculated into a fermentation medium with Polygonatum sibiricum powder as the sole carbon source for anaerobic fermentation to obtain Polygonatum sibiricum fermentation broth. The fermentation broth of Polygonatum was subjected to solid-liquid separation, and the supernatant was collected; The supernatant was subjected to fractional alcohol precipitation to collect the polysaccharide precipitate, which was then dried to obtain crude Polygonatum polysaccharide. The crude polysaccharide of Polygonatum was purified by ion exchange chromatography and gel filtration chromatography in sequence. The fraction corresponding to the target polysaccharide peak was collected and dried to obtain low molecular weight Polygonatum fructan.

2. The preparation method according to claim 1, characterized in that, The anaerobic fermentation conditions were as follows: inoculum size 5% (v / v), culture temperature 37 ℃, initial pH 7.0, strictly anaerobic culture, and fermentation time 12 h.

3. The preparation method according to claim 1, characterized in that, The fractional alcohol precipitation process includes: concentrating the fermentation supernatant, adding anhydrous ethanol to a final concentration of 40% (v / v), allowing it to stand at 4 ℃, centrifuging to remove the precipitate, adding anhydrous ethanol to the supernatant to a final concentration of 80% (v / v), allowing it to stand at 4 ℃, centrifuging to collect the polysaccharide precipitate.

4. The preparation method according to claim 1, characterized in that, The ion exchange chromatography was performed using a DEAE-52 ion exchange column, specifically including: dissolving crude polysaccharide from Polygonatum odoratum and loading it onto a DEAE-52 ion exchange column; sequentially eluting with deionized water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl solutions; detecting the polysaccharide content using the phenol-sulfuric acid method; and collecting the fraction corresponding to the main peak based on the elution curve; the flow rate of the gradient elution was 10 mL / min.

5. The preparation method according to claim 1, characterized in that, The gel filtration chromatography is Sephadex G-75 gel column chromatography, which specifically includes: dissolving the polysaccharide component purified by ion exchange chromatography and loading it onto a Sephadex G-75 gel column, using deionized water as the mobile phase for isocratic elution at a flow rate of 1 mL / min, detecting the polysaccharide content using the phenol-sulfuric acid method, and collecting the fraction corresponding to the main peak according to the elution curve.

6. A low molecular weight Polygonatum fructan, characterized in that, It is prepared by any one of the preparation methods described in claims 1-5.

7. The low molecular weight Polygonatum fructan according to claim 6, characterized in that, The weight-average molecular weight of the low molecular weight Polygonatum fructan is 4.850 × 10⁻⁶. 3 Da, with a number-average molecular weight of 3.570 × 10⁻⁶. 3 Da has a polydispersity index of 1.358; and its monosaccharide composition contains 94.32% fructose, 2.88% glucose, and 2.80% mannose.

8. The use of the low molecular weight Polygonatum fructans according to claim 6 or 7 in the preparation of drugs or functional foods for regulating intestinal flora.

9. The application according to claim 8, characterized in that, The regulation of gut microbiota includes at least one of the following functions: (a) Enhance the species richness and community diversity of the gut microbiota, including increasing the Chao1 index, the Observed OTUs index, and the Shannon index; (b) Regulate the gut microbiota community structure to shift the microbiota structure in the disease state toward the normal direction; (c) Increase the relative abundance of Actinobacteria and Bacteroidetes at the phylum level, and decrease the relative abundance of Firmicutes; (d) Increase the relative abundance of Bifidobacteriaceae and Muribacaceae at the family level; (e) Increase the relative abundance of Bifidobacterium and Muribacula at the genus level, and decrease the relative abundance of Broutella.

10. A functional product for regulating intestinal flora, characterized in that, It comprises the low molecular weight Polygonatum fructans as described in claim 6 or 7 and a food- or pharmaceutically acceptable carrier or excipient.

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