A non-starch uniform polysaccharide of radix pseudostellariae and a preparation method and application thereof

Abstract

The present application provides a kind of Pseudostellaria heterophylla non-starch uniform polysaccharide and its preparation method and application, belong to natural substance extraction technical field.The Pseudostellaria heterophylla non-starch uniform polysaccharide is divided into five kinds, molecular weight is 2124Da, 85900Da, 72970Da, 35820Da and 20300Da respectively, all have three spiral structures.The Pseudostellaria heterophylla non-starch uniform polysaccharide with molecular weight of 2124Da includes the following molar ratio of glucose, galactose and mannose: 95.47:2.15:2.37, and its repeatable primary structure unit includes the following sugar residues: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, 1,4,6-Glcp.The Pseudostellaria heterophylla non-starch uniform polysaccharide of the present application has excellent prebiotic activity and simple preparation method, and the obtained Pseudostellaria heterophylla non-starch uniform polysaccharide has stable yield, high purity and stable structure, and is suitable for industrial scale production.

Description

Technical Field

[0001] This invention provides a non-starch homogeneous polysaccharide from Codonopsis pilosula, its preparation method, and its application, belonging to the field of natural substance extraction technology. Background Technology

[0002] Prince ginseng is the dried tuberous root of the plant *Codonopsis pilosula*, belonging to the Caryophyllaceae family. It is known for its effects of "tonifying qi and strengthening the spleen, promoting body fluids and moistening the lungs," and is clinically used for symptoms such as spleen deficiency and fatigue, loss of appetite, post-illness weakness, qi and yin deficiency, spontaneous sweating and thirst, and dry cough due to lung dryness. It is a tonic suitable for all ages.

[0003] However, the following problems also exist: the tuber of Codonopsis pilosula contains a large amount of starch, and the polysaccharides obtained by previous preparation methods have low solubility, which interferes with the structural characterization results; there is a lack of suitable extraction and purification processes to prepare Codonopsis pilosula non-starch homogeneous polysaccharides with clear chemical structures and uniform composition.

[0004] Chinese patent CN111647091B discloses an active hexacarbon aldehyde oligosaccharide from *Codonopsis pilosula*, its preparation method, and its applications. The method involves alcohol extraction, water extraction, alcohol precipitation, enzyme-Sevage method for protein removal, and purification using a DEAE-52 anion exchange column to collect products with molecular weights in the range of 1000 Da to 2000 Da, thus obtaining the active hexacarbon aldehyde oligosaccharide. The molecular weight of this active hexacarbon aldehyde oligosaccharide is 1476.6185 Da, and its monosaccharide composition is mannose, glucose, and galactose (molar ratio of 0.076:0.814:2.600). This active hexacarbon aldehyde oligosaccharide exhibits anti-inflammatory and antioxidant activities, as well as strong reducing power. However, the study only investigated the effects of the active hexacarbon aldehyde oligosaccharide on intestinal diseases and did not delve into specific gut microbiota.

[0005] Xie Meilin et al. published a study in *Food Industry Technology* detailing the purification, composition, and protective effect of a *Codonopsis pilosula* polysaccharide against LPS-damaged Raw264.7 macrophages. The extracted polysaccharide had a purity of 80.52% and included galactose, D-mannose, rhamnose, arabinose, D-anhydrous glucose, D-glucuronic acid, and D-lactouronic acid. It showed a protective effect against Raw264.7 macrophages. However, this *Codonopsis pilosula* polysaccharide did not remove starch, and the tubers contain a large amount of starch. Therefore, the polysaccharide obtained by this method had low solubility, which interfered with the structural characterization results. Consequently, the article did not specify the exact structure and composition, and the molecular weight was given as a range. It remains a crude polysaccharide, not a homogeneous one. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a non-starch homogeneous polysaccharide from *Codonopsis pilosula*, its preparation method, and its applications. This preparation method is simple and efficient, simultaneously removing starch from the polysaccharide, thus improving purity and reducing interference from structural characterization. The obtained non-starch homogeneous polysaccharide exhibits stable yield, high purity, and stable structure, making it suitable for industrial-scale production while also possessing good intestinal beneficial properties.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a non-starch homogeneous polysaccharide of *Codonopsis pilosula*, the monosaccharide comprising glucose, galactose, and mannose in the following molar ratio: 95.47:2.15:2.37; the molecular weight of the non-starch homogeneous polysaccharide is 2124 Da; the non-starch homogeneous polysaccharide has a triple helix structure; the repeatable primary structural units of the non-starch homogeneous polysaccharide include the following sugar residues: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, 1,4,6-Glcp, with the following linkage formula:

[0009]

[0010] Secondly, this invention provides a non-starch homogeneous polysaccharide from *Codonopsis pilosula*, the monosaccharide composition of which includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose in the following molar ratio: 1.79:11.32:1.45:2.60:2.45:31.99:48.40; the molecular weight of the non-starch homogeneous polysaccharide is 85900 Da; the non-starch homogeneous polysaccharide has a triple helix structure; the non-starch homogeneous polysaccharide has a reproducible first-order structure. The structural units include the following sugar residues: T-Araf, T-Rhap, 1,2-Araf, T-Glcp, T-GlcpA, T-Galp, 1,2-Galp, 1,2,3,5-Araf, 1,3,6-Manp, 1,3,6-Glcp, 1,4,6-Galp, 1,2,3,4-Galp, 1,2,3,4-GalpA, 1,2,3,6-Galp, and 1,2,3,6-GalpA, with the following linkage formula: Figure 1 As shown.

[0011] Thirdly, the present invention provides a non-starch homogeneous polysaccharide of *Codonopsis pilosula*, the monosaccharide composition of which includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratio: 13.04:15.18:0.82:19.83:51.14; the molecular weight of the non-starch homogeneous polysaccharide of *Codonopsis pilosula* is 72970 Da; the non-starch homogeneous polysaccharide of *Codonopsis pilosula* has a triple helix structure; the repeatable primary structural unit of the non-starch homogeneous polysaccharide of *Codonopsis pilosula* includes the following sugar residues: T-Rhap, 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,2-Galp, 1,2,3-Araf, 1,3,5-Araf, 1,2,3-Galp, 1,3,4-Rhap, 1,2,3,4-Rhap, 1,2,3,5-Araf.

[0012] Fourthly, the present invention provides a non-starch homogeneous polysaccharide of *Codonopsis pilosula*, the monosaccharide composition of which includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratio: 22.58:24.15:3.00:16.30:33.97; the molecular weight of the non-starch homogeneous polysaccharide of *Codonopsis pilosula* is 35820 Da; the non-starch homogeneous polysaccharide of *Codonopsis pilosula* has a triple helix structure; the repeatable primary structural unit of the non-starch homogeneous polysaccharide of *Codonopsis pilosula* includes the following sugar residues: 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,2-Galp, 1,2,3-Araf, 1,3,5-Araf, 1,3,4-Rhap, 1,2,4-Rhap, 1,2,3,4-Glcp, 1,2,3,4-Rhap, 1,2,3,5-Araf.

[0013] Fifthly, this invention provides a non-starch homogeneous polysaccharide from *Codonopsis pilosula*, the monosaccharide composition of which comprises rhamnose, galacturonic acid, glucose, galactose, and arabinose in the following molar ratio: 24.75:45.64:2.19:11.37:16.04; the molecular weight of the non-starch homogeneous polysaccharide is 20300 Da; the non-starch homogeneous polysaccharide has a triple helix structure; the non-starch homogeneous polysaccharide has a repeatable... The hierarchical structural units include the following sugar residues: 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,4-Rhap, 1,2,3-Araf, 1,3,5-Araf, 1,2,3-GalpA, 1,3,4-Rhap, 1,2,4-Rhap, 1,2,3,4-Glcp, 1,2,3,4-Rhap, and 1,2,3,5-Araf.

[0014] Sixthly, the present invention provides a method for preparing the above-mentioned non-starch homogeneous polysaccharide of *Codonopsis pilosula*, comprising the following steps:

[0015] (1) Extract the Codonopsis pilosula in ethanol, filter to obtain a precipitate, dry the precipitate, and obtain Codonopsis pilosula residue;

[0016] (2) Take the dregs of Codonopsis pilosula obtained in step (1), add water to extract it, and obtain Codonopsis pilosula water extract;

[0017] (3) After removing starch from the water extract of Codonopsis pilosula obtained in step (2), centrifuge and concentrate it, take the supernatant, add ethanol for alcohol precipitation, centrifuge again, collect the precipitate, and obtain crude polysaccharide of Codonopsis pilosula.

[0018] (4) The crude polysaccharide of Codonopsis pilosula obtained in step (3) is separated and purified, eluted, and the eluents corresponding to different gradient elution peaks are collected and concentrated to obtain Codonopsis pilosula non-starch homogeneous polysaccharide.

[0019] Specifically, the extraction described in step (1) is reflux extraction.

[0020] Specifically, the volume percentage of ethanol in step (1) of the Codonopsis pilosula non-starch homogeneous polysaccharide is 70-85%.

[0021] Specifically, in step (1) of the non-starch homogeneous polysaccharide of Codonopsis pilosula, the ratio of Codonopsis pilosula to ethanol is 1g:10mL-1g:30mL.

[0022] Specifically, the extraction temperature of the non-starch homogeneous polysaccharide of Codonopsis pilosula in step (1) is 85-90℃, the time is 1-4h / time, and a total of 1-3 extractions are performed.

[0023] Specifically, the extraction of the non-starch homogeneous polysaccharide from Codonopsis pilosula in step (2) is reflux extraction.

[0024] Specifically, the ratio of the ginseng residue to water in step (2) is 1g:10mL-1g:30mL.

[0025] Specifically, the extraction temperature in step (2) is 80-90℃, the extraction time is 1-4h / h / time, and the extraction is performed 1-3 times in total.

[0026] Specifically, the starch removal in step (3) involves adding amylase until the enzyme activity is 1-2 U / mL, hydrolyzing at a temperature of 80-90℃, and hydrolyzing for 0.5-2 hours.

[0027] Preferably, the amylase is α-amylase.

[0028] Specifically, in step (3), the ethanol concentration of the alcohol precipitation is 70-90% by volume, the temperature is 4-8℃, and the time is 12h.

[0029] Preferably, the purification in step (4) uses an anion exchange chromatography column.

[0030] Specifically, the elution in step (4) is: elution with NaCl deionized water solutions of different concentrations.

[0031] Specifically, the different concentrations are:

[0032] A. Elution with deionized water; to obtain the non-starch homogeneous polysaccharide of Codonopsis pilosula described in the first aspect;

[0033] B. Elute with 0.1M NaCl deionized water to obtain the non-starch homogeneous polysaccharide of Codonopsis pilosula described in the second aspect;

[0034] C. Elute with 0.2M NaCl deionized water to obtain a homogeneous non-starch polysaccharide mixture of Codonopsis pilosula.

[0035] Preferably, step (4) further includes separating and purifying the non-starch homogeneous polysaccharide of Codonopsis pilosula obtained by eluting with 0.1M NaCl deionized water, wherein the purification is performed using a gel column.

[0036] Specifically, step (4) further includes separating and purifying the non-starch homogeneous polysaccharide mixture of Codonopsis pilosula to obtain the non-starch homogeneous polysaccharides of Codonopsis pilosula described in the third to fifth aspects, wherein the purification is performed using a gel column.

[0037] Preferably, the concentration in step (4) further includes desalting.

[0038] Seventhly, the present invention provides an application of a non-starch homogeneous polysaccharide of Codonopsis pilosula in the preparation of intestinal probiotic foods and health products.

[0039] Preferably, the intestinal probiotics include BO bacteria, BT bacteria, BL bacteria, and LGG bacteria.

[0040] Definitions and Explanations

[0041] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense.

[0042] The BO bacteria mentioned in this invention are Bacteroides ovatu.

[0043] The BT bacteria mentioned in this invention are Bacteroides thetaiotaomicro.

[0044] The BL bacteria mentioned in this invention are Bifidobacterium longum.

[0045] The LGG bacterium mentioned in this invention is Lactobacillus rhamnosus.

[0046] The technical effects achieved by this invention are:

[0047] (1) The preparation process of the present invention is mild, simple to operate and environmentally friendly. The resulting Codonopsis pilosula non-starch homogeneous polysaccharide has high purity, and the total content of neutral sugar and uronic acid can reach more than 85%.

[0048] (2) The non-starch homogeneous polysaccharide of Codonopsis pilosula prepared by removing starch with amylase has a clear structure, high yield and high solubility.

[0049] (3) The non-starch homogeneous polysaccharide of Codonopsis pilosula of the present invention has good intestinal probiotic activity and has application potential in the preparation of intestinal probiotic foods and health products. Attached Figure Description

[0050] Figure 1 This is a PHP-2a connection.

[0051] Figure 2 The ultraviolet spectrum of non-starch homogeneous polysaccharide from Codonopsis pilosula.

[0052] Figure 3 The image shows the HPGPC diagram of non-starch homogeneous polysaccharides from *Codonopsis pilosula*, where A represents non-starch homogeneous crude polysaccharide; B represents PHP-1; C represents PHP-2a; D represents PHP-3a; E represents PHP-3b; and F represents PHP-3c.

[0053] Figure 4 UPLC / Q-TOF-MS chromatograms of monosaccharide standards mixed with PHP-1, PHP-2a, PHP-3a, PHP-3b, and PHP-3c are shown. The mixed standards in the order of 1-8 are: mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose.

[0054] Figure 5 Infrared spectra of PHP-1, PHP-2a, PHP-3a, PHP-3b, and PHP-3c.

[0055] Figure 6 The Congo red assay is for non-starch homogeneous polysaccharides of Codonopsis pilosula, where A is PHP-1, B is PHP-2a, C is PHP-3a, D is PHP-3b, and E is PHP-3C.

[0056] Figure 7The effects of PHP-1, PHP-2a, PHP-3a, PHP-3b, and PHP-3c on the growth of BO, BT, BL, and LGG bacteria (* indicates P < 0.05 compared to the Blank group, ** indicates P < 0.01 compared to the Blank group, and *** indicates P < 0.001 compared to the Blank group).

[0057] Figure 8 The figures show the in vitro experiments and fermentation characteristics of non-starch homogeneous polysaccharides from *Codonopsis pilosula*. In the figures, A represents the pH value after fermentation, B represents the reducing sugar content after fermentation, C represents the neutral sugar content after fermentation, D represents the carbohydrate consumption rate after fermentation, E represents the molecular weight change after fermentation, and F represents the change in monosaccharide composition at different time points during fecal fermentation (a, b, and c indicate significant differences between different treatment groups at the same time point, P < 0.05). Detailed Implementation

[0058] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0059] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0060] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. 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 this invention pertains.

[0061] The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0062] Example 1: Preparation of non-starch homogeneous polysaccharide from Codonopsis pilosula

[0063] (1) Take 150g of dried Codonopsis pilosula powder and add it to a 70% ethanol solution at a material-to-liquid ratio of 1:20. Extract at 90℃ for 2 hours. Extract three times in total. Collect the Codonopsis pilosula powder, evaporate the ethanol to remove it, and obtain Codonopsis pilosula residue.

[0064] (2) Take 100g of Codonopsis pilosula residue, add it to water at a material-to-liquid ratio of 1:10, and heat and reflux extract it three times at 90℃ for 2 hours each time. Combine the water extracts and concentrate them to a crude drug concentration of 0.5g / mL to obtain Codonopsis pilosula water extract concentrate.

[0065] (3) Add α-amylase to the concentrated aqueous extract of Codonopsis pilosula to maintain the final enzyme activity at 1 U / mL, and perform enzymatic hydrolysis at 90℃ for 0.5 h to obtain the hydrolysate; use I2 / KI reagent to detect the hydrolysate until the starch is completely removed, centrifuge, collect the supernatant to obtain the aqueous extract of Codonopsis pilosula after starch removal; alcohol precipitation, add anhydrous ethanol to the aqueous extract of Codonopsis pilosula after starch removal to make the final concentration 90% by volume, perform alcohol precipitation at 4℃ for 12 h, centrifuge, collect the precipitate to obtain the non-starch homogeneous crude polysaccharide of Codonopsis pilosula.

[0066] (4) Purification: The non-starch homogeneous crude polysaccharide of Codonopsis pilosula was dissolved in deionized water and then separated and purified using a DEAE-52 anion exchange column. The eluent obtained by eluting with deionized water was collected and the non-starch homogeneous polysaccharide of Codonopsis pilosula was hereinafter referred to as PHP-1. The non-starch homogeneous polysaccharide of Codonopsis pilosula was eluted with 0.1M NaCl deionized water solution and the non-starch homogeneous polysaccharide of Codonopsis pilosula was hereinafter referred to as PHP-2. The non-starch homogeneous polysaccharide of Codonopsis pilosula was eluted with 0.2M NaCl deionized water solution and the non-starch homogeneous polysaccharide of Codonopsis pilosula was hereinafter referred to as PHP-3.

[0067] (4) Dissolve PHP-2 by sonication, centrifuge and load the supernatant into a Focurose CL-6B (2.5×100cm) gel column, elute with deionized water at a flow rate of 4mL / 15min, and test the purity of each eluent by HPGFC-ELSD. Combine the high-purity eluents and freeze-dry to obtain the non-starch homogeneous polysaccharide of Codonopsis pilosula, hereinafter referred to as PHP-2a.

[0068] (5) Dissolve PHP-3 by sonication, centrifuge and load the supernatant into a Focurose CL-6B (2.5×100cm) gel column, elute with deionized water at a flow rate of 4mL / 15min, and test the purity and type of each eluent by HPGFC-ELSD. Combine the high-purity eluents of the same type and freeze-dry to obtain three non-starch homogeneous polysaccharides of Codonopsis pilosula, hereinafter referred to as PHP-3a, PHP-3b and PHP-3c.

[0069] I. Physicochemical properties of non-starch homogeneous polysaccharides from Codonopsis pilosula

[0070] (1) Determination of carbohydrate content in non-starch homogeneous polysaccharides from Codonopsis pilosula

[0071] The carbohydrate content of the non-starch homogeneous polysaccharide from *Codonopsis pilosula* prepared in Example 1 was determined using the phenol-sulfuric acid method. The absorbance was measured at 495 nm using a UV spectrophotometer. The carbohydrate content in PHP-1 was 89.15 ± 2.75%. The total carbohydrate content was determined using both the phenol-sulfuric acid method and the m-hydroxybiphenyl method. The carbohydrate contents of PHP-2a, PHP-3a, PHP-3b, and PHP-3c were 93.54 ± 2.12%, 91.9 ± 4.35%, 95.01 ± 3.55%, and 85.82 ± 1.89%, respectively.

[0072] (2) Ultraviolet spectral analysis of non-starch homogeneous polysaccharides from Codonopsis pilosula

[0073] Ultraviolet spectra of five non-starch homogeneous polysaccharides from *Codonopsis pilosula* were scanned in the wavelength range of 200-400 nm. No obvious absorption peaks were observed at 260 nm and 280 nm. Figure 2 This indicates that the five types of non-starch homogeneous polysaccharides from Codonopsis pilosula do not contain nucleic acids or proteins.

[0074] (3) Homogeneity determination of non-starch homogeneous polysaccharides from Codonopsis pilosula

[0075] The homogeneity of five non-starch homogeneous polysaccharides from Codonopsis pilosula was determined by high performance liquid chromatography-gel permeation chromatography combined with evaporative light detector (HPGFC-ELSD).

[0076] Chromatographic conditions: Polysep-GFC-P4000 column (300×7.8mm, 35×7.8mm); mobile phase: ultrapure water; flow rate: 0.4mL / min; injection volume: 10μL; column temperature: 30℃; ELSD detector parameters: N2 pressure 30psi, drift tube temperature 100℃, nebulizer heating power 60%, gain value 10.

[0077] HPGFC-ELSD analysis showed that, compared with the homogeneous crude polysaccharide of *Codonopsis pilosula* (a type of ginseng), the five homogeneous non-starch polysaccharides of *Codonopsis pilosula* exhibited single symmetrical peaks, such as... Figure 3 This indicates that it is a homogeneous polysaccharide.

[0078] II. Chemical Structure Identification

[0079] (1) Determination of molecular weight of non-starch homogeneous polysaccharide from Codonopsis pilosula

[0080] The dried, non-starch homogeneous polysaccharide sample of *Codonopsis pilosula* was dissolved in 0.9% NaCl aqueous solution to a concentration of 2 mg / mL. The solution was then filtered through a 0.45 μm filter to obtain the test solution. Dextran with different molecular weights (Mw = 6300, 9800, 22000, 49400, 107000 Da) was prepared using 0.9% NaCl water at a concentration of 2 mg / mL. The molecular weight of the *Codonopsis pilosula* non-starch homogeneous polysaccharide was determined using high-performance gel permeation chromatography-refractive index detection (HPSEC-RID). The mobile phase was 0.9% NaCl solution, and the flow rate was 0.5 mL / min. The retention time of the above samples was measured using HPSEC-RID. A size exclusion curve equation was plotted with the logarithm of the dextran molecular weight as the ordinate and the retention time as the abscissa. The molecular weight of the *Codonopsis pilosula* non-starch homogeneous polysaccharide was calculated based on the size exclusion curve equation. The results are shown in Table 1.

[0081] Table 1. Molecular weight of non-starch homogeneous polysaccharides from *Codonopsis pilosula*

[0082] PHP-1 PHP-2a PHP-3a PHP-3b PHP-3c molecular weight 2124Da 85900Da 72970Da 35820Da 20300Da

[0083] (2) Monosaccharide composition of non-starch homogeneous polysaccharides from Codonopsis pilosula

[0084] The monosaccharide composition of non-starch homogeneous polysaccharides from *Codonopsis pilosula* was determined by pre-column PMP derivatization combined with UPLC / Q-TOF-MS. 1.0 mg of *Codonopsis pilosula* non-starch homogeneous polysaccharide was accurately weighed and placed in a clean, dry, stoppered test tube. 4 M TFA solution was added, and the mixture was incubated in an oil bath at 120 °C for 2 h, followed by nitrogen blowing to dryness, yielding a completely acid-hydrolyzed sample. Then, 0.5 mL of ammonia (25% by mass) and 0.5 mL of PMP-methanol solution (0.5 M) were added to the completely acid-hydrolyzed polysaccharide sample. After thorough mixing, the mixture was reacted at 70 °C for 30 min, followed by nitrogen blowing to dryness. The solution was reconstituted with 1 mL of methanol (LC-MS grade), centrifuged at 12000 rpm for 10 min, and the supernatant was collected for LC-MS analysis.

[0085] Derivatization of monosaccharide standards: Accurately weigh 1.0 mg of monosaccharide standards (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and fucose) into a stoppered test tube. Add 0.5 mL of ammonia and 0.5 mL of LPMP-methanol solution (0.5 M), mix thoroughly, and react at 70 °C for 30 min. Then, remove to dryness by nitrogen blowing. Redissolve in 1 mL of liquid mass spectrometry grade methanol, centrifuge at 12000 rpm for 10 min, and collect the supernatant for LC-MS analysis.

[0086] UPLC / Q-TOF-MS analysis: A Kinetex C18 100A column (50 × 2.1 mm, 1.7 μm) was used at 25 °C. Gradient elution was performed using a mobile phase of 0.05% acetic acid-20 mM ammonium acetate (phase A)-acetonitrile (phase B): 0–10 min, 15% B; 10–16 min, 15%–17% B; 16–19 min, 17%–70% B. The injection volume was 2 μL, and the flow rate was 0.4 mL / min. Acquisition was performed in positive ion mode, with a mass acquisition range of 420–600 Da.

[0087] Compared with mixed monosaccharide standard derivatives, PHP-1 mainly contains glucose (95.47%), with small amounts of mannose (2.37%) and galactose (2.15%). Figure 4 .

[0088] Compared with mixed monosaccharide standard derivatives, PHP-2a mainly contains mannose (1.79%), rhamnose (11.32%), glucuronic acid (1.45%), galacturonic acid (2.60%), glucose (2.45%), galactose (31.99%), and arabinose (48.40%). Figure 4 .

[0089] Compared with the mixed monosaccharide standard derivatives, PHP-3a mainly contains rhamnose (13.04%), galacturonic acid (15.18%), glucose (0.82%), galactose (19.83%), and arabinose (51.14%); PHP-3b mainly contains rhamnose (22.58%), galacturonic acid (24.15%), glucose (3.00%), galactose (16.30%), and arabinose (33.97%); PHP-3c mainly contains rhamnose (24.75%), galacturonic acid (45.64%), glucose (2.19%), galactose (11.37%), and arabinose (16.04%). Figure 4 .

[0090] (3) Characteristic group analysis of non-starch homogeneous polysaccharides from Codonopsis pilosula

[0091] Fourier transform infrared spectroscopy was used to analyze the characteristic groups of non-starch homogeneous polysaccharides from *Codonopsis pilosula*. Two mg of dried non-starch homogeneous polysaccharide was precisely weighed and added to an appropriate amount of dried KBr (spectral grade) powder. The mixture was then finely ground in a dry agate mortar, vacuum-pressed, and the concentration of the non-starch homogeneous polysaccharide was determined at 4000-400 cm⁻¹. -1 Characteristic peaks within the range.

[0092] FT-IR spectrum of PHP-1 Figure 5The sample exhibited typical polysaccharide characteristic absorption peaks at 3394.38 and 2920.84 cm⁻¹. -1 The broad peak at 1610 cm⁻¹ is attributed to the stretching vibrations of -OH and -CH₃. Because PHP-1 is highly hygroscopic, [the peak at 1610 cm⁻¹ is likely a hygroscopic peak]. -1 The left and right peaks are attributed to the shear vibration of bound water. (At 1000-1200 cm⁻¹) -1 The absorption between them is attributed to the stretching vibration of the COC glycosidic bond on the pyranose ring. 1146.09 cm⁻¹ -1 The band at 853.16 cm⁻¹ is considered to be a result of the formation of polysaccharide glycosidic bonds. -1 The absorption peak at this point indicates the presence of α-glycosidic bonds in PHP-1.

[0093] FT-IR spectrum of PHP-2a Figure 5 The sample exhibited typical polysaccharide characteristic absorption peaks. (3419.70 cm⁻¹) -1 The broad peak at 2924.76 cm⁻¹ belongs to -OH. -1 The absorption peak at 1324.21 cm⁻¹ is attributed to the stretching vibration of CH, while the bending vibration peak appears at 1324.21 cm⁻¹. -1 Nearby. 1000-1200cm -1 The absorption peak is attributed to the symmetric stretching vibrations of COC and COH. 1615.60 cm⁻¹ -1 The strong absorption peak at 894.14 cm⁻¹ is attributed to the stretching vibration of C=O, indicating the presence of uronic acid. -1 The absorption peak at 856.03 cm⁻¹ indicates the presence of a β-glycosidic bond. -1 The absorption peak at that point indicates the presence of an α-glycosidic bond.

[0094] FT-IR spectra of PHP-3a, PHP-3b and PHP-3c Figure 5 It showed a similar characteristic absorption peak for polysaccharides. 3426.12 cm⁻¹ -1 The broad peak at 2932.84 cm⁻¹ belongs to the -OH stretching vibration. -1 The absorption peak at 1334.33 cm⁻¹ is attributed to the stretching vibration of CH, while the bending vibration peak appears at 1334.33 cm⁻¹. -1 Nearby. 1000-1200cm -1 The presence of three absorption peaks indicates the presence of a pyranose ring. (1743.19 cm⁻¹) -1 The peak at 1615.60 cm⁻¹ indicates the stretching vibrations of C=O and CO in the ester group (-COO-R) of the polysaccharide. -1 The strong absorption peak at 892.54 cm⁻¹ is attributed to the stretching vibration of C=O. -1 The absorption peak at 823.13 cm⁻¹ indicates the presence of a β-glycosidic bond. -1 The absorption peak at that point indicates the presence of an α-glycosidic bond.

[0095] (4) Analysis of glycosidic bond types in non-starch homogeneous polysaccharides of Codonopsis pilosula

[0096] The glycosidic bond types of non-starch homogeneous polysaccharides from *Codonopsis pilosula* were analyzed using gas chromatography-mass spectrometry (GC-MS). The polysaccharide samples underwent complete methylation. Acidic polysaccharides (PHP-2a, PHP-3a, PHP-3b, and PHP-3c) also required carboxyl reduction. After acid hydrolysis, reduction, and acetylation, the fully methylated polysaccharide samples yielded partially methylated adipol acetates (PMAAs). GC-MS analysis was performed according to the parameters shown in Table 2.

[0097] Table 2 GC-MS Condition Parameters

[0098] condition Operating parameters instrument Trace 1300-ISQ QD chromatographic column Agilent DB-5MS (3m×0.25mm, 0.25μm) carrier gas helium Flow rate 1mL / min Injection volume 2μL Program heating Hold at 60℃ for 1 minute, then increase the temperature to 280℃ at a rate of 5℃ / min and hold for 1 minute. Quality Scan Range 40-400Da

[0099] Based on retention time and mass fragmentation, chromatographic peaks were assigned by comparing the CCRC database with literature. The molar percentage of each derivative was calculated by dividing the peak area in the total ion chromatogram by the molecular weight of PMAAs. The molar ratio of mannose, glucose, and galactose obtained from PHP-1 methylation analysis was 2.31:95.92:1.77, which was basically consistent with the molar ratio of mannose, glucose, and galactose obtained from monosaccharide composition analysis (2.37:95.47:2.15). The results showed that PHP-1 mainly contains five sugar residue linkage forms, as shown in Table 3: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, and 1,4,6-Glcp. PHP-1 has a high degree of branching (DB = 47%), indicating that PHP-1 is a heteropolysaccharide with a highly branched structure.

[0100] Table 3. Methylation analysis of major glycosidic bonds in PHP-1

[0101]

[0102]

[0103] Note: NT, NB, and NL are the numbers of terminal residues, branched residues, and linear residues, respectively.

[0104] Methylation analysis revealed that PHP-2a contains 15 types of glycosidic bonds, as shown in Table 4. The content of various monosaccharides analyzed by methylation was similar to that detected by UPLC-MS (Table 5), indicating that the methylation reaction of this polysaccharide was essentially complete. The calculated branching degree (DB) of PHP-2a was 58.66%, classifying it as a highly branched polysaccharide.

[0105] Table 4. Methylation analysis of major glycosidic bonds in PHP-2a

[0106]

[0107]

[0108] Table 5 Comparison of PHP-2a methylation analysis and monosaccharide composition analysis results

[0109] Monosaccharide names methylation Monosaccharide composition Man 2.73 1.79 Rha 10.88 11.32 GlcAc 1.17 1.45 GalAc 2.85 2.60 Glc 3.26 2.45 Gal 30.70 31.99 Ara 48.41 48.40

[0110] The content of various monosaccharides analyzed by methylation was similar to that detected by UPLC-MS (Table 6), indicating that the methylation reaction of these three polysaccharides was basically complete. The methylation analysis results showed that PHP-3a, PHP-3b, and PHP-3c had complex glycosidic bond types, possessing 12 different types of glycosidic bonds (Table 7). All three exhibited high branching, with PHP-3a showing the highest branching degree (66.85%), consistent with the result above indicating that PHP-3a had the highest branching degree based on sugar ratio.

[0111] Table 6 Comparison of methylation analysis and monosaccharide composition analysis results for PHP-3a, PHP-3b, and PHP-3c

[0112]

[0113] Table 7. Methylation analysis of major glycosidic bonds in PHP-3a, PHP-3b, and PHP-3c.

[0114]

[0115] (5) NMR signal attribution of non-starch homogeneous polysaccharides from Codonopsis pilosula

[0116] Accurately weigh 60 mg of non-starch homogeneous polysaccharide from *Codonopsis pilosula*, dissolve it completely in 600 μL of D₂O, and then freeze-dry to replace the active hydrogen. Repeat this process three times to completely replace the active hydrogen. Add 600 μL of D₂O to the dried polysaccharide sample and dissolve it completely. Centrifuge and collect the supernatant. Analyze the activity of the non-starch homogeneous polysaccharide from *Codonopsis pilosula* on a Bruker Advance III 500 MHz NMR spectrometer at 298 K. 1 H NMR, 13 C NMR and two-dimensional spectroscopy (including C NMR and two-dimensional spectroscopy) 1 H- 1 (H COSY, HSQC, HMBC, and NOESY spectra). NMR spectra were analyzed using MestReNova 12.0 software.

[0117] Combining data from monosaccharide composition, glycosidic bond type, 2D NMR spectroscopy, and relevant literature, the C / H chemical shifts of all monosaccharide residues in PHP-1 were obtained (Table 8). 1H and 13 CNMR spectra can identify the α- or β-configurations of isomeric hydrogens (H1) and isomeric carbons (C1) in sugar residues. In most cases, the α-configuration isomer region appears at δ 5.1–5.8 ppm and δ 98–103 ppm (isomeric carbon), while the β-configuration corresponds to δ 4.3–4.8 ppm and δ 103–106 ppm (isomeric carbon). PHP-1 exhibits multiple distinct anomeric hydrogen (H1) signals in the δ 4.5–5.5 ppm anomeric hydrogen region. A large number of hydrogen signals (H2–H6) are concentrated in the δ 3.0–4.5 ppm region, with significant signal overlap, making assignment difficult. 1 Compared to the H NMR spectrum, PHP-1's 13 The C NMR spectrum has fewer spectral lines. The anomeric carbon region in the δ90-110 ppm region has five distinct signal peaks at δ92.46 ppm, δ96.78 ppm, δ98.70 ppm, δ99.75 ppm and δ99.87 ppm, which correspond to the C1 of residues A, B, C, D and E, respectively.

[0118] COSY and HSQC spectra were used to analyze other signals in 1 H and 13 Assignment in 1C NMR. Taking sugar residue A→2)-α-Galp-(1→) as an example. COSY spectroscopy determined the cross peaks to be δ 5.22 / 3.49ppm, δ 3.49 / 3.73ppm, δ 3.73 / 3.19ppm, δ 3.19 / 3.42ppm, and δ 3.42 / 3.70ppm. δ 5.22ppm corresponds to H-1 of residue A, and δ 3.49ppm, δ 3.73ppm, δ 3.19ppm, δ 3.42ppm, and δ 3.70ppm correspond to H-2, H-3, H-4, H-5, and H-6 of residue A, respectively. HSQC spectroscopy determined the cross peaks.

[0119] δ5.22 / 92.46ppm, δ3.49 / 73.38ppm, δ3.73 / 70.45ppm, δ3.19 / 71.83ppm, δ3.42 / 70.41ppm and δ3.70 / 60.52ppm correspond to H-1 / C-1, H-2 / C-2, H-3 / C-3, H-4 / C-4, H-5 / C-5 and H-6 / C-6 of A, respectively.

[0120] HMBC and NOESY spectra were used to determine the glycosyl residues, backbone, and substitution sites of polysaccharides. HMBC spectra can be used to calculate bonds between adjacent sugar residues, reflecting long-range coupling of approximately two or three bond distances between protons and carbons. NOESY spectra simultaneously reflect cross-peaks within and between sugar residues. NOESY spectra reflect the correlations between sugar residues in PHP-1 (Table 9). The cross-peaks of 5.19 / 3.57 ppm reflect a long-range correlation between H-1 of sugar residue E and H-3 of sugar residue D; 5.19 / 5.37 ppm reflect a long-range correlation between H-1 of sugar residue E and H-1 of sugar residue D; and 5.19 / 3.36 ppm reflect a long-range correlation between H-1 of sugar residue E and H-3 of sugar residue C. The sequence between residues C, D, and E was established as: →3,6)-α-Manp-(1→3)-α-Glcp -(1→3)-α-Glcp-(1→α-Glcp). 5.37 / 3.57ppm reflects a long-range correlation between H-1 and H-3 of sugar residue D, 5.37 / 3.83ppm reflects a long-range correlation between H-1 of sugar residue D and H-6 of sugar residue C, 5.37 / 3.49ppm reflects a long-range correlation between H-1 of sugar residue D and H-2 of sugar residue A, and 3.57 / 3.64ppm reflects a long-range correlation between H-1 of sugar residue D and H-2 of sugar residue A. There is a long-range correlation between H-1 and H-4 of sugar residue B; 4.94 / 3.95 ppm reflects a long-range correlation between H-1 of sugar residue C and H-6 of sugar residue B; 4.63 / 3.73 ppm reflects a long-range correlation between H-1 and H-4 of sugar residue B; therefore, 5.22 / 3.57 ppm reflects a long-range correlation between H-1 of sugar residue A and H-3 of sugar residue D. In the HMBC spectrum, δ 5.37 / 75.82 ppm... The cross peaks at m and δ4.94 / 73.53 ppm represent the correlations between H-1 of residue D and C-4 of residue B, and H-1 of residue C and C-6 of residue B, respectively. The cross peak at δ4.63 / 75.82 ppm indicates a correlation between H-1 of residue B and C-4 of residue B. The HMBC spectrum reflects the correlations between sugar residues in PHP-1 (Table 10). Based on this, the structure of PHP-1 was obtained by linking residues A, B, C, D, and E:

[0121]

[0122] The PHP-1 main chain consists of T-α-Glcp-(1→、→3)-α-Glcp-(1→、→3,6)-α-Manp-(1→、

[0123] →4,6)-β-Glcp-(1→、→2)-α-Galp-(1→) are composed of five glycosidic bonds, with the branches originating from...

[0124] →3,6)-α-Manp-(1→ of C-3 and →4,6)-β-Glcp-(1→ of C-3, C-6 extended to Glc.

[0125] Table 8 PHP-1 1 H and 13 Summary of C NMR chemical shifts (unit: ppm)

[0126]

[0127]

[0128] Table 9. Correlation among sugar residues in PHP-1

[0129]

[0130] Table 10. Correlation among sugar residues in PHP-1

[0131] Glycosyl residues Proton Proton correlation A →2)-α-Galp-(1→ H-1(5.22) 77.06 (D; C-3) B →4,6)-β-Glcp-(1→ H-1(4.63) 75.82 (B; C-4) C →3,6)-α-Manp-(1→ H-1(4.94) 73.53(B; C-6) D →3)-α-Glcp-(1→ H-1(5.37) 75.82 (B; C-4), 74.49 (A; C-2) E T-α-Glcp-(1→ H-1(5.19) 77.06(D;C-3),99.75(D;C-1),76.74(C;C-3)

[0132] The precise structural information of PHP-2a was further analyzed using nuclear magnetic resonance spectroscopy. Combining monosaccharide composition, glycosidic bond type, COSY spectrum, and HSQC spectrum, the C / H chemical shifts of all monosaccharide residues were assigned (Table 11). Taking residue AT-Galp-(1→) as an example, the COSY spectrum determined the cross peaks to be δ5.01 / 3.78ppm, δ3.78 / 3.99ppm, δ3.99 / 3.41ppm, δ3.41 / 3.64ppm, and 3.64 / 3.73ppm. δ5.01ppm corresponds to H-1 of residue A, and δ3.78ppm, δ3.99ppm, δ3.41ppm, δ3.64ppm, and δ3.73ppm correspond to... The H-2, H-3, H-4, H-5, and H-6 residues of A were determined by HSQC spectroscopy. The cross peaks were δ5.01 / 99.66ppm, δ3.78 / 66.32ppm, δ3.99 / 69.15ppm, δ3.41 / 71.75ppm, and δ3.73 / 61.17ppm, corresponding to H-1 / C-1, H-2 / C-2, H-3 / C-3, H-4 / C-4, H-5 / C-5, and H-6 / C-6 of A, respectively.

[0133] HMBC and NOESY spectra were used to determine the glycosyl residues, backbone, and substitution sites of polysaccharides. Table 12 summarizes the correlations between glycosyl residues of PHP-2a in the NOESY spectra. The cross peaks of 5.18 / 4.08 ppm reflect a long-range correlation between H-1 of glycosyl residue L and H-2 of glycosyl residue K, and 5.01 / 3.87 ppm reflect a long-range correlation between H-1 of glycosyl residue A and H-5 of glycosyl residue K. The sequences between residues A, K, and L were established as: T-α-Galp(1→5)-α-1,2-Araf-(3→1)-Araf-(2→5). 5.01 / 3.80 ppm reflects a long-range correlation between H-1 of glycosyl residue A and H-2 of glycosyl residue N, 4.42 / 3.87 ppm reflects a long-range correlation between H-1 of glycosyl residue D and H-5 of glycosyl residue K, and 5.12 / 4.10 ppm reflects a long-range correlation between H-1 of glycosyl residue I and H-5 of glycosyl residue N. The presence of long-range correlations between 2 indicates the presence of repeating sugar residue I units within PHP-2a. 5.09 / 4.29 ppm reflects a long-range correlation between H-1 of sugar residue H and H-3 of sugar residue F. 4.70 / 3.75 ppm reflects a long-range correlation between H-1 of sugar residue B and H-2 of sugar residue B, indicating the presence of repeating sugar residue B units within PHP-2a. 3.75 / 5.12 ppm reflects a long-range correlation between H-2 of sugar residue B and H-1 of sugar residue I. 4.70 / 3.75 ppm reflects a long-range correlation between H-1 of sugar residue F and H-2 of sugar residue I. 4.48 / 4.01 ppm reflects a long-range correlation between H-1 of sugar residue C and H-3 of sugar residue B.

[0134] The correlations between sugar residues in PHP-2a in the HMBC spectrum are shown in Table 13. The cross-peaks at δ5.01 / 69.26ppm and δ5.01 / 84.47ppm represent the correlations between H-1 of residue A and C-5 of residue K, and H-1 of residue A and C-2 of residue N, respectively. The cross-peak at δ4.63 / 75.82ppm indicates a correlation between H-1 of residue B and C-4 of residue B. Based on this, the structure of PHP-2a is derived through the connections between sugar residues as follows: Figure 1 .

[0135] The main chain of PHP-2a is composed of T-α-Galp, 1,2,3,5-α-Araf, 1,2-α-Araf and 1,2-β-Galp glycosidic bonds, while the side chains are mostly composed of T-α-Galp, T-β-Rhap and T-α-Araf.

[0136] Table 11 PHP-2a 1 H and 13 Summary of C NMR chemical shifts (unit: ppm)

[0137]

[0138]

[0139] Table 12 Correlation between sugar residues in PHP-2a

[0140]

[0141]

[0142] Table 13 Correlation between sugar residues in PHP-2a

[0143]

[0144] (6) Congo red test of non-starch homogeneous polysaccharide from Codonopsis pilosula

[0145] Congo red solution (50 μM) was first mixed in equal volume with a homogeneous non-starch polysaccharide solution of *Codonopsis pilosula* (0.5 mg / mL). Then, NaOH solution (1 M) was added to produce a series of mixed solutions with final NaOH concentrations ranging from 0.1 to 0.5 M. The UV absorption in the 400-600 nm range was measured, and Cp was plotted. NaOH With the maximum absorption wavelength λ max The relationship diagram. In dilute alkaline solution, the λ of the triple-helicaled gel polysaccharide (curdlan)... max A significant redshift occurs from 495nm to 508nm; with C NaOH As concentration increases, λ max Decrease. The λ of the non-starch homogeneous polysaccharide solution of *Codonopsis pilosula* decreased. max As the alkali concentration increases, a λ-reactance relationship with curdlan occurs. max The consistent trend of change indicates that the non-starch homogeneous polysaccharide of *Codonopsis pilosula* has a triple helix structure. Figure 6 ).

[0146] III. In vitro experiments on non-starch homogeneous polysaccharides from Codonopsis pilosula

[0147] (1) Preparation of culture medium

[0148] Preparation of GAM medium: Weigh 4.9g of GAM medium and dissolve it in 100mL of deionized water that has been boiled to remove oxygen. Autoclave (121℃, 15min). After cooling, add sterile heme chloride (1mL, 5mg / mL) and vitamin K1 (1mL, 0.1%). Preparation of MM medium: Accurately weigh 1.360 g KH2PO4, 87.75 g NaCl, 925.11 ng MgCl2, 112.319 mg (NH4)2SO4, 48.464 g L-cysteine, 3.103 g L-histidine, and 38.927 ng FeSO4·7H2O into a 150 mL Erlenmeyer flask. Dissolve the contents in 100 mL of ultrapure water, adjust the pH to 7.4, add 100 μL of resazurin indicator, autoclave (121 °C, 15 min), cool, and then add sterile heme chloride (100 μL, 5 mg / mL), vitamin K3 (100 μL, 1 mg / mL), and vitamin B12 (5 μL, 0.1 mg / mL). Mix well and set aside.

[0149] (2) Effects of different concentrations of non-starch homogeneous polysaccharide from Codonopsis pilosula on the in vitro activity of BO, BT, BL and LGG bacteria.

[0150] BO, BT, BL, and LGG bacteria were cultured anaerobicly in vitro on GAM medium at 37°C for 24 h. Obvious bacterial precipitate was observed, and the supernatant was discarded after centrifugation (3000 rpm, 5 min). The bacterial precipitate was washed with MM medium, and the bacterial concentration was diluted to an OD600 value of 0.1-0.2. PHP-1, PHP-2a, PHP-3a, PHP-3b, and PHP-3c were prepared at concentrations of 2.5 mg / mL, 5 mg / mL, and 10 mg / mL, respectively, as the sole carbon source for the culture medium. Sterile water (Blank) served as a blank control. 100 μL of diluted BO and BT bacterial suspensions and 100 μL of different concentrations of *Codonopsis pilosula* non-starch homogeneous polysaccharide solution or Blank solution were added to each 96-well plate. Five replicates were used for each sample. The plates were anaerobically cultured at 37°C for 72 h, and the bacterial concentration was observed at different time points using a microplate reader to plot growth curves.

[0151] (3) Experimental Results

[0152] Growth curves of *Codonopsis pilosula* non-starch homogeneous polysaccharides PHP-1, PHP-2a, PHP-3a, PHP-3b, and PHP-3c as the sole carbon source for *BO*, *BT*, *BL*, and *LGG* bacteria were observed. It was found that different concentrations of these polysaccharides promoted the growth of the strains. The best growth-promoting effect was observed at a concentration of 10 mg / mL on *BO*, *BT*, *BL*, and *LGG* bacteria. Figure 7Among them, PHP-1 has a better growth-promoting effect on BL and LGG bacteria than other polysaccharides; PHP-3b has a better growth-promoting effect on BO bacteria than other polysaccharides.

[0153] IV. Fermentation Characteristics Test of Non-Starch Homogeneous Polysaccharides from Codonopsis pilosula

[0154] (1) Preparation of human feces

[0155] Fresh stool samples were collected from three healthy volunteers (aged 20-25, no antibiotic or probiotic treatment for at least three months, and normal body mass index). Volunteers, wearing masks and gloves, used sterile samplers to collect a specific weight of fresh stool into sterile stool collection containers, which were immediately sealed and placed in anaerobic culture bags. Equal amounts of stool from each volunteer were mixed on a laminar flow hood. Empty tubes were weighed beforehand, and stool was added to the tubes, weighed again, and then diluted to 32% (w / v). For example, 16g of the mixed stool was placed in a sterile 50mL centrifuge tube and immediately diluted with 50mL of sterile phosphate buffer (0.1M, pH 7.0-7.2), vortexed for 1 min to obtain a stool suspension (32%, w / v). The stool suspension was centrifuged (265×g, room temperature, 5 min) to remove food residue, and the supernatant was used as a stool slurry for further experiments. A sterile low-nutrient fermentation medium was prepared for use. The in vitro fermentation experiment consisted of three groups: a blank control group (BLANK) containing no other carbon source, a positive control group (INL) containing inulin (100.0 mg), and an experimental group containing non-starch homogeneous polysaccharide of Codonopsis pilosula (100.0 mg). Each group was tested in three parallel experiments.

[0156] The non-starch homogeneous polysaccharide of *Codonopsis pilosula* used in the fermentation characteristic test was the non-starch homogeneous crude polysaccharide (cPHP) of *Codonopsis pilosula* from step (3) of Example 1. Prepare an appropriate amount of sterile tubes. Add 2.5 mL of fecal slurry (32%, w / v) to 10.0 mL of low-nutrient fermentation medium for the BLANK group, INL group, and experimental group, respectively. Cover the top of the liquid with a 1 cm thick layer of liquid paraffin, seal the tube openings with silicone stoppers, and then place all the tubes into an anaerobic gas-generating bag. Add the anaerobic gas-generating bag and anaerobic indicator, and start in vitro simulated colonic fermentation in a 37℃ constant temperature incubator. The entire fermentation process was carried out at 37℃, and 2.5 mL samples were taken at 0, 6, 12, and 24 h of fermentation. The fermentation product was flash-frozen and stored at -80℃, and fermentation was stopped at 24 h.

[0157] (2) Detection of physicochemical properties of fermentation products

[0158] The pH of the fermentation products at different culture time points was measured using a pH meter. The reducing sugar content of the fermentation products at different culture time points was determined using the DNS method. The molecular weight changes of the fermentation products at different culture time points were determined using HPLC-ELSD. The monosaccharide composition of the fermentation products at different culture time points was determined using PMP derivatization.

[0159] (3) The short-chain fatty acid and lactic acid contents of the supernatant of fermentation products at different culture time points were determined by pre-column derivatization UPLC / Q-TOF-MS:

[0160] Preparation and derivatization of the mixed reference solution: 11.42 μL of acetic acid, 14.82 μL of propionic acid, 17.62 μL of n-butyric acid, 22.00 μL of valeric acid, 22.00 μL of isovaleric acid, and 11.68 μL of lactic acid were diluted to 20 mL with 50% acetonitrile-water to prepare a 10 mM mixed reference solution as the stock solution. The stock solution was then diluted in half to create a series of gradients. 100 μL of each concentration of the mixed standard was added to 50 μL of 200 mM 3-NPH-50% acetonitrile-water and 50 μL of 120 mM EDC-50% acetonitrile-water (containing 6% pyridine). After vortexing, the mixture was derivatized at 37 °C for 30 min, followed by rapid cooling in an ice-water bath for 1 min. The solution was then diluted to 1 mL with 10% acetonitrile, vortexed, and centrifuged at 12000 rpm for 10 min. The supernatant was collected and stored at -20 °C. Before injection analysis, 400 μL of the internal standard derivative and 600 μL of the mixed standard derivative were mixed to obtain the mixed reference test solution.

[0161] Fermentation product derivatization: 100 μL of the supernatant from the fermentation products at different culture time points after centrifugation at 4℃ was added to 100 μL of acetonitrile and mixed well to obtain the sample solution. 100 μL of the sample solution was then added to 50 μL of 200 mM 3-NPH-50% acetonitrile / water and 50 μL of 120 mM EDC-50% acetonitrile / water (containing 6% pyridine). The derivatization reaction was carried out at 37℃ for 30 min, followed by rapid cooling in an ice-water bath for 1 min. The solution was diluted to 1 mL with 10% acetonitrile, vortexed, and centrifuged at 12000 rpm for 10 min. The supernatant was then stored at -20℃. Before sample analysis, 400 μL of the internal standard derivative and 600 μL of the fermentation product derivative were mixed to obtain the test solution.

[0162] UPLC / Q-TOF-MS analysis: A BEH C18 column (50×2.1 mm, 1.7 μm) was used, with a column temperature of 40℃ and an autosampler temperature of 4℃. The injection volume was 2 μL, and the flow rate was 0.35 mL / min. Gradient elution was performed using a mobile phase of 0.1% formic acid water (A) - acetonitrile containing 0.1% formic acid (B): 0-2 min, 10%-15% B; 2-5 min, 15%-30% B; 5-6 min, 30%-45% B; 6-7 min, 45%-55% B; 7-8 min, 55%-100% B; 8-9 min, 100% B. ESI ion source was used, with negative ion mode, and the mass acquisition range was 100-600 Da (m / z).

[0163] (4) Experimental Results

[0164] The pH changes during the 24-hour fermentation of non-starch homogeneous polysaccharides from *Codonopsis pilosula* showed that, 6 hours after the start of fermentation, the pH of the non-starch homogeneous polysaccharide group decreased significantly, from 6.86±0.15 to 4.85±0.24. Figure 8 (A). At the start of fermentation, the neutral sugar content of the non-starch homogeneous polysaccharide group of *Codonopsis pilosula* was 3.24 ± 0.24 mg / mL, and at the end it was 0.35 ± 0.03 mg / mL. Figure 8 (C). Within the first 12 hours of fermentation, the sugar consumption rate reached 85.47%, and the sugar content decreased slowly in the later stages of fermentation, with the final consumption rate reaching 90.72%. Figure 8 (D). The non-starch homogeneous polysaccharide of the present invention can be efficiently utilized by intestinal flora.

[0165] The results of the molecular weight change show ( Figure 8 The peak shape of the non-starch homogeneous polysaccharide of Codonopsis pilosula (E) continued to shift towards the peak shape of the fermentation medium. At 12h and 24h of fermentation, the peaks of the non-starch homogeneous polysaccharide group of Codonopsis pilosula were basically the same, indicating that the non-starch homogeneous polysaccharide of Codonopsis pilosula could be largely degraded by intestinal microorganisms within the first 12h of fermentation. This result is consistent with the results of carbohydrate consumption rate.

[0166] Short-chain fatty acids (SCFAs) are the main products of intestinal microorganisms fermenting carbohydrates that are not easily digested. As fermentation proceeds, the types and contents of SCFAs in the non-starch homogeneous polysaccharide group of Codonopsis pilosula increase, and the short-chain fatty acids obtained are mainly acetic acid, propionic acid and butyric acid.

[0167] At 12 h of fermentation, the total SCFAs in the *Codonopsis pilosula* non-starch homogeneous polysaccharide group reached its highest level (38.08±0.52 mM), significantly higher than the blank control group (24.06±0.39 mM), and even higher than the total SCFAs produced by the INL group (36.23±0.32 mM). Specifically, the butyric acid content produced by the fermentation of *Codonopsis pilosula* non-starch homogeneous polysaccharide (7.38±1.21 mM) was significantly higher than that of the INL group (5.53±0.55 mM). At 24 h of fermentation, the total SCFAs content in the *Codonopsis pilosula* non-starch homogeneous polysaccharide group decreased (34.36±0.32 mM), a change corresponding to the increase in pH value during the later stages of fermentation.

[0168] The concentration of SCFAs is considered an important indicator of microbial activity, suggesting that the non-starch homogeneous polysaccharide of *Codonopsis pilosula* stimulates the production of SCFAs in the intestinal flora. SCFAs can lower the pH of the large intestine, thereby inhibiting the growth of harmful pathogens, affecting the activity of microbial enzymes, and preventing colorectal cancer. Butyric acid, a component of SCFAs, is a major nutrient for intestinal epithelial cells, primarily produced and absorbed in the colon, maintaining colonic cell stability and regulating intestinal flora imbalance. It can be used for the prevention and treatment of irritable bowel syndrome, colitis, acute and chronic diarrhea, and colorectal cancer. Therefore, the non-starch homogeneous polysaccharide of *Codonopsis pilosula* has good preservative and application value in regulating intestinal microbiota and maintaining intestinal health.

[0169] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A non-starch homogeneous polysaccharide of Radix Pseudoginseng, characterized in that, The monosaccharide composition comprises glucose, galactose and mannose in a molar ratio of 95.47:2.15:2.37; The non-starch uniform polysaccharide of the Radix Pseudoginseng has a molecular weight of 2124 Da; The non-starch uniform polysaccharide of the Radix Pseudoginseng has a triple helix structure; The repeatable primary structure unit of the non-starch uniform polysaccharide of the Radix Pseudoginseng comprises the following sugar residues: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, 1,4,6-Glcp; The structural formula of the non-starch uniform polysaccharide of the Radix Pseudoginseng is: ； The preparation method of the non-starch uniform polysaccharide of the Radix Pseudoginseng comprises the following steps: (1) extracting the Radix Pseudoginseng in ethanol, filtering to obtain a precipitate, drying the precipitate to obtain Radix Pseudoginseng dregs; (2) taking the Radix Pseudoginseng dregs obtained in step (1), adding water to extract to obtain a Radix Pseudoginseng water extract; (3) removing starch from the Radix Pseudoginseng water extract obtained in step (2), centrifuging and concentrating, taking the supernatant, adding ethanol for alcohol precipitation, centrifuging and collecting the precipitate to obtain crude polysaccharide of Radix Pseudoginseng; (4) separating and purifying the crude polysaccharide of Radix Pseudoginseng obtained in step (3), eluting with deionized water, collecting the eluate, and concentrating to obtain the non-starch uniform polysaccharide of Radix Pseudoginseng.

2. The method for preparing the non-starch homogeneous polysaccharide of Radix Pseudoginseng according to claim 1, characterized in that, comprises the following steps: (1) extracting the Radix Pseudoginseng in ethanol, filtering to obtain a precipitate, drying the precipitate to obtain Radix Pseudoginseng dregs; (2) taking the Radix Pseudoginseng dregs obtained in step (1), adding water to extract to obtain a Radix Pseudoginseng water extract; (3) removing starch from the Radix Pseudoginseng water extract obtained in step (2), centrifuging and concentrating, taking the supernatant, adding ethanol for alcohol precipitation, centrifuging and collecting the precipitate to obtain crude polysaccharide of Radix Pseudoginseng; (4) separating and purifying the crude polysaccharide of Radix Pseudoginseng obtained in step (3), eluting with deionized water, collecting the eluate, and concentrating to obtain the non-starch uniform polysaccharide of Radix Pseudoginseng.

3. The preparation method according to claim 2, characterized in that, In step (3), the starch removal is performed by adding amylase to an enzyme activity of 1-2 U / mL, enzyme hydrolysis at a temperature of 80-90℃ for 0.5-2 h.

4. Use of the non-starch uniform polysaccharide of the Radix Pseudoginseng of claim 1 or prepared by the preparation method of any one of claims 2-3 in the preparation of an intestinal probiotic food; The intestinal probiotic is BL bacteria and / or LGG bacteria.

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