A method for quality control of traditional Chinese medicine polysaccharides based on the utilization spectrum of Bacteroides polysaccharides

Abstract

This invention belongs to the field of polysaccharide quality control technology, specifically relating to a method for quality control of traditional Chinese medicine polysaccharides based on the utilization spectrum of Bacteroides polysaccharides. The method described in this invention achieves polysaccharide quality control by measuring the utilization of polysaccharides by Bacteroides. This method can not only distinguish polysaccharides from different sources and preparation methods, but also provide potential pharmacodynamic and mechanistic information. Furthermore, different polysaccharides can induce differential growth in Bacteroides species, revealing that prebiotic polysaccharides can improve the growth of Bacteroides species, exhibiting potential commercial value.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide quality control technology, specifically relating to a method for quality control of traditional Chinese medicine polysaccharides based on the utilization spectrum of Bacteroides polysaccharides. Background Technology

[0002] Traditional Chinese medicine (TCM) polysaccharides and their derivatives possess a wide range of effects, including enhancing immune function, anti-tumor activity, anti-oxidation, anti-inflammation, antiviral activity, neuroprotection, hypoglycemia, lipid regulation, and gut microbiota regulation. Numerous studies have shown that TCM polysaccharides exert their effects by regulating the composition and metabolism of the gut microbiota. For example, oyster polysaccharides can regulate the gut microbiota, increase the concentrations of propionic acid and butyric acid, affect TLR signaling pathways, increase the tumor inhibition rate of 5-fluorouracil, and improve the mental state of mice. Aloe polysaccharides alter the composition of the gut microbiota, increasing the production of SCFAs and reducing the abundance of harmful bacteria. Astragalus polysaccharides can promote the growth of acetic acid-producing desulfurovibrio and alleviate non-alcoholic fatty liver disease (NAFLD) in mice. Ficus pumila fruit polysaccharides can improve gut microbiota dysbiosis in mice fed a high-fat diet (HFD) and improve obesity. Mushroom polysaccharides from Ganoderma lucidum and Poria cocos can significantly enrich gut commensal bacteria associated with anti-obesity, SCFA production, polysaccharide degradation, and lactic acid production.

[0003] However, due to the complexity and chemical diversity of polysaccharides, including those from traditional Chinese medicine (TCM), quality control remains a bottleneck in the development of novel polysaccharide drugs. Currently, some chemical composition-based quality control methods exist for TCM polysaccharides, such as colorimetric determination of total sugar content, high-performance liquid chromatography (HPLC) determination of molecular weight and distribution, and HPLC determination of monosaccharide composition. However, these physical and chemical methods still have limitations in polysaccharide quality control, failing to reflect the relationship between the high-dimensional structure and activity of polysaccharides. In fact, the biological activity of polysaccharides is closely related to their molecular structure.

[0004] The polysaccharide utilization profiles of Bacteroidetes in traditional Chinese medicine can reflect the differences in the physicochemical properties of polysaccharides. Most monosaccharides in food are absorbed in the small intestine, while polysaccharides, which are not directly absorbed by the host, enter the intestine. To adapt to this polysaccharide environment, intestinal symbiotic bacteria, especially Bacteroides, have evolved a large number of polysaccharide utilization loci (PULs). It is estimated that more than one-quarter of the Bacteroidetes genome encodes PULs. The Bacteroides polymorpha VPI-5482 strain possesses 88 PULs, used for the degradation of various polysaccharides. Bacteroidetes species have a high prevalence and relative abundance in the human gut. Furthermore, human gut Bacteroides are also targets for many traditional Chinese medicine polysaccharides. For example, ginseng polysaccharides are enriched in Bacteroides vulgatus and Parabacteroides distasonis, increasing the response rate to PD-1 / PD-L1 immunotherapy; cordyceps polysaccharides are enriched in the gut symbiotic Parabacteroides goldsteinii, improving obesity and metabolic disorders. Although existing technologies have published numerous studies on the correlation between polysaccharides and Bacteroides species, their polysaccharide utilization sites, and polysaccharide utilization profiles, how to utilize the Bacteroides polysaccharide utilization profile for polysaccharide quality control remains a challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for quality control of traditional Chinese medicine polysaccharides based on the utilization spectrum of Bacteroides polysaccharides. The method achieves quality control of polysaccharides by measuring the utilization of traditional Chinese medicine polysaccharides by Bacteroides.

[0006] This invention provides a polysaccharide quality control method based on the utilization profile of Bacteroides polysaccharides, comprising the following steps:

[0007] Bacteroides strains were inoculated into mBMM medium containing polysaccharides and cultured anaerobically. The cell density of Bacteroides was measured every 12–24 hours to obtain cell density at multiple time points. The cell density was expressed as OD0.05. 600 Value measurement;

[0008] A Bacteroides polysaccharide utilization profile was constructed using data on Bacteroides cell density at the aforementioned multiple time points;

[0009] The quality of polysaccharides is determined based on the utilization profile of the Bacteroides polysaccharides. The determination includes: if the similarity of the utilization profiles of two or more Bacteroides polysaccharides is higher, the monosaccharide composition of the two or more polysaccharides is more similar.

[0010] The correlation between Bacteroides and polysaccharides was confirmed based on the utilization spectrum of Bacteroides polysaccharides, thereby achieving quality control of polysaccharides;

[0011] The mBMM medium contains the following components dissolved in each 1L of water: 50mL mineral 3B solution, 2.0g NaHCO3, 1.0g L-cysteine ​​hydrochloride, 1.5mL FeSO4·7H2O solution with a concentration of 2.78mg / mL, 1.0mL heme chloride solution with a concentration of 5mg / mL, 100μL vitamin K3 solution with a concentration of 5mg / mL, 100μL vitamin B12 solution with a concentration of 0.05mg / mL, 20mg L-methionine, and 31mg histidine.

[0012] The mineral 3B solution dissolves the following components per 100 mL of water: 1.8 g KH2PO4, 1.8 g NaCl, 40 mg MgCl2·6H2O, 39 mg CaCl2, 2 mg CoCl2·6H2O, 20 mg MnCl2·4H2O, 1 g NH4Cl and 500 mg Na2SO4.

[0013] Preferably, the polysaccharide-containing mBMM medium comprises polysaccharide and mBMM medium, wherein the concentration of the traditional Chinese medicine polysaccharide in the mBMM medium is 0.1-1 wt.%.

[0014] Preferably, the anaerobic culture is carried out under a mixed atmosphere, which includes 85% N2, 10% H2 and 5% CO2; the temperature of the anaerobic culture is 37°C and the time is 48-72 hours.

[0015] Preferably, the Bacteroidetes strain is inoculated using a Bacteroidetes PBS solution, wherein the volume ratio of the Bacteroidetes PBS solution to the polysaccharide-containing mBMM medium is 1:100 to 1:1, and the OD of the Bacteroidetes PBS solution is... 600 The value ranges from 0.1 to 1.

[0016] Preferably, the Bacteroidetes species include *Parabacteroides merda*, *Bacteroides dorei*, *Bacteroides vulgatus*, *Bacteroides bouchesdurhonensis*, *Bacteroides cellulosilyticus*, *Bacteroides finegoldii*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Parabacteroides johnsonii*, *Parabacteroides goldsteinii*, *Bacteroides eggerthii*, *Bacteroides faecis*, *Bacteroides clarus*, *Bacteroides oleiciplenus*, *Parabacteroides distasonis*, *Bacteroides thetaiotaomicron*, and *Bacteroides masilia*. One or more of the following: *Bacteroides massiliensis*, *Bacteroides uniformis*, *Bacteroides caccae*, *Bacteroides stercoris*, *Bacteroides faecichinchillae*, *Bacteroides sartorii*, *Bacteroides salyersiae*, *Bacteroides intestinalis*, *Bacteroides xylanisolvens*, *Parabacteroides faecis*, and *Bacteroides clarus*.

[0017] Preferably, the method for constructing the Bacteroides polysaccharide utilization profile is as follows: the OD values ​​of Bacteroides cells at the multiple time points are... 600 Values ​​are used as a polysaccharide utilization profile.

[0018] Preferably, the polysaccharide quality control method further includes a positive control and a negative control, wherein the culture medium in the positive control is mBMM medium containing glucose, and the culture medium in the negative control is mBMM medium containing sterile water.

[0019] Preferably, the glucose-containing mBMM medium comprises glucose and mBMM medium, wherein the concentration of glucose in the mBMM medium is 0.1 to 1 wt.%.

[0020] The mBMM culture medium containing sterile water comprises sterile water and mBMM culture medium, wherein the concentration of sterile water in the mBMM culture medium is 0.1–1 wt.%.

[0021] Preferably, the polysaccharide includes polysaccharides derived from traditional Chinese medicine.

[0022] This invention provides a Bacteroidetes strain, which includes one or more strains listed in the table below. The strain number, Latin name, and preservation number of the Bacteroidetes strain are shown in Table 1.

[0023] Beneficial effects:

[0024] This invention provides a polysaccharide quality control method based on the utilization spectrum of Bacteroides polysaccharides, comprising the following steps: inoculating Bacteroides strains into mBMM medium containing traditional Chinese medicine polysaccharides for anaerobic culture, measuring the Bacteroides cell density every 12 hours to obtain the Bacteroides cell density at multiple time points, wherein the cell density is expressed as OD. 600The data on Bacteroides cell density at multiple time points are used to construct a Bacteroides polysaccharide utilization profile. Based on this profile, the quality of the polysaccharides is determined, including: the higher the similarity of the utilization profiles of two or more polysaccharides, the more similar the monosaccharide composition of the two or more polysaccharides; the correlation between Bacteroides and polysaccharides is confirmed based on the utilization profile, thus achieving polysaccharide quality control; the mBMM culture medium, based on 1L of water as the solvent, also includes 50mL of mineral 3B solution, 2.0g NaHCO3, 1.0g L-cysteine ​​hydrochloride, 1.5mL of FeSO4·7H2O solution with a concentration of 2.78mg / mL, 1.0mL of heme chloride solution with a concentration of 5mg / mL, 100μL of vitamin K3 solution with a concentration of 5mg / mL, 100μL of vitamin B12 solution with a concentration of 0.05mg / mL, 20mg L-methionine, and 31mg histidine; the mineral 3B solution, based on 100mL of water as the solvent, also includes 1.8g... The ingredients are KH₂PO₄, 1.8 g NaCl, 40 mg MgCl₂·6H₂O, 39 mg CaCl₂, 2 mg CoCl₂·6H₂O, 20 mg MnCl₂·4H₂O, 1 g NH₄Cl, and 500 mg Na₂SO₄. The method described in this invention achieves quality control of polysaccharides by determining the utilization spectrum of polysaccharides by Bacteroides. This method can not only distinguish polysaccharides from different sources and preparation methods but also provide potential pharmacodynamic and mechanistic information. Furthermore, different polysaccharides can induce differential growth in Bacteroides species, revealing that prebiotic polysaccharides can improve the growth of Bacteroides species, possessing potential commercial value.

[0025] Biological Preservation Information

[0026] The Bacteroidetes strains described in Table 1 below were deposited in April 2024 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain names, Latin names, and accession numbers are recorded in Table 1. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 This is the workflow of the quality control method for traditional Chinese medicine polysaccharides based on the polysaccharide utilization spectrum of Bacteroides in Example 1;

[0029] Figure 2 This is a graph showing the prevalence and relative abundance of Bacteroides in the human gut microbiota in Example 1;

[0030] Figure 3This is a graph showing the test results of the basal culture medium in Example 1;

[0031] Figure 4 This is a graph showing the quality control results of traditional Chinese medicine polysaccharides based on monosaccharide composition in Example 1;

[0032] Figure 5 The utilization spectrum of traditional Chinese medicine polysaccharides from Bacteroides in Example 1;

[0033] Figure 6 This is a cluster analysis diagram of the utilization spectrum of Bacteroides polysaccharides in Example 1;

[0034] Figure 7 This is a cluster analysis diagram of the monosaccharide composition in Example 1;

[0035] Figure 8 The graph shows the correlation analysis results between the distance of monosaccharide composition and the distance of Bacteroidetes utilization spectrum in Example 1.

[0036] Figure 9 The graph shows the evaluation results of technical repeatability (A) and batch repeatability (B) in Example 1.

[0037] Figure 10 The graph shows the evaluation results of the Bacteroides polysaccharide utilization spectrum distance (A) and monosaccharide composition distance (B) in Example 1. Detailed Implementation

[0038] This invention provides a polysaccharide quality control method based on the utilization profile of Bacteroides polysaccharides, comprising the following steps:

[0039] Bacteroides strains were inoculated into mBMM medium containing polysaccharides and cultured anaerobically. The cell density of Bacteroides was measured every 12–24 hours to obtain cell density at multiple time points. The cell density was expressed as OD0.05. 600 Value measurement;

[0040] A Bacteroides polysaccharide utilization profile was constructed using data on Bacteroides cell density at the aforementioned multiple time points;

[0041] The quality of polysaccharides is determined based on the utilization profile of the Bacteroides polysaccharides, including: the higher the similarity of the utilization profiles of two or more Bacteroides polysaccharides, the more similar the quality of the two or more polysaccharides.

[0042] The correlation between Bacteroides and polysaccharides was confirmed based on the utilization spectrum of Bacteroides polysaccharides, thereby achieving quality control of polysaccharides;

[0043] The mBMM medium contains the following components dissolved in each 1L of water: 50mL mineral 3B solution, 2.0g NaHCO3, 1.0g L-cysteine ​​hydrochloride, 1.5mL FeSO4·7H2O solution with a concentration of 2.78mg / mL, 1.0mL heme chloride solution with a concentration of 5mg / mL, 100μL vitamin K3 solution with a concentration of 5mg / mL, 100μL vitamin B12 solution with a concentration of 0.05mg / mL, 20mg L-methionine, and 31mg histidine.

[0044] The mineral 3B solution dissolves the following components per 100 mL of water: 1.8 g KH2PO4, 1.8 g NaCl, 40 mg MgCl2·6H2O, 39 mg CaCl2, 2 mg CoCl2·6H2O, 20 mg MnCl2·4H2O, 1 g NH4Cl and 500 mg Na2SO4.

[0045] In this invention, Bacteroides strains are inoculated into mBMM medium containing polysaccharides for anaerobic culture. The cell density of Bacteroides is measured every 12–24 hours to obtain the cell density at multiple time points. The cell density is expressed as OD0.05. 600 The cell density of the Bacteroides described in this invention is preferably measured every 12 hours.

[0046] In this invention, the Bacteroidetes species preferably include, but are not limited to, *Parabacteroides merdae*, *Bacteroides dorei*, *Bacteroides vulgatus*, *Bacteroides bouchesdurhonensis*, *Bacteroides cellulosilyticus*, *Bacteroides finegoldii*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Parabacteroides johnsonii*, *Parabacteroides goldsteinii*, *Bacteroides eggerthii*, *Bacteroides faecis*, *Bacteroides clarus*, *Bacteroides oleiciplenus*, and *Parabacteroides*. One or more of the following: Bacteroides distasonis, Bacteroides thetaiotaomicron, Bacteroides massiliensis, Bacteroides uniformis, Bacteroides caccae, Bacteroides stercoris, Bacteroides faecichinchillae, Bacteroides sartorii, Bacteroides salyersiae, Bacteroides intestinalis, Bacteroides xylanisolvens, Parabacteroides faecis, and Bacteroides clarus.Further preferred species include *Parabacteroides merda*, *Bacteroides dorei*, *Bacteroides vulgatus*, *Bacteroides bouchesdurhonensis*, *Bacteroides cellulosilyticus*, *Bacteroides finegoldii*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Parabacteroides johnsonii*, *Parabacteroides goldsteinii*, *Bacteroides eggerthii*, *Bacteroides faecis*, *Bacteroides clarus*, *Bacteroides oleiciplenus*, *Parabacteroides distasonis*, *Bacteroides thetaiotaomicron*, and *Bacteroides masilia*. The species include *Bacteroides massiliensis*, *Bacteroides uniformis*, *Bacteroides caccae*, *Bacteroides stercoris*, *Bacteroides faecichinchillae*, *Bacteroides sartorii*, *Bacteroides salyersiae*, *Bacteroides intestinalis*, *Bacteroides xylanisolvens*, *Parabacteroides faecis*, and *Bacteroides clarus*, more preferably the species listed in Table 1. The polysaccharides of this invention preferably include traditional Chinese medicine polysaccharides, which preferably include one or more of Astragalus polysaccharides, Dendrobium polysaccharides, Ganoderma lucidum polysaccharides, ginseng polysaccharides, and Lycium barbarum polysaccharides, more preferably Astragalus polysaccharides, Dendrobium polysaccharides, Ganoderma lucidum polysaccharides, ginseng polysaccharides, and Lycium barbarum polysaccharides.The 28 Bacteroides mentioned above are core bacterial species in the human gut. Based on the correlation between polysaccharides and the reproduction and activity of these 28 Bacteroides, the quality control of polysaccharides can be achieved by observing the growth of specific Bacteroides and the specificity of the polysaccharide utilization spectrum. The preferred method for controlling the quality of polysaccharides includes: observing the regulatory effect of the target polysaccharide on the growth of the same specific strain in vivo and in vitro. When the growth trend of the specific strain remains consistent in both in vivo and in vitro culture environments, it indicates that the quality of the target polysaccharide meets the conventional requirements in the field; otherwise, the quality does not meet the conventional requirements. The in vitro culture conditions are preferably performed according to the conventional culture conditions for the specific strain. For example, if ginseng polysaccharide promotes the growth of the key bacterium *Bacteroides vulgatus* in vivo, and ginseng polysaccharide is observed to promote the growth of *Bacteroides vulgatus* in vitro, the quality of the ginseng polysaccharide is considered acceptable.

[0047] In this invention, the Bacteroidetes strain is preferably inoculated using a Bacteroidetes PBS solution. The volume ratio of the Bacteroidetes PBS solution to the polysaccharide-containing mBMM medium is preferably 1:100 to 1:1, more preferably 1:10. The OD of the Bacteroidetes PBS solution is... 600 The value is preferably 0.1 to 1, and more preferably 1.

[0048] In this invention, the polysaccharide-containing mBMM medium preferably comprises polysaccharide and mBMM medium, wherein the concentration of polysaccharide in the mBMM medium is 0.1–1 wt.%, more preferably 0.5 wt.%. The anaerobic culture of this invention is preferably carried out under mixed atmosphere conditions, wherein the mixed atmosphere preferably comprises 85% N2, 10% H2, and 5% CO2; the temperature of the anaerobic culture is preferably 37°C, and the time is preferably 48–72 h, more preferably 48 h. The anaerobic culture of this invention is preferably carried out in an anaerobic workstation, the specific characteristics of which are not particularly limited, and a conventional anaerobic workstation in the art can be used. When carrying out the anaerobic culture in the anaerobic workstation of this invention, it is preferred that three biological replicates be set up for each strain of Bacteroides.

[0049] The polysaccharide quality control method of the present invention further includes a positive control and a negative control. The culture medium in the positive control is preferably an mBMM medium containing glucose, and the culture medium in the negative control is preferably an mBMM medium containing sterile water. The mBMM medium containing glucose preferably comprises glucose and mBMM medium, and the concentration of glucose in the mBMM medium is preferably 0.1–1 wt.%, more preferably 0.5 wt.%. The mBMM medium containing sterile water preferably comprises sterile water and mBMM medium, and the concentration of sterile water in the mBMM medium is preferably 0.1–1 wt.%, more preferably 0.5 wt.%. The positive and negative controls of the present invention serve to ensure whether the test strain grows normally and to ensure that the basal culture medium does not grow, respectively, to eliminate the influence of external errors on the experiment.

[0050] After obtaining the data on Bacteroides cell density at the multiple time points, this invention constructs a Bacteroides polysaccharide utilization profile using the data on Bacteroides cell density at the multiple time points. The preferred method for constructing the Bacteroides polysaccharide utilization profile of this invention is to use the OD values ​​of 28 Bacteroides strains grown in a medium supplemented with 0.1–1 wt.% traditional Chinese medicine polysaccharides in mBMM as the polysaccharide utilization profile; more preferably, it is the OD values ​​after growth in a medium supplemented with 0.5 wt.% traditional Chinese medicine polysaccharides.

[0051] After constructing the Bacteroides polysaccharide utilization spectrum, the quality of the polysaccharides is determined based on the Bacteroides polysaccharide utilization spectrum, which includes: the higher the similarity of the Bacteroides polysaccharide utilization spectra of two or more polysaccharides, the more similar the monosaccharide composition of the two or more polysaccharides;

[0052] The correlation between Bacteroides and polysaccharides was confirmed based on the utilization spectrum of Bacteroides polysaccharides, thereby achieving quality control of polysaccharides.

[0053] This invention preferably determines the similarity of polysaccharides based on the Euclidean distance between two or more polysaccharides. When the Euclidean distance is greater than 0.4, it indicates that the two or more polysaccharides are different polysaccharides with different monosaccharide compositions. When the Euclidean distance is between 0 and 0.4, it indicates that the two or more polysaccharides have similar compositions and cannot be distinguished. The Euclidean distance mentioned in this invention is preferably calculated using the R-package vegan method based on the Bacteroides polysaccharide utilization spectrum data.

[0054] This invention is preferably based on the correlation between polysaccharides and the reproduction and activity of the above 28 Bacteroides species. The quality control of polysaccharides can be achieved by observing the growth of specific Bacteroides and the specificity of the polysaccharide utilization spectrum. For example, if polysaccharides promote the growth of the key bacterium Bacteroides vulgatus in vivo, the quality of polysaccharides is considered to be acceptable as long as the growth of Bacteroides vulgatus is observed in vitro.

[0055] This invention provides a Bacteroidetes strain, which includes one or more strains listed in the table below. The strain number, Latin name, and preservation number of the Bacteroidetes strain are shown in Table 1 and will not be repeated here.

[0056] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] A quality control method for polysaccharides from traditional Chinese medicine based on the polysaccharide utilization spectrum of Bacteroidetes (BacPUS) includes the following steps:

[0059] First, let's introduce the BacPUS workflow ( Figure 1 ):

[0060] 1) Modified BMM medium (mBMM) was added to 96-well shallow plates in an anaerobic workstation (the gas mixture consisted of 85% N2, 10% H2, and 5% CO2). The mBMM medium supplemented with 0.5 wt.% (0.5 g / 100 mL, the same below) ddH2O served as a negative control; the mBMM medium supplemented with 0.5 wt.% glucose served as a positive control; and the mBMM medium supplemented with 0.5 wt.% traditional Chinese medicine polysaccharide served as the experimental group. Each group had three biological replicates. The traditional Chinese medicine polysaccharide was sterilized by filtration through a 0.22 μm filter after being incubated at 40℃ for 20 min before being mixed with the modified BMM medium.

[0061] 2) Next, the bacterial culture from the cryopreservation tubes was inoculated into BHIS medium at a volume ratio of 1:100 and incubated in an anaerobic incubator at 37°C for 24 hours. The cultured bacteria were washed twice with anaerobic PBS, and the OD of the bacterial PBS solution was measured. 600 Adjust the concentration to 0.1. Then, inoculate the bacteria into 96-well plates at a ratio of 1:10 and incubate at 37°C. Measure and record the growth OD every 12 hours using a microplate reader. 600 Measurements were taken over 48 hours.

[0062] 1.1 Analysis of the prevalence and relative abundance of Bacteroides

[0063] 1.1.1 Test Methods

[0064] Twenty-eight Bacteroidetes were selected from the previously unpublished Gutbacteria Isolate Collection (GBIC), including 23 Bacteroidetes and 5 Parabacteroidetes (Table 1). 16,282 healthy human metagenomics were screened from publicly available metagenomic data, representing the global healthy human gut microbiome, by searching for criteria defined in the Human Gut Microbiota Database (GMrepo) (experiment type = "metagenomics", sequencing quality status = "good running", host age > 5, country = "non-empty", recent antibiotic use = "no", and phenotype = "healthy". Species with a prevalence greater than 30% and a mean relative abundance greater than 0.1% were defined as core species.

[0065] Table 1 Bacteroidetes species

[0066]

[0067]

[0068] Note: The absence of a Chinese name is marked with "————"; the accession number refers to the accession number deposited at the China General Microbiological Culture Collection Center of the China Association for the Preservation and Management of Microbial Cultures.

[0069] 1.1.2 Experimental Results

[0070] To assess the prevalence and relative abundance of Bacteroidetes species in the major taxonomic composition of the human gut microbiota, this invention collected publicly available 16S rRNA gene amplicon datasets (n = 16282) from GMRepo. It was found that 71.1% (22 / 31) of the Bacteroidetes species had a prevalence greater than 30%, and 84.1% (26 / 31) had a relative abundance greater than 0.1%. These results indicate that most species in BacPUS are core species of the human gut microbiota (species with a prevalence >30% and a relative abundance >0.1% are defined as core species of the human gut). Figure 2 (Table 3).

[0071] 1.2 Culture medium test

[0072] 1.2.1 Test Methods

[0073] This invention prepared three different basal culture media: modified BMM medium (mBMM) supplemented with 0.5% glucose, modified YCFA medium (mYCFA), and modified M9 medium (mM9). Detailed compositions of each medium can be found in Tables 2–9.

[0074] Table 2 BHIS culture medium

[0075]

[0076] Table 3 Modified BMM medium

[0077]

[0078] Note: Items without a product number are marked with "————".

[0079] Table 4 Mineral element 3B solution

[0080]

[0081] Table 5 Modified YCFA medium

[0082]

[0083]

[0084] Note: Add 1 mL of Vitamin Solution II (see table below) after sterilization.

[0085] Items without a product number are marked with "————".

[0086] Table 6 Vitamin Solution I

[0087]

[0088] Table 7 Vitamin Solution II

[0089]

[0090] Table 8 Modified mM9 solution

[0091]

[0092]

[0093] Note: Items without a product number are marked with "————".

[0094] Table 9 M9 solution (5X)

[0095]

[0096] Table 10 PBS solutions (10X)

[0097]

[0098] Using an anaerobic workstation with a gas mixture of 85% N2, 10% H2, and 5% CO2, the modified basal medium was aliquoted into 96-well plates. The bacterial cultures were washed twice with anaerobic PBS, and the OD values ​​of the bacterial solutions were adjusted. 600Adjust the temperature to 0.1, then inoculate the bacteria from Table 1 into 96-well plates at a ratio of 1:10 and incubate at 37°C. Measure and record the growth OD every 12 hours using a microplate reader. 600 Measurements were taken over 72 hours.

[0099] 1.2.2 Experimental Results

[0100] To screen for suitable basal culture media for the growth of Bacteroides strains, this invention tested three basal culture media, including modified BMM (mBMM) supplemented with 0.5% glucose, modified YCFA (mYCFA), and modified M9 (mM9). By measuring the growth curves of 31 Bacteroides strains under these basal media, this invention found that mBMM and mYCFA generally support the growth of Bacteroides species. Figure 3 Compared to mYCFA, mBMM is a synthetic culture medium with well-defined components and more stable polysaccharide utilization profile measurements.

[0101] 1.3 Quality control of traditional Chinese medicine polysaccharides based on Bacteroides polysaccharide utilization profile

[0102] 1.3.1 Test Methods

[0103] Preparation of polysaccharides from traditional Chinese medicine: Eight batches of medicinal materials, including Astragalus membranaceus, ginseng, Ganoderma lucidum 1, Ganoderma lucidum 2, Dendrobium nobile 1, Dendrobium nobile 2, Lycium barbarum 1, and Lycium barbarum 2, were collected for polysaccharide extraction (origin and batch information are shown in Table 11). For Astragalus membranaceus, ginseng, Ganoderma lucidum 1, Dendrobium nobile 1, and Lycium barbarum 1, the medicinal materials were soaked in 5-8 times their volume of deionized water for 1 hour, followed by decoction extraction for 2 hours. The filtrate was filtered, and the extraction was repeated three times. The filtrates were combined and adjusted to 80% alcohol content with anhydrous ethanol, stored at 4°C overnight, and precipitates were obtained. These precipitates were then freeze-dried to obtain polysaccharides Aps, GPs, GLPs, DPs, and LBPs. Two slices of wolfberry, two slices of Ganoderma lucidum, two slices of Dendrobium, and two slices of wolfberry were soaked in 5-8 times the amount of deionized water for 1 hour, then decocted and extracted for 2 hours. The filtrate was filtered and extracted a total of three times. The filtrates were combined and adjusted to 80% alcohol content with anhydrous ethanol. The mixture was stored at 4°C overnight to obtain a precipitate. After redissolving the precipitate, substances with a molecular weight less than 3500 were removed by dialysis. The precipitate was then freeze-dried to obtain polysaccharides GLPs, DPs, and LBPs.

[0104] Table 11. Origin and batch information of eight batches of medicinal decoction pieces and polysaccharide numbers.

[0105]

[0106] Quality control of traditional Chinese medicine polysaccharides based on monosaccharide composition: The monosaccharide composition of polysaccharide samples was analyzed using a high-performance liquid chromatography (HPLC) system. Specific parameters were configured as follows: mobile phase: phosphate buffer (0.1 mol / L) + acetonitrile; flow rate: 0.6 mL / min; single sample volume: 20 μL; column: Cosmosil 5C18-PAQ 4.6ID × 250 mm; column temperature: 40℃; detector: ultraviolet (UV) detector. After setting and verifying accuracy, the HPLC system was started, and the instrument was preheated, tubing flushed, column equilibrated, and detector calibrated according to specifications. Monosaccharide standards (arabinose, fructose, galacturonic acid, galactose, glucose, glucuronic acid, mannose, rhamnose, xylose) were prepared into 1 mg / mL solutions and injected into the HPLC system for calibration. The peak retention times of each known monosaccharide sample were recorded. Polysaccharide samples that had undergone complete acid hydrolysis and derivatization pretreatment (…) were then analyzed. Figure 4 Eight polysaccharides were analyzed in a liquid chromatography system. The pretreatment process, including complete acid hydrolysis and derivatization, involved dissolving 1 mg of each of the eight polysaccharides in 200 μL of water, filtering the solution through a 0.22 μm filter until clear, and then analyzing the samples in the liquid chromatography system. The types of monosaccharides were determined by comparing their retention times with those of standards, and the peak area ratio of each monosaccharide was calculated to obtain the proportion of each monosaccharide. The results are shown below. Figure 4 As shown.

[0107] Quality control of traditional Chinese medicine polysaccharides based on BacPUS: Modified BMM medium was added to 96-well plates in an anaerobic workstation (85% N2, 10% H2, and 5% CO2). The medium was supplemented with 0.5% ddH2O (negative control), 0.5% glucose (positive control), or 0.5% traditional Chinese medicine polysaccharides (experimental group). Next, the bacteria were washed twice with anaerobic PBS, and the OD of the bacterial solution was determined. 600 Adjust the concentration to 0.1. Then, inoculate the bacteria into 96-well plates at a ratio of 1:10 and incubate at 37°C. Measure and record the growth OD every 12 hours using a microplate reader. 600 Measurements were taken over 48 hours.

[0108] 1.3.2 Experimental Results

[0109] Monosaccharide composition analysis ( Figure 4Table 12 shows that Astragalus polysaccharides (APs), Ginseng polysaccharides (GPs), and Ganoderma lucidum polysaccharides (GLPs_1 and GLPs_2) are mainly composed of glucose (92.8%, 93.1%, 72.2%, and 68.3%, respectively). Dendrobium polysaccharides (DPs_1 and DPs_2) are mainly composed of mannose (81.1% and 78.7%, respectively). Lycium barbarum polysaccharides (LBPs_1) are mainly composed of 36.9% arabinose, 22.6% galactose, and 23.4% galactonic acid, while Lycium barbarum polysaccharides (LBPs_2) are mainly composed of 16.0% glucose, 33.5% galactose, and 39% galactonic acid.

[0110] Table 12 Monosaccharide composition and content

[0111] ％ Man GlcA Rha GalA Glc Gal Xyl Ara Fuc LBPs_1 2.8 0 5.2 23.4 7.7 22.6 0 36.9 0 LBPs_2 3 1.1 5.2 39 16 33.5 0 1.7 0 GLPs_1 4.6 3.6 0 0 72.2 0 12.5 0 6.3 GLPs_2 7.5 2 0 0 68.3 13.7 0 1.5 6.9 APs 0 0 0 3 92.8 0 2.5 1.7 0 GPs 0 0 0 0 93.1 0 0 2.9 1.6 DPs_1 81.1 0 0 0 16.9 0 0 0 0 DPs_2 78.7 0 0 0 19.8 0 0 0 0

[0112] To characterize the utilization spectrum of Bacteroides to polysaccharides from different sources and preparation methods, this invention selected 28 Bacteroides strains and cultured them in a medium using eight kinds of Bacteroides as the sole carbon source. This invention found significant differences in the utilization spectrum of different Bacteroides. Ginseng polysaccharides (GPs) supported the growth of multiple Bacteroides species, while Dendrobium polysaccharides (DPs_1 and DPs_2) specifically supported the growth of Bacteroides uniformis. Figure 5 ).

[0113] By analyzing the polysaccharide utilization profile ( Figure 6 ) and monosaccharides ( Figure 7 Cluster analysis (using the R package pheatmap with the clustering method "complete") revealed that polysaccharides with similar monosaccharide compositions exhibit more similar Bacteroidetic utilization profiles, such as DPs_1 and DPs_2. In other cases, polysaccharides with similar monosaccharide compositions have different Bacteroidetic utilization profiles, such as APs and GPs.

[0114] Next, this invention performed a correlation analysis between the distance of monosaccharide composition and the distance of Bacteroides polysaccharide utilization spectrum (the software used for the distance of monosaccharide composition and the distance of Bacteroides polysaccharide utilization spectrum was R (version 4.2.0) package vegan (version 2.6.4), and the distance was calculated using the "Euclidean" method; the software used for the correlation analysis was R (version 4.2.0) package ggpubr (version 0.6.0), and the correlation analysis method was "spearman"). Unexpectedly, this invention found almost no correlation between them, indicating that polysaccharides with similar monosaccharide compositions do not have similar Bacteroides utilization spectra ( Figure 8 , Figure 10The reason for this phenomenon may be that polysaccharides in traditional Chinese medicine with similar monosaccharide compositions have different glycobonding and high-dimensional structures. These results indicate that the method based on the utilization profile of Bacteroides polysaccharides can distinguish polysaccharides that are difficult to distinguish by chemical or physical methods. Furthermore, the high-dimensional structure of polysaccharides is a factor affecting the utilization profile of Bacteroides polysaccharides in traditional Chinese medicine. Therefore, evaluating the utilization profile of Bacteroides polysaccharides can reflect the differences in the high-dimensional structure of polysaccharides to a certain extent, and thus be used for the quality control of traditional Chinese medicine polysaccharides.

[0115] Furthermore, polysaccharide quality control can be achieved by observing the growth of specific bacteria and the specificity of the polysaccharide utilization spectrum. For example, if GPs can promote the growth of the key bacterium Bacteroides vulgatus (Bv) in vivo, then as long as GPs are observed to promote the growth of Bv in vitro, the quality of GPs is considered to be up to standard.

[0116] BacPUS-based quality control of traditional Chinese medicine polysaccharides can distinguish between polysaccharides with similar monosaccharide compositions. For example, based on the quality control method of monosaccharide composition, the distance between APs and GPs was calculated to be 0.04. Figure 10 This indicates that the monosaccharide compositions of the two are extremely similar and indistinguishable. However, based on the BacPUS-based quality control method for polysaccharides in traditional Chinese medicine, the calculated distance between APs and GPs is 1.2 (…). Figure 10 This can significantly distinguish between the two polysaccharides.

[0117] 1.4 Assessment of technical repeatability and batch repeatability

[0118] 1.4.1 Test Methods

[0119] Growth OD between experiments involving three techniques and two batches 600 Correlation analysis was performed to assess the technical repeatability and batch repeatability of BacPUS.

[0120] 1.4.2 Experimental Results

[0121] We observed that the technical repeatability was low (R = 0.96, p < 2.2e16). Figure 9 (A), and batch repeatability (R = 0.91, p < 2.2e16) Figure 9 The high correlation between B and B indicates that the BacPUS-based method for quality control of traditional Chinese medicine polysaccharides is robust.

[0122] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A polysaccharide quality control method based on the utilization profile of Bacteroides polysaccharides, comprising the following steps: Bacteroides strains were inoculated into mBMM medium containing polysaccharides and cultured anaerobically. The cell density of Bacteroides was measured every 12–24 hours to obtain cell density at multiple time points. The cell density was expressed as OD0.

05. 600 Value measurement; A Bacteroides polysaccharide utilization profile was constructed using data on Bacteroides cell density at the aforementioned multiple time points; The quality of the polysaccharides is determined based on the Bacteroides polysaccharide utilization profile, and the determination includes: The higher the similarity of the utilization profiles of two or more Bacteroidetes polysaccharides, the more similar the monosaccharide composition of the two or more polysaccharides. The correlation between Bacteroides and polysaccharides was confirmed based on the utilization spectrum of Bacteroides polysaccharides, thereby achieving quality control of polysaccharides; The mBMM medium contains the following components dissolved in each 1L of water: 50mL mineral 3B solution, 2.0g NaHCO3, 1.0g L-cysteine ​​hydrochloride, 1.5mL FeSO4·7H2O solution with a concentration of 2.78mg / mL, 1.0mL heme chloride solution with a concentration of 5mg / mL, 100μL vitamin K3 solution with a concentration of 5mg / mL, 100μL vitamin B12 solution with a concentration of 0.05mg / mL, 20mg L-methionine, and 31mg histidine. The mineral 3B solution dissolves the following components per 100 mL of water: 1.8 g KH2PO4, 1.8 g NaCl, 40 mg MgCl2·6H2O, 39 mg CaCl2, 2 mg CoCl2·6H2O, 20 mg MnCl2·4H2O, 1 g NH4Cl and 500 mg Na2SO4.

2. The polysaccharide quality control method according to claim 1, characterized in that, The polysaccharide-containing mBMM medium comprises polysaccharide and mBMM medium, wherein the concentration of polysaccharide in the mBMM medium is 0.1–1 wt.%.

3. The polysaccharide quality control method according to claim 1 or 2, characterized in that, The anaerobic culture was carried out under a mixed atmosphere, which included 85% N2, 10% H2 and 5% CO2; the temperature of the anaerobic culture was 37°C and the time was 48 to 72 hours.

4. The polysaccharide quality control method according to claim 1, characterized in that, The Bacteroidetes strain was inoculated using a Bacteroidetes PBS solution, wherein the volume ratio of the Bacteroidetes PBS solution to mBMM medium containing polysaccharides was 1:100 to 1:1, and the OD of the Bacteroidetes PBS solution was... 600 The value ranges from 0.1 to 1.

5. The polysaccharide quality control method according to claim 1, characterized in that, The Bacteroides species mentioned include *Parabacteroides merda*, *Bacteroides dorei*, *Bacteroides vulgatus*, *Bacteroides bouchesdurhonensis*, *Bacteroides cellulosilyticus*, *Bacteroides finegoldii*, *Bacteroides fragilis*, *Bacteroides ovatus*, *Parabacteroides johnsonii*, *Parabacteroides goldsteinii*, *Bacteroides eggerthii*, *Bacteroides faecis*, *Bacteroides clarus*, *Bacteroides oleiciplenus*, *Parabacteroides distasonis*, *Bacteroides thetaiotaomicron*, and *Bacteroides masilia*. One or more of the following: *Bacteroides massiliensis*, *Bacteroides uniformis*, *Bacteroides caccae*, *Bacteroides stercoris*, *Bacteroides faecichinchillae*, *Bacteroides sartorii*, *Bacteroides salyersiae*, *Bacteroides intestinalis*, *Bacteroides xylanisolvens*, *Parabacteroides faecis*, and *Bacteroides clarus*.

6. The polysaccharide quality control method according to claim 1, characterized in that, The method for constructing the Bacteroides polysaccharide utilization profile is as follows: The OD values ​​of Bacteroides cells at the multiple time points are... 600 Values ​​are used as a polysaccharide utilization profile.

7. The polysaccharide quality control method according to claim 1, characterized in that, The polysaccharide quality control method also includes a positive control and a negative control. The culture medium for the positive control is mBMM medium containing glucose, and the culture medium for the negative control is mBMM medium containing sterile water.

8. The polysaccharide quality control method according to claim 7, characterized in that, The glucose-containing mBMM medium comprises glucose and mBMM medium, wherein the concentration of glucose in the mBMM medium is 0.1–1 wt.%. The mBMM culture medium containing sterile water comprises sterile water and mBMM culture medium, wherein the concentration of sterile water in the mBMM culture medium is 0.1–1 wt.%.

9. The polysaccharide quality control method according to claim 1, characterized in that, The polysaccharides include those derived from traditional Chinese medicine.

10. A species of Bacteroidetes, characterized in that, The Bacteroidetes species include one or more species listed in the table below. The strain number, Latin name, and preservation number of the Bacteroidetes species are shown in the table below:

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