Application of acid polysaccharide from tribonema to preparation of functional food or feed containing prebiotics

Abstract

The invention discloses application of acid polysaccharide from tribonema to preparation of functional food or feed containing prebiotics, and the acid polysaccharide is prepared by the following method: (1) adding dried tribonema powder into water, stirring and extracting, and collecting supernate; (2) adding the residues into water, stirring and extracting, collecting supernate, merging the supernate, concentrating, deproteinizing, precipitating and collecting precipitate; dissolving the precipitate in water, dialyzing, and freeze-drying; (3) dissolving the tribonema crude polysaccharide in water, and adding absolute ethyl alcohol for first-stage precipitation; adding absolute ethyl alcohol into supernate obtained after the first-stage precipitation to carry out second-stage precipitation; adding absolute ethyl alcohol into supernate obtained after the second-stage precipitation to carry out third-stage precipitation; (4) testing the total sugar content of each stage of precipitate; and (5) dissolving the precipitate with the highest total sugar content in water, and carrying out ultrafiltration treatment by adopting a polyether sulfone membrane to obtain the tribonema-derived acidic polysaccharide. The polysaccharide disclosed by the invention has prebiotic activity.

Description

Technical Field

[0001] This invention belongs to the field of prebiotic technology, and more specifically, relates to the application of an acidic polysaccharide derived from *Hylocereus undatus* in the preparation of functional foods or feeds containing prebiotics. Background Technology

[0002] With the continuous advancement of the "Healthy China" strategy and the "dual carbon" goals, the development of green and sustainable food resources has become an important direction for the functional food industry. In recent years, maintaining the balance of the gut microbiota has been widely recognized as closely related to human health and plays a crucial role in the prevention of various diseases. Prebiotics, as functional food ingredients that regulate gut microbiota, generally refer to a class of carbohydrates that can resist gastrointestinal digestion and can be selectively utilized by specific beneficial microorganisms in the colon. Their main functions include promoting the proliferation of beneficial bacteria, inhibiting the growth of harmful bacteria, and inducing the production of short-chain fatty acids (SCFAs), playing a key physiological role in immune regulation and metabolic improvement.

[0003] Currently, most commonly used prebiotics are derived from edible plants, such as inulin and fructooligosaccharides. However, these traditional raw materials are limited by factors such as land use, planting season, and climate conditions, making it difficult to meet the growing demand for diversified, efficient, and sustainable prebiotic products.

[0004] Microalgae, with their advantages of high photosynthetic efficiency, rapid growth, and independence from arable land resources, are increasingly being regarded as a potential new source of prebiotics. Most microalgae are rich in carbohydrates, including soluble polysaccharides and cellulose. Furthermore, algal polysaccharides typically possess various biological activities such as antioxidant, immunomodulatory, and antitumor effects, and their structures (e.g., sulfation modifications) differ significantly from those of terrestrial plant polysaccharides, endowing them with stronger functionality and prebiotic application potential. Therefore, in recent years, research based on microalgae extracts has become a research hotspot in the prebiotic field. Summary of the Invention

[0005] The purpose of this invention is to provide an application of acidic polysaccharides derived from *Phyllostachys edulis* in the preparation of functional foods or feeds containing prebiotics. The acidic polysaccharides derived from *Phyllostachys edulis* of this invention exhibit good intestinal regulatory function and have prebiotic activity.

[0006] To achieve the above objectives, the present invention provides an application of an acidic polysaccharide derived from *Hylocereus undatus* in the preparation of functional foods or feeds containing prebiotics. This acidic polysaccharide is prepared by a method comprising the following steps:

[0007] (1) Add dried yellow algae powder to water, then stir to extract, centrifuge and collect the supernatant;

[0008] (2) Add the residue after centrifugation in step (1) to water, then stir to extract, centrifuge to collect the supernatant, combine the supernatant collected by centrifugation with the supernatant obtained by centrifugation in step (1), then concentrate, remove protein, precipitate with alcohol and centrifuge to collect the precipitate; finally, dissolve the centrifuged precipitate in water, dialyze and freeze dry to obtain crude polysaccharide of *Hygrophora spp.*

[0009] (3) Dissolve the crude polysaccharide of *Hygrophytes macrantha* in water, add anhydrous ethanol for the first-stage precipitation, centrifuge to collect the first-stage precipitate, and freeze-dry it; then add anhydrous ethanol to the supernatant obtained after centrifugation following the first-stage precipitation for the second-stage precipitation, centrifuge to collect the second-stage precipitate, and freeze-dry it; finally add anhydrous ethanol to the supernatant obtained after centrifugation following the second-stage precipitation for the third-stage precipitation, centrifuge to collect the third-stage precipitate, and freeze-dry it.

[0010] (4) Test the total sugar content of each grade of precipitate obtained after freeze-drying in step (3);

[0011] (5) Dissolve the precipitate with the highest total sugar content after freeze-drying in water, and then perform ultrafiltration using a polyethersulfone (PES) membrane. Freeze-dry the collected retentate to obtain the acidic polysaccharide derived from *Phyllostachys edulis*. The molecular weight cutoff (MWCO) of the polyethersulfone membrane is in the range of 0.5-3 kDa. The conditions for ultrafiltration include: pressure of 0.1-0.3 MPa and stirring speed of 250-350 rpm.

[0012] According to the present invention, preferably, in step (1), the algal species of Tribonema used to prepare the Tribonema powder is Tribonema sp. GXU A10.

[0013] According to the present invention, preferably, in step (1), the ratio of the dried yellow algae powder to water is 1:(15-30)g / mL;

[0014] The stirring extraction was carried out in a water bath at 50-70℃;

[0015] The centrifugation conditions are: rotation speed of 3500-4500 rpm, time of 10-20 min, and temperature of 3-5℃.

[0016] According to the present invention, preferably, in step (2), the ratio of the residue after centrifugation in step (1) to water is 1:(10-20)g / mL;

[0017] The stirring extraction was carried out in a water bath at 50-70℃;

[0018] The centrifugation conditions for the supernatant collected by centrifugation are: rotation speed of 3500-4500 rpm, time of 10-20 min, and temperature of 3-5℃.

[0019] Concentrate to 5%-15% of the original volume.

[0020] According to the present invention, preferably, in step (2), the protein is deproteinized using Sevag reagent;

[0021] Alcohol precipitation was performed using anhydrous ethanol; the precipitation temperature was 3-5℃ and the time was 8-16h.

[0022] The centrifugation conditions for collecting the precipitate were: 7500-8500 rpm, 10-20 min, and 3-5℃.

[0023] The dialysis time is 48-75 hours.

[0024] According to the present invention, preferably, in step (3):

[0025] During the first-stage precipitation, the temperature is 3-5℃ and the time is 8-16h; the ratio of crude polysaccharide to water is 1:(50-150)g / mL; the centrifugation conditions are: 7500-8500rpm, 10-20min, and 3-5℃; the volume percentage of anhydrous ethanol is 35-45% based on the total volume of crude polysaccharide, water, and anhydrous ethanol.

[0026] When performing the second-stage precipitation, the volume percentage of anhydrous ethanol is 55-65% based on the total volume of the supernatant obtained by centrifugation after the first-stage precipitation and anhydrous ethanol; the temperature of the second-stage precipitation is 3-5℃, and the time is 8-16h; the centrifugation conditions are: speed of 7500-8500rpm, time of 10-20min, and temperature of 3-5℃.

[0027] When performing the third-stage precipitation, the volume percentage of anhydrous ethanol is 75-85% based on the total volume of the supernatant obtained by centrifugation after the second-stage precipitation and anhydrous ethanol; the temperature of the third-stage precipitation is 3-5℃, and the time is 8-16h; the centrifugation conditions are: rotation speed of 7500-8500rpm, time of 10-20min, and temperature of 3-5℃.

[0028] According to the present invention, preferably, in step (4), the freeze-dried precipitates obtained in step (3) are dissolved in water to prepare polysaccharide solutions corresponding to each precipitate, and then the total sugar content of the polysaccharide solutions corresponding to each precipitate is obtained by the phenol-sulfuric acid method; wherein, the concentration of the polysaccharide solutions corresponding to each precipitate is 0.5-1.5 mg / mL; the method of obtaining the total sugar content of the polysaccharide solutions corresponding to each precipitate by the phenol-sulfuric acid method specifically includes the following steps:

[0029] First, using D-glucose as a standard, the absorbance (OD) values ​​of D-glucose aqueous solutions with different concentration gradients in the range of 0-100 mg / mL were tested at 490 nm. 490 This allows us to obtain the standard curve y = 9.965x + 0.015(R), and thus the standard curve y = 9.965x + 0.015(R). 2 =0.999); where y represents the absorbance value at 490nm and x represents the D-glucose concentration; then, the absorbance value of the polysaccharide solution corresponding to each grade of precipitate at 490nm is tested, and the total sugar content of each grade of precipitate after freeze-drying obtained in step (3) is calculated based on the standard curve.

[0030] According to the present invention, preferably, in step (5), the precipitate with the highest total sugar content after freeze-drying is dissolved in water, and the concentration of the resulting polysaccharide solution is 1-3 mg / mL.

[0031] The technical solution of the present invention has the following beneficial effects:

[0032] Possessing excellent prebiotic properties and broad application potential: The acidic polysaccharide derived from *Phyllostachys edulis* in this invention exhibits good resistance to digestion during in vitro simulated digestion. In an in vitro fecal fermentation model, it can significantly promote the proliferation of probiotics such as *Bifidobacterium* and *Parabacteroides*, while maintaining a low pH environment and enhancing the production of SCFAs (such as acetic acid, propionic acid, and butyric acid), demonstrating good prebiotic activity.

[0033] The purification process of this invention is simple and efficient, which is conducive to industrialization: This invention establishes a rapid purification method for polysaccharides from *Hylocereus flavomarginata* that does not rely on column chromatography. It adopts ethanol fractionation precipitation combined with membrane separation technology, which significantly simplifies the operation process and shortens the purification time. The resulting acidic polysaccharides from *Hylocereus flavomarginata* have a single symmetrical molecular weight distribution peak and high purity, which is convenient for large-scale preparation and application.

[0034] With a novel structure and significant potential for biological functions, the acidic polysaccharide derived from *Phyllostachys edulis* is a novel acidic polysaccharide rich in glucuronic acid and sulfate groups and possessing β-glycosidic bonds. Its structural characteristics differ from those of previously reported *Phyllostachys edulis* polysaccharides, endowing it with unique functional properties and providing a new direction for the high-value utilization of algal resources.

[0035] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0036] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the invention.

[0037] Figure 1 The ultraviolet-visible absorption spectra of the polysaccharides from *Hygrophytes flavomarginata* prepared by fractionation with different ethanol concentrations according to Preparation Example 1 of the present invention are shown.

[0038] Figure 2 The graph shows the effect of different membrane pore sizes on the polysaccharide recovery rate and content during the TP80 ultrafiltration process of polysaccharide precipitation from *Phyllostachys edulis* according to the preparation examples and comparative preparation examples of the present invention.

[0039] Figure 3A , Figure 3B , Figure 3C The molecular weight diagram, monosaccharide composition diagram and infrared spectrum of the purified polysaccharide TP80-M from *Hygrophytes macrantha* according to Preparation Example 1 of the present invention are shown respectively.

[0040] Figure 4A The 1H NMR spectrum of the purified polysaccharide TP80-M from *Hygrophytes lucida* according to Preparation Example 1 of the present invention is shown.

[0041] Figure 4B A structural hypothesis of the purified polysaccharide TP80-M from *Hygrophytes lucida* according to Preparation Example 1 of the present invention is shown.

[0042] Figure 5A The diagram shows the reduction sugar release of the purified polysaccharide TP80-M from *Pterocarya stenoptera* prepared according to Example 1 of the present invention during simulated digestion in the oral cavity, stomach, and small intestine.

[0043] Figure 5B The graph shows the molecular weight (B) changes of the purified polysaccharide TP80-M from *Hygrophytes lucida* prepared according to Example 1 of the present invention during simulated digestion in the oral cavity, stomach, and small intestine.

[0044] Figure 6 The diagram shows the promoting effects of the purified polysaccharide TP80-M from *Pterocarya stenoptera* prepared according to Example 1 of the present invention on in vitro fermentation of intestinal flora growth (A), polysaccharide degradation (B), culture medium pH (C), and short-chain fatty acid (SAFC) generation (D).

[0045] Figures 7A-7BThe diagram shows the effect of treatment with the purified polysaccharide TP80-M from *Hylocereus undatus* according to Preparation Example 1 of the present invention on the composition of the human gut microbiota after 48 hours of in vitro fermentation. Among them, Figure 7A A diagram illustrating the influence of phylum-level microbial composition; Figure 7B The diagram shows the influence of microbial composition at the genus level; CK represents the control group; INL represents inulin, which is the positive control group. Detailed Implementation

[0046] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0047] The invention is further illustrated below through test examples:

[0048] The *Tribonema* species used in the following preparation examples and comparative preparation examples was *Tribonema* sp. GXU-A10, which has been disclosed in patent application (publication number: CN116218679A) and was donated by Guangxi University, the applicant of CN116218679A. The dried *Tribonema* powder was obtained by drying in a constant temperature oven at 50°C, and its protein, lipid, and carbohydrate contents were 12.68%, 22.68%, and 53.20% (on dry weight), respectively.

[0049] Preparation Example 1

[0050] This preparation example provides a method for preparing acidic polysaccharides derived from *Hygrophytes maculatus*, as detailed below:

[0051] (1) Extraction and deproteinization of crude polysaccharides: Weigh 100g of dried *Hylocereus flavomarginata* powder, add 2L of deionized water (solid-liquid ratio 1:20, w / v = g / mL), and extract in a 70℃ water bath with stirring for 3h. The extract was centrifuged (4000rpm, 4℃, 15min) and the supernatant was collected. The residue was extracted once more (specifically, the centrifuged residue was added to deionized water at a solid-liquid ratio of 1:10 (w / v = g / mL), and extracted in a 70℃ water bath with stirring for 3h; the extract was centrifuged (4000rpm, 4℃, 15min) and the supernatant was collected). The supernatants collected from the two centrifugations were combined and concentrated to 10% of the original volume by rotary evaporation at 50℃.

[0052] The concentrated solution was mixed with Sevag reagent (chloroform:n-butanol = 4:1, v / v) at a volume ratio of 1:3, stirred at room temperature for 20 min, and allowed to stand for separation. The supernatant was then collected. This process was repeated until no flocculent material was found in the intermediate layer. Four volumes of anhydrous ethanol were added to the deproteinized solution, and the mixture was allowed to stand at 4 °C for 12 h to precipitate. The precipitate was collected by centrifugation (8000 rpm, 4 °C, 15 min), dissolved in pure water, dialyzed for 72 h, and freeze-dried to obtain crude polysaccharide from *Hylocereus undatus*, with a yield of 10.30 g / 100 g dry algae powder.

[0053] 2) Ethanol fractionation and precipitation

[0054] First-stage precipitation: Dissolve 1g of crude polysaccharide in 100mL of deionized water (solid-to-liquid ratio 1:100, w / v = g / mL), add anhydrous ethanol to adjust to anhydrous ethanol volume percentage of 40% (based on the total volume of crude polysaccharide, water and anhydrous ethanol), let it stand at 4℃ for 12h to precipitate, centrifuge (8000rpm, 4℃, 15min) to collect the precipitate and freeze-dry it. The freeze-dried precipitate is named TP40.

[0055] Second-stage precipitation: Anhydrous ethanol was added to the supernatant obtained by centrifugation after the first-stage precipitation to adjust the anhydrous ethanol volume percentage to 60% (based on the total volume of the supernatant obtained by centrifugation after the first-stage precipitation and anhydrous ethanol). The mixture was allowed to stand at 4°C for 12 hours to precipitate, then centrifuged (8000 rpm, 4°C, 15 min) to collect the precipitate and freeze-dried. The freeze-dried precipitate was named TP60.

[0056] Third-stage precipitation: Anhydrous ethanol was added to the supernatant obtained after centrifugation following the second-stage precipitation to adjust the anhydrous ethanol volume percentage to 80% (based on the total volume of the supernatant obtained after centrifugation following the second-stage precipitation and anhydrous ethanol). The mixture was allowed to stand at 4°C for 12 hours to precipitate, then centrifuged (8000 rpm, 4°C, 15 min) to collect the precipitate and freeze-dried. The freeze-dried precipitate was named TP80.

[0057] Subsequently, TP40, TP60, and TP80 were dissolved in deionized water to prepare TP40 polysaccharide solutions, TP60 polysaccharide solutions, and TP80 polysaccharide solutions, each with a concentration of 1 mg / mL. Then, using D-glucose as a standard, the absorbance (OD) values ​​of D-glucose aqueous solutions at different concentration gradients within the range of 0-100 mg / mL were determined using the phenol-sulfuric acid method at 490 nm. 490 The standard curve y = 9.965x + 0.015(R) was obtained. 2=0.999), where y represents the absorbance value at 490 nm and x represents the D-glucose concentration in the standard curve; finally, the absorbance values ​​of 1 mg / mL TP40 polysaccharide solution, TP60 polysaccharide solution and TP80 polysaccharide solution at 490 nm were tested using the phenol-sulfuric acid method, and the total sugar content of TP40, TP60 and TP80 was calculated based on the standard curve.

[0058] The results are as follows: the total sugar contents of TP40, TP60, and TP80 were 39.20% (wt%), 39.93% (wt%), and 57.22% (wt%), respectively. Among them, the TP80 fraction had the highest total sugar content. Simultaneously, it exhibited the lowest UV-Vis absorption peak at 255 nm (see...). Figure 1 The presence of this number indicates that the content of protein impurities is relatively low.

[0059] In summary, TP80, with its high polysaccharide content and low protein impurity level, was selected as the target component for subsequent membrane separation and purification.

[0060] 3) Membrane separation and purification

[0061] The TP80 component was dissolved in deionized water to prepare a polysaccharide solution with a concentration of 2 mg / mL (volume 20 mL). Ultrafiltration was performed using a polyethersulfone (PES) membrane with a molecular weight cutoff (MWCO) of 1 kDa at a pressure of 0.2 ± 0.02 MPa and a stirring speed of 300 rpm. The retentate was freeze-dried to obtain an acidic polysaccharide derived from *Phyllostachys edulis*, which was named TP80-M.

[0062] Comparative Preparation Example 1

[0063] The only difference between this comparative preparation example and preparation example 1 is step 3), as follows:

[0064] Membrane separation and purification: The TP80 component was dissolved in deionized water to prepare a polysaccharide solution with a concentration of 2 mg / mL (volume 20 mL). Ultrafiltration was performed using a polyether sulfone (PES) membrane with a pore size of 5 kDa at a pressure of 0.2 ± 0.02 MPa and a stirring speed of 300 rpm. The retentate was freeze-dried to obtain the polysaccharide sample.

[0065] Comparative Preparation Example 2

[0066] The only difference between this comparative preparation example and preparation example 1 is step 3), as follows:

[0067] Membrane separation and purification: The TP80 component was dissolved in deionized water to prepare a polysaccharide solution with a concentration of 2 mg / mL (volume 20 mL). Ultrafiltration was performed using a polyether sulfone (PES) membrane with a pore size of 10 kDa at a pressure of 0.2 ± 0.02 MPa and a stirring speed of 300 rpm. The retentate was freeze-dried to obtain the polysaccharide sample.

[0068] Test Example 1

[0069] The total sugar content of the polysaccharides prepared in Preparation Example 1 and Comparative Preparation Examples 1-2 is tested; details are as follows:

[0070] TP80-M, the polysaccharide sample of Comparative Preparation Example 1, and the polysaccharide sample of Comparative Preparation Example 2 were dissolved in deionized water to prepare polysaccharide solutions with a concentration of 1 mg / mL. Then, using D-glucose as a standard, the absorbance (OD) of D-glucose aqueous solutions with different concentration gradients in the range of 0-100 mg / mL was determined at 490 nm using the phenol-sulfuric acid method. 490 The standard curve y = 9.965x + 0.015(R) was obtained. 2 =0.999), where y represents the absorbance value at 490 nm and x represents the D-glucose concentration in the standard curve; finally, the absorbance value of the above 1 mg / mL polysaccharide solution at 490 nm was tested by the phenol-sulfuric acid method, and the total sugar content of each polysaccharide sample was calculated based on the standard curve.

[0071] like Figure 2 As shown, when using a 10kDa membrane, due to the larger pore size, some target polysaccharides may permeate into the filtrate, leading to a decrease in recovery rate (13.47%, g / 100g TP80) and no significant improvement in purity (13.54%, wt%). Using a 5kDa membrane significantly improved polysaccharide purity (67.30%, wt%), but because its molecular weight is close to the membrane pore size, there is still a certain risk of permeation under pressure, resulting in unsatisfactory recovery efficiency (20.72%, g / 100g TP80). In summary, using a 1kDa membrane ensured a high recovery rate (48.22%, g / 100g TP80) while significantly improving polysaccharide purity. The total sugar content of the obtained TP80-M sample reached 73.68% (wt%), which represents the optimal purification conditions determined in this study.

[0072] Test Example 2

[0073] The chemical composition of the acidic polysaccharide (TP80-M) derived from *Phyllostachys edulis* prepared in Preparation Example 1 was analyzed, and the results are as follows:

[0074] 1) The content of uronic acid was determined using the sulfuric acid-carbazole method, with glucuronic acid as the standard. 1 mL of 0.2-1 mg / mL glucuronic acid aqueous solution was added to 2 mL of concentrated sulfuric acid. The mixture was heated in an 85℃ water bath for 20 min, cooled to room temperature, and then 2 mL of carbazole solution (0.1% by mass, using 95% ethanol as solvent) was added. After mixing, the absorbance was measured at 530 nm to establish a standard curve: y = 1.305x + 0.075(R²). 2 =0.992), where y represents the absorbance value at 530 nm wavelength and x represents the uronic acid concentration in the standard curve. Following the same procedure, replace the glucuronic acid aqueous solution with a 1 mg / mL TP80-M aqueous solution and measure the OD. 530 Based on the absorbance, the uronic acid content in TP80-M can be calculated to be 34.48% according to the standard curve.

[0075] 2) The sulfate content was determined using the barium sulfate precipitation method. Anhydrous potassium sulfate was used as the standard. A 0.6 mg / mL potassium sulfate aqueous solution was prepared. 0.1–0.5 mL of the standard solution was taken and diluted to 0.5 mL with 1 mol / L hydrochloric acid aqueous solution. Then, 7.5 mL of trichloroacetic acid aqueous solution (3% m / v) and 2 mL of barium chloride-gelatin aqueous solution (1% barium chloride and 0.5% gelatin) were added sequentially. The mixture was stirred and reacted for 15 min. The absorbance was measured at 360 nm, and a standard curve was established: y = 0.467x - 0.004 (R²). 2 =0.995), where y represents the absorbance value at 360 nm wavelength and x represents the sulfate concentration in the standard curve. Following the same procedure, replace the potassium sulfate aqueous solution with a 1 mg / mL TP80-M aqueous solution and measure the OD. 360 Based on the absorbance and the standard curve, the sulfate content in TP80-M can be calculated to be 16.88%.

[0076] 3) TP80-M molecular weight analysis

[0077] The molecular weight and distribution uniformity of TP80-M were analyzed by gel permeation chromatography (GPC).

[0078] Weigh 20 mg of TP80-M sample, dissolve it in deionized water to prepare a polysaccharide solution with a mass concentration of 2 mg / mL, filter it through a 0.45 μm filter membrane, and then inject it for analysis.

[0079] Instrumentation and chromatographic conditions: An Agilent 1260 gel permeation chromatograph equipped with an Agilent RID G1362A refractive index detector was used, and the chromatographic column was a Waters Ultrahydrogel series (300×7.8mm). The mobile phase was a 0.1 mol / L NaNO3 aqueous solution, the flow rate was set to 1 mL / min, and the column temperature was maintained at 40 °C. Polyethylene glycol (PEG) of different molecular weights was used as standards (Mw = 10). 2 -10 5 Da), establish a standard curve.

[0080] The results showed that the GPC spectrum of TP80-M exhibited a single and symmetrical peak shape, indicating good sample homogeneity; its polydispersity index (Mw / Mn) was 1.45, and the estimated average molecular weight was 4.79 kDa. Figure 3A The results showed that TP80-M obtained after ethanol fractionation and membrane separation purification had high purity and structural homogeneity.

[0081] 4) Monosaccharide composition analysis of TP80-M

[0082] The monosaccharide composition of purified polysaccharide TP80-M was analyzed by high performance liquid chromatography-1-phenyl-3-methyl-5-pyrazolone derivatization (HPLC-PMP).

[0083] Weigh 4 mg of TP80-M sample, add 2 mL of trifluoroacetic acid (TFA, 2 mol / L), seal, and hydrolyze at 121 °C for 2 h. After hydrolysis, remove residual TFA by vacuum drying and redissolve in 1 mL of deionized water to prepare a polysaccharide hydrolysate with a mass concentration of 4 mg / mL.

[0084] The above hydrolysate was taken and 200 μL of sodium hydroxide aqueous solution (0.6 mol / L) and 400 μL of PMP derivatizing reagent (0.5 mol / L) were added sequentially. The reaction was carried out at 70 °C for 60 min to complete derivatization. After the reaction, 400 μL of hydrochloric acid aqueous solution (0.3 mol / L) was added for neutralization. To remove unreacted PMP, the mixture was extracted multiple times with chloroform until the lower extract phase was colorless. Finally, the upper aqueous phase was filtered through a 0.22 μm filter membrane for HPLC analysis.

[0085] The HPLC determination conditions were as follows: An HPLC system (EClassical 3200, Elact Analytical Instruments Ltd.) equipped with an Elite C18 column (250 mm × 4.6 mm, 5 μm) was used, and the detection wavelength was set to 250 nm. Mobile phase A was acetonitrile, and mobile phase B consisted of potassium dihydrogen phosphate (6 g) and triethylamine (5 mL) dissolved in ultrapure water, diluted to 1 L. The flow rate was set to 0.8 mL / min, the column temperature to 33 °C, and the injection volume to 10 μL.

[0086] A standard curve is established by comparing the retention times and peak areas of monosaccharide standards such as glucose, galactose, and arabinose, and then the molar proportion of each monosaccharide component in the sample is calculated. Figure 3B As shown, the main monosaccharide composition of TP80-M is: glucuronic acid (67.18%), galacturonic acid (23.54%), fucose (3.86%), mannose (2.50%), glucose (2.21%), and galactose (0.72%). The results indicate that TP80-M is a typical acidic polysaccharide, primarily composed of glucuronic acid and galacturonic acid. Furthermore, the detection of fucose suggests the possible presence of a sulfated ester structure, consistent with the aforementioned sulfate group content determination results.

[0087] 5) Infrared spectral analysis of TP80-M

[0088] The functional group structure of the TP80-M sample was characterized using Fourier transform infrared spectroscopy (FT-IR). The sample was prepared using the potassium bromide pellet method, and the scanning wavelength range was 4000–400 cm⁻¹. -1 .like Figure 3C As shown, in addition to the characteristic absorption peaks of typical polysaccharides, TP80-M also exhibits the following key absorption characteristics: 1650 cm⁻¹ -1 The presence of a distinct C=O stretching vibration absorption peak near the molecule indicates the presence of a carboxyl group (-COOH), suggesting the presence of a uronic acid structure; the peak at 888.76 cm⁻¹... -1 The characteristic absorption peak near 618 cm⁻¹ is attributed to the fingerprint absorption of β-glycosidic bonds, indicating that the main chain or branches of TP80-M contain β-glycosidic linkages; furthermore, the characteristic absorption peak near 618 cm⁻¹ is attributed to the fingerprint absorption of β-glycosidic bonds. -1 The absorption peak at that location corresponds to the characteristic absorption of the sulfate group, further indicating that TP80-M is a sulfated acidic polysaccharide.

[0089] In summary, the FT-IR results verified at the functional group level that TP80-M has structural features such as carboxyl groups, sulfate groups, and β-glycosidic bonds, which corresponds to the aforementioned chemical composition analysis and monosaccharide composition analysis results.

[0090] 6) Analysis of the glycosidic bond linkage mode of TP80-M:

[0091] To elucidate the glycosidic bond linkage of TP80-M, uronic acid reduction, methylation, hydrolysis of the methylation product, and derivatization were performed sequentially. The structure was then analyzed using gas chromatography-mass spectrometry (GC-MS).

[0092] ①Uronic acid reduction: Weigh 40 mg of TP80-M sample and dissolve it in deionized water to prepare a solution with a mass concentration of 2 mg / mL. Add 600 mg of 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride (EDC), and slowly add 0.01 mol / L hydrochloric acid aqueous solution dropwise under magnetic stirring to adjust the pH of the reaction system to 4.75. React for 2 h. Then, add 15 mL of 2 mol / L sodium borohydride aqueous solution dropwise over 45 min, adjust the pH to 7.0 with 4 mol / L hydrochloric acid aqueous solution, and continue the reaction for 2 h. Place the reaction solution in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze for 48 h, and then freeze-dry for subsequent methylation analysis.

[0093] ② Methylation treatment: Weigh 20 mg of the reduced polysaccharide sample and dissolve it in 2 mL of anhydrous dimethyl sulfoxide (DMSO, dried over anhydrous sodium sulfate for 24 h). Dissolve using sonication. Under nitrogen protection, add 30 mg of dry NaOH powder and mix again by sonication. Place the reaction system in an ice-water bath for 10 min, then slowly add 2 mL of iodomethane. Seal and sonicate for 50 min. After the reaction is complete, add 1 mL of deionized water and 3 mL of chloroform to terminate the reaction. After standing and separating the layers, collect the lower organic phase and wash it three times with deionized water. The obtained organic phase is dried over anhydrous sodium sulfate for 24 h, filtered, and then evaporated to dryness under reduced pressure. Repeat the above methylation steps three times to ensure complete methylation.

[0094] ③ Hydrolysis and Derivatization of Methylated Products: The dried methylated products were dissolved in 4 mL of trifluoroacetic acid (TFA, 2 mol / L) and hydrolyzed at 105 °C for 6 h. Residual TFA was removed by nitrogen purging after hydrolysis. 5 mL of deionized water and 15 mg of sodium borohydride were added, and the reaction was carried out at room temperature in the dark for 12 h. The reaction was then terminated with glacial acetic acid until all bubbles disappeared. After drying, the resulting sample was washed 3-4 times with chromatographic grade methanol and residual solvent was removed by nitrogen purging. 2 mL of pyridine and 2 mL of acetic anhydride were added, and the reaction was carried out at 95 °C for 30 min. 2 mL of deionized water was added to the reaction solution to terminate the reaction, followed by extraction with 2 mL of dichloromethane. This process was repeated twice, and the organic phases were combined, dried, filtered (using a 0.22 μm organic phase filter membrane), and then used for GC-MS analysis.

[0095] ④ GC-MS Analysis Conditions: An Agilent 7890 / 7000GCMS-QQQ gas chromatography-mass spectrometry system was used, with a TR-5MS capillary column (30m × 0.25mm × 0.5μm). The temperature program was as follows: initial temperature 160℃, hold for 2 min; increase to 210℃ at 2℃ / min, hold for 2 min; then increase to 240℃ at 5℃ / min, hold for 5 min. Nitrogen was used as the carrier gas at a flow rate of 1.0 mL / min; the ion source temperature was set to 250℃, and the injection volume was 1 μL.

[0096] ⑤ Results Analysis: The GC-MS spectra were compared with the mass spectrometry database of the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences and the database of the Complex Carbohydrate Research Center (CCRC). Five typical partially methylated aldosterone acetates (PMAAs) were detected: 2,4,6-Me3-β-D-Glcp, 2,3,6-Me3-α-D-Manf, 2,4-Me2-β-D-Glcp, 2,3,5,6-Me4-α-D-Galf, and 1,3,4-Me3-β-L-Fucf.

[0097] The above results indicate that the glycosidic bond linkages of TP80-M include: (1→3)-linked Glcp, (1→4)-linked Manf, →3,6-linked GlcAp, →4-Galf(1→), and →2,5-Fucf, with the molar percentages of each structural unit being 9.54%, 9.12%, 41.73%, 28.74%, and 10.87%, respectively.

[0098] 7) NMR structure analysis of TP80-M:

[0099] To further verify the glycosidic bond linkage mode of TP80-M polysaccharide, its fine structure was analyzed using nuclear magnetic resonance (NMR) technology.

[0100] Take 30 mg of the freeze-dried TP80-M sample, dissolve it in an appropriate amount of deuterated water (D2O), and acquire its one-dimensional proton spectrum using a Bruker AVANCE III 500MHz nuclear magnetic resonance spectrometer at room temperature. 1 H NMR spectrum.

[0101] The 1H NMR spectrum of TP80-M shows almost no absorption peaks in the chemical shift region δ = 2.00–3.00 ppm, indicating no significant protein residue. A small peak F (1.23 ppm) appears in the region δ = 1.00–1.20 ppm, which is a characteristic signal of fucose (Fucf), while the main hydrogen proton signals are concentrated in the region δ = 3.00–5.50 ppm. Figure 4A As shown.

[0102] Five distinct anomeric hydrogen signals were detected in the anomeric hydrogen region (δ = 4.50–5.50 ppm), labeled A (4.75 ppm), B (5.29 ppm), C (5.24 ppm), D (4.55 ppm), and E (4.68 ppm), indicating that TP80-M contains five different types of sugar residues. According to chemical shift rules, anomeric hydrogen signals with δ > 4.90 ppm generally belong to the α-configuration, while signals with δ < 4.90 ppm are mostly of the β-configuration.

[0103] Based on the aforementioned methylation analysis and monosaccharide composition results, the following classifications were made: B (5.29 ppm) belongs to →4-Galf-(1→)(α-configuration); C (5.24 ppm) belongs to (1→4)-linked Manf (α-configuration), where mannose has a stronger shielding effect than galactose and a slightly lower chemical shift; A (4.75 ppm) belongs to →3,6-GlcAp (β-glucuronic acid); E (4.68 ppm) belongs to (1→3)-linked Glcp (β-glucose); D (4.55 ppm) may be a different configuration of β-Glcp or GlcAp; F (1.23 ppm) is the methyl absorption peak of fucose (→2,5-Fucf).

[0104] Integral analysis of the above signals yielded the following integral ratios for the five anomeric hydrogens: β-GlcAp H-1: α-Galf H-1: β-Fucf H-1: α-Manf H-1: β-Glcp H-1 = 11.02: 5.37: 4.32: 3.43: 3.42. This ratio is highly consistent with the results of methylation PMAA analysis, indicating that TP80-M has a β-1,3-GlcAp backbone with highly C-6 branched structure, with side chains mainly composed of α-1,4-Galf, and non-reducing terminators of β-L-Fucf, α-Manf, and a small amount of β-Glcp.

[0105] In summary, based on NMR, methylation, and monosaccharide composition analysis results, the structural framework of TP80-M is presumed to be: -β-GlcAp-(1→3)-[→6]-β-GlcAp-(1→3)-, with its side chains extending from (1→4)-Galf-(1→) and further connected to residues such as (1→4)-Manf, (2→5)-Fucf, and (1→3)-Glcp. Possible structural models of TP80-M are as follows: Figure 4B As shown.

[0106] Test Example 3: Evaluation of the in vitro simulated gastrointestinal digestive properties and prebiotic activity of purified polysaccharide TP80-M from *Hylocereus undatus*.

[0107] (1) In vitro simulation of gastrointestinal digestion

[0108] This experiment used a commercially available simulated digestive fluid system. Oral fluid (R27906), gastric fluid (R30386), and small intestinal fluid (R22156) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0109] Weigh 100 mg of TP80-M powder and dissolve it in 10 mL of deionized water to prepare a polysaccharide solution with a mass concentration of 10 mg / mL. Then add 10 mL of artificial saliva and place the solution in a 37°C constant temperature shaking water bath (120 rpm) for 30 min to simulate the oral digestive process.

[0110] Gastric digestion simulation: 10 mL of oral digestive products was mixed with an equal volume of artificial gastric juice (containing sodium chloride, dilute hydrochloric acid and pepsin), the pH of the mixture was adjusted to 2.0, and the mixture was reacted in a constant temperature shaking water bath (120 rpm) at 37 °C for 3 h.

[0111] Small intestinal digestion simulation: 10 mL of gastric digestion products were mixed with 10 mL of artificial small intestinal fluid (containing trypsin and 0.1% porcine bile salts), the pH was adjusted to 7.0, and the mixture was reacted for 3 h in a constant temperature shaking water bath (120 rpm) at 37 °C.

[0112] Throughout the simulated digestion process, samples were collected every 10 minutes (oral digestion stage) or every 0.5 hours (gastrointestinal digestion stage), and immediately placed in a boiling water bath for 5 minutes to terminate the enzyme activity reaction. After centrifugation, the supernatant was collected, and the reducing sugar content was determined using the 3,5-dinitrosalicylic acid method (DNS method).

[0113] Method for quantifying reducing sugars: Using D-glucose as the standard, prepare a series of standard solutions ranging from 0 to 0.12 mg / mL. Take 2 mL of the standard solution, add 4 mL of DNS reagent, heat in a boiling water bath for 5 min, cool, and dilute to 10 mL. Measure the absorbance at 540 nm and plot a standard curve for quantitative analysis of the sample.

[0114] Molecular weight change analysis: The supernatants before and after digestion were added with 4 times the volume of anhydrous ethanol to precipitate polysaccharides. After centrifugation, the precipitates were collected, reconstituted, and dialyzed (MWCO = 1000 Da). After freeze-drying, the digested samples were obtained, and their molecular weight changes were analyzed by gel permeation chromatography (GPC).

[0115] The results are as follows Figure 5AAs shown, in the simulated oral cavity stage, the initial reducing sugar content was 0.0972 mg / mL, and remained relatively stable thereafter, indicating that TP80-M is not easily degraded in a salivary environment. In the first 0.5 hours of gastric digestion, the reducing sugar content slightly increased to 0.1018 mg / mL, presumably due to slight hydrolysis of some glycosidic bonds in a strongly acidic environment. In the simulated small intestine stage, the reducing sugar content rose to 0.1232 mg / mL at the end of digestion, indicating that enzymes in the small intestinal digestive fluid could act on some glycosidic bond structures. Despite this, the total increase in reducing sugar released by TP80-M throughout the entire simulated digestion process was only 0.0260 mg / mL, approximately one-quarter of its theoretical maximum release, indicating its good resistance to gastrointestinal digestion. Furthermore, GPC results showed no significant change in the main peak molecular weight of TP80-M before and after digestion. Figure 5B This further corroborates its structural stability.

[0116] In summary, TP80-M exhibits strong tolerance in simulated oral, gastric, and small intestinal environments, demonstrating the potential to cross the gastrointestinal barrier and successfully reach the colon, providing theoretical support for its utilization by the gut microbiota as a prebiotic in the colon.

[0117] (2) Prebiotic activity assessment

[0118] 1) Preparation of fermentation culture medium and fecal microbial inoculum

[0119] Add 2.0g yeast extract, 2.0g peptone, 1.0g NaHCO3, 0.25g bile salts, 0.50g cysteine-HCl, 0.05g NaCl, 0.02g K2HPO4, 0.02g KH2PO4, 0.01g heme chloride, 0.01g MgSO4, 0.005g CaCl2, 0.5g resazurite, 15μL vitamin K, and 1mL Tween 80 to 1L of distilled water to prepare the basal fermentation medium. Adjust the pH to 7.2 with 0.1mol / L hydrochloric acid for later use.

[0120] Fresh fecal samples were obtained from four healthy volunteers aged 20-25 (2 males and 2 females) who had not used antibiotics in the two weeks prior to sampling, had normal diets, and no history of gastrointestinal diseases. In a laminar flow hood, 3g of fresh fecal matter was taken from each volunteer, mixed with 30mL of sterile saline, and centrifuged (4℃, 2204rpm, 5min). The supernatant was collected. Equal volumes of the four fecal supernatants were mixed and placed in a 37℃ anaerobic incubator for activation culture, serving as the inoculum for subsequent anaerobic fermentation of intestinal microorganisms.

[0121] 2) Fermentation Samples and System Setup

[0122] TP80-M was dissolved in the basal fermentation medium to a final concentration of 7.5 mg / mL, and used as the carbon source for the treatment group. Each fermentation system consisted of 10 mL of medium containing TP80-M and 1 mL of activated fecal microbiota solution. Before fermentation began, all media and consumables were pre-treated in an anaerobic incubator (gas composition: 5% H2 + 5% CO2 + 90% N2) to ensure the system's anaerobic properties.

[0123] The experiment was divided into three groups: treatment group (TP80-M): using TP80-M as the sole carbon source; positive control group (INU): using inulin as the carbon source; and blank control group (BLK): using ultrapure water mixed with fecal inoculum without adding any carbon source. The fermentation time was 48 hours.

[0124] 3) Detection of fermentation-related indicators

[0125] ① Bacterial OD 600 Measurement: During the in vitro anaerobic fermentation process, samples were taken every 6 hours, and the absorbance of the fermentation broth at a wavelength of 600 nm was measured using a spectrophotometer to indirectly reflect the growth dynamics of the microbial community.

[0126] like Figure 6 As shown in Figure A, the OD of the TP80-M processing group 600 The OD value gradually increased with prolonged fermentation time, indicating that it can effectively promote the proliferation and activity of intestinal microorganisms as a carbon source. Throughout the fermentation cycle, the OD value of the TP80-M group... 600 The OD values ​​of the TP80-M group were consistently higher than those of the blank control group, indicating that TP80-M possesses good fermentability and prebiotic potential. At the end of fermentation (48 hours), the OD values ​​of the TP80-M group were significantly higher than those of the control group. 600 The value reached 1.18, equivalent to 86.76% of the level in the inulin treatment group and 1.82 times that of the control group, further confirming its promoting effect on microbial growth.

[0127] ② Determination of polysaccharide utilization: During fermentation, samples were taken every 6 hours, the fermentation broth was centrifuged, and the supernatant was collected. The total sugar content was determined using the phenol-sulfuric acid method. The initial mass of TP80-M was defined as 100%, and the proportion of remaining sugar in the culture medium at different time points was calculated to assess the degradation and utilization of TP80-M.

[0128] like Figure 6 As shown in Figure B, TP80-M and the positive control inulin exhibited similar utilization trends, both being gradually degraded by gut microbiota during fermentation. Particularly at 48 hours, the residual sugar content of TP80-M decreased to 36.27%, indicating good microbial metabolic activity. This result is consistent with its high OD... 600The trend of value change is consistent, further verifying that TP80-M has good prebiotic potential.

[0129] ③ Determination of pH of fermentation medium: Samples were taken every 6 hours. After centrifugation, the supernatant of the fermentation broth was collected and its pH value was measured to reflect the accumulation of metabolic products such as organic acids during the fermentation process.

[0130] The results showed that in the early stage of cultivation (0h), the pH of the culture medium in the TP80-M treatment group was significantly lower than that in the control group, which is presumably related to the fact that it is an acidic polysaccharide rich in structural units such as glucuronic acid and galacturonic acid. With prolonged fermentation time, the pH of the TP80-M group continued to decrease, reaching 5.20 at 48h, a decrease of 2.05 from the initial value. This decrease was significantly greater than that in the blank control group (ΔpH=1.01), but lower than that in the positive control inulin group (ΔpH=3.21). These results indicate that TP80-M can be metabolized by intestinal microorganisms during in vitro anaerobic fermentation to produce a certain amount of short-chain fatty acids (SCFAs), succinic acid, and other acidic products, leading to a decrease in the pH of the culture medium, further confirming its potential prebiotic activity.

[0131] ④ Determination of short-chain fatty acid content: Fermentation broth samples were collected every 6 hours, and 500 μL of supernatant was collected by centrifugation. This supernatant was then mixed with an equal volume of 25% metaphosphoric acid solution for protein precipitation and sample acidification. Subsequently, 20 μL of internal standard solution (2 mg / mL aqueous solution of 2-ethylbutyric acid) was added, and the mixture was vortexed thoroughly for 5 min. After centrifugation (4℃, 10000 rpm, 10 min), the mixture was filtered through a 0.22 μm filter membrane, and the resulting supernatant was used for gas chromatography (GC) analysis.

[0132] Gas chromatography analysis was performed using a Fuli GC9720Plus gas chromatograph equipped with a flame ionization detector (FID) and a DB-FFAP capillary column. SCFAs were separated using a temperature-programmed method. The chromatographic conditions were as follows: initial temperature 90℃, maintained for 6 min; temperature increased to 200℃ at a rate of 10℃ / min, maintained for 6 min; carrier gases were hydrogen (flow rate 30 mL / min) and dry air (flow rate 300 mL / min).

[0133] The quantitative detection of CFAs was based on a mixed stock solution of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, prepared at a concentration of 0.4 mg / mL. This stock solution was then diluted with 25% metaphosphoric acid to a series of standard solutions ranging from 0.025 to 0.2 mg / mL. The following standard curve was established: Acetic acid, y = 64762x - 1149.7, R 2 =0.99; propionic acid, y = 114587x - 2544.4, R 2 =0.98; butyric acid, y = 108238x - 2784.7, R 2=0.98; Isobutyric acid, y = 100057x - 2449.9, R 2 =0.98; Valeric acid, y = 91700x - 1833.3, R 2 =0.99; isovaleric acid, y = 109000x - 2587.6, R 2 =0.99.

[0134] The chromatographic retention times and peak areas of the target substances in fermentation samples at different time points were analyzed, and the concentrations of SCFAs in each group of samples were calculated using the aforementioned standard curve. The results are as follows: Figure 6 As shown in Figure C. Based on the peak retention time and peak area of ​​the target analytes in the fermentation samples at each time point, and combined with the corresponding standard curve, the concentrations of SCFAs were calculated, and the results are shown in Figure C. Figure 6 As shown in C.

[0135] During fermentation, SCFAs continued to accumulate in all treatment groups, mainly including acetic acid and propionic acid. Acetic acid accounted for approximately 60-70% of the total SCFAs, while propionic acid accounted for 10-20%. In the early stage of fermentation (6-12 h), the acetic acid content was in the following order: positive control group (inulin) > TP80-M group > blank group. In the later stage (12-48 h), the acetic acid concentration in the TP80-M group increased significantly, reaching 6.80±0.53 mmol / L at 48 h, which was close to the level of the inulin group.

[0136] Furthermore, TP80-M demonstrated superior butyrate production, with a higher butyrate concentration than the inulin group, indicating that TP80-M is more conducive to providing energy substrates for colonic epithelial cells and potentially supports the stability of intestinal barrier function. Although the levels of isomeric SCFAs (such as isobutyric acid, valeric acid, and isovaleric acid) were low, they showed an increasing trend in all groups, which may regulate intestinal flora homeostasis and inhibit pathogen growth through synergistic effects.

[0137] At the 48-hour fermentation endpoint, the total SCFAs concentration in the TP80-M group was 28.56±1.43 mmol / L, which was basically the same as that in the positive control inulin group (29.96±1.49 mmol / L), and increased by 139.60% compared with the blank group (11.92±0.60 mmol / L).

[0138] In summary, the results indicate that the purified polysaccharide TP80-M from *Hygrophytes maculatus* possesses good fermentability and SCFA generation capacity, and its prebiotic activity is comparable to that of commercially available typical prebiotic inulin, demonstrating its potential application value as a functional dietary ingredient.

[0139] 4) Effects of purified polysaccharide TP80-M from *Phyllostachys edulis* on fecal microbial community composition.

[0140] To further evaluate the prebiotic activity of purified polysaccharide TP80-M from *Phyllostachys edulis*, the gut microbiota composition of the TP80-M group, the positive control group (INL), and the blank control group (CK) after 48 hours of fermentation was analyzed using an in vitro fermentation system.

[0141] The gut microbiota structure determination process included: total DNA extraction, PCR amplification of the target region, addition of specific barcode tags, sample library construction and quantification, library quality control, sequencing, and bioinformatics analysis. Sample DNA extraction was performed using the NovaSeq 6000SP Reagent kit. After passing quality control, amplification was performed using 16S rRNA V3-V4 region-specific primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC), with three biological replicates. PCR products were validated by 2% agarose gel electrophoresis, purified using magnetic beads, and library concentration was analyzed using Qubit quantification and an Agilent 2100 Bioanalyzer. Qualified libraries were sequenced using the NovaSeq 6000 platform with a paired-end PE250 strategy. The obtained raw sequences underwent quality control using QIIME2, and feature sequence annotation was performed using a pre-trained Naive Bayes classifier based on the Greengenes database (version 13.8) with a confidence level set to 0.8. Simultaneously, spiked insertion sequences were identified, and the relative abundance of each ASV in each sample was calculated by combining read counts and copy numbers. Alpha and Beta diversity analyses of the microbial community were performed using R language to systematically assess the richness, evenness, and structural differences of each microbial community. All sequencing and analysis procedures were technically supported by Shanghai Haoweitai Biotechnology Co., Ltd.

[0142] like Figure 7A As shown, at the phylum classification level, the original fecal microbiota mainly consisted of Pseudomonadota, Bacillota, Fusobacteriia, and Bacteroidia. After 48 hours of fermentation, the relative abundance of Pseudomonadota in the TP80-M group decreased from 30.92% to 22.08%, and Fusobacteriia significantly decreased from 23.70% to 0.10%; Bacteroidia significantly increased to 47.86%, and Bacillota also increased to 25.56%. Significant enrichment of Bacteroidia was observed in both the TP80-M and inulin groups, suggesting that it can efficiently degrade complex carbohydrates such as TP80-M, consistent with the rapid substrate consumption of TP80-M during fermentation.

[0143] Further analysis at the genus level revealed ( Figure 7BTP80-M significantly promoted the proliferation of various beneficial bacteria genera, including Parabacteroides, Bacteroides, Phascolarctobacterium, and Bifidobacterium, while inhibiting the proliferation of Escherichia-Shigella and Fusobacterium. The relative abundance of Parabacteroides increased from 2.63% to 25.43%; this genus is an important acetic acid and succinic acid-producing bacterium. Phascolarctobacterium and Bifidobacterium were also enriched in the TP80-M group; the former produces succinic acid, which helps maintain intestinal barrier function, while the latter can produce SCFAs through carbohydrate fermentation, exerting a probiotic effect.

[0144] Meanwhile, both TP80-M and INL treatments significantly reduced the abundance of potentially harmful bacteria. Specifically, Escherichia coli-Shigella decreased to 19.64% and 24.87%, respectively, while Fusobacterium decreased to 0.10% and 0.12%. This reduction in such bacteria is often considered an important indicator of how prebiotics promote gut microbiota health.

[0145] The mechanism was determined to be that TP80-M-enriched Bacteroides and Parabacteroides synthesize large amounts of SCFAs and antimicrobial metabolites during fermentation, thereby reducing the local pH value in the intestine and inhibiting the growth of harmful bacteria; at the same time, competition among bacterial communities for substrates may also lead to changes in their structure.

[0146] In summary, TP80-M can serve as an excellent carbon source, effectively promoting the enrichment of functional probiotics and inhibiting the proliferation of harmful bacteria, thereby optimizing the intestinal microecological structure and demonstrating good prebiotic functional potential.

[0147] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. The application of an acidic polysaccharide derived from *Phyllostachys edulis* in the preparation of functional foods or feeds containing prebiotics, characterized in that... The acidic polysaccharide derived from *Phyllostachys edulis* was prepared by a method including the following steps: (1) Add dried yellow algae powder to water, then stir to extract, centrifuge and collect the supernatant; (2) Add the residue after centrifugation in step (1) to water, then stir to extract, centrifuge to collect the supernatant, combine the supernatant collected by centrifugation with the supernatant obtained by centrifugation in step (1), then concentrate, remove protein, precipitate with alcohol and centrifuge to collect the precipitate; finally, dissolve the centrifuged precipitate in water, dialyze and freeze dry to obtain crude polysaccharide of *Hygrophora spp.* (3) Dissolve the crude polysaccharide of *Hygrophytes macrantha* in water, add anhydrous ethanol for the first-stage precipitation, centrifuge to collect the first-stage precipitate, and freeze-dry it; then add anhydrous ethanol to the supernatant obtained after centrifugation following the first-stage precipitation for the second-stage precipitation, centrifuge to collect the second-stage precipitate, and freeze-dry it; finally add anhydrous ethanol to the supernatant obtained after centrifugation following the second-stage precipitation for the third-stage precipitation, centrifuge to collect the third-stage precipitate, and freeze-dry it. (4) Test the total sugar content of each grade of precipitate obtained after freeze-drying in step (3); (5) Dissolve the precipitate with the highest total sugar content after freeze-drying in water, and then perform ultrafiltration using a polyethersulfone membrane. Freeze-dry the collected residue to obtain the acidic polysaccharide derived from *Phyllostachys edulis*. The molecular weight cutoff of the polyethersulfone membrane is 0.5-3 kDa. The ultrafiltration conditions include a pressure of 0.1-0.3 MPa and a stirring rate of 250-350 rpm.

2. The application according to claim 1, wherein, In step (1), the algal species of Tribonema used to prepare the Tribonema powder is Tribonema sp. GXU A10.

3. The application according to claim 1, wherein, In step (1), the ratio of the dried yellow algae powder to water is 1:(15-30)g / mL; The stirring extraction was carried out in a water bath at 50-70℃; The centrifugation conditions are: rotation speed of 3500-4500 rpm, time of 10-20 min, and temperature of 3-5℃.

4. The application according to claim 1, wherein, In step (2), the ratio of the residue after centrifugation in step (1) to water is 1:(10-20)g / mL; The stirring extraction was carried out in a water bath at 50-70℃; The centrifugation conditions for the supernatant collected by centrifugation are: rotation speed of 3500-4500 rpm, time of 10-20 min, and temperature of 3-5℃. Concentrate to 5%-15% of the original volume.

5. The application according to claim 1, wherein, In step (2), the protein is removed using Sevag reagent; Alcohol precipitation was performed using anhydrous ethanol; the precipitation temperature was 3-5℃ and the time was 8-16h. The centrifugation conditions for collecting the precipitate were: 7500-8500 rpm, 10-20 min, and 3-5℃. The dialysis time is 48-75 hours.

6. The application according to claim 1, wherein, In step (3): During the first-stage precipitation, the temperature is 3-5℃ and the time is 8-16h; the ratio of crude polysaccharide to water is 1:(50-150)g / mL; the centrifugation conditions are: 7500-8500rpm, 10-20min, and 3-5℃; the volume percentage of anhydrous ethanol is 35-45% based on the total volume of crude polysaccharide, water, and anhydrous ethanol. When performing the second-stage precipitation, the volume percentage of anhydrous ethanol is 55-65% based on the total volume of the supernatant obtained by centrifugation after the first-stage precipitation and anhydrous ethanol; the temperature of the second-stage precipitation is 3-5℃, and the time is 8-16h; the centrifugation conditions are: speed of 7500-8500rpm, time of 10-20min, and temperature of 3-5℃. When performing the third-stage precipitation, the volume percentage of anhydrous ethanol is 75-85% based on the total volume of the supernatant obtained by centrifugation after the second-stage precipitation and anhydrous ethanol; the temperature of the third-stage precipitation is 3-5℃, and the time is 8-16h; the centrifugation conditions are: rotation speed of 7500-8500rpm, time of 10-20min, and temperature of 3-5℃.

7. The application according to claim 1, wherein, In step (4), the freeze-dried precipitates obtained in step (3) are dissolved in water to prepare polysaccharide solutions corresponding to each precipitate. Then, the total sugar content of the polysaccharide solutions corresponding to each precipitate is obtained by the phenol-sulfuric acid method. The concentration of the polysaccharide solutions corresponding to each precipitate is 0.5-1.5 mg / mL. The specific steps for obtaining the total sugar content of the polysaccharide solutions corresponding to each precipitate by the phenol-sulfuric acid method are as follows: First, using D-glucose as a standard, the absorbance (OD) values ​​of D-glucose aqueous solutions with different concentration gradients in the range of 0-100 mg / mL were tested at 490 nm. 490 This allows us to obtain the standard curve y = 9.965x + 0.015(R), and thus the standard curve y = 9.965x + 0.015(R). 2 =0.999); where y represents the absorbance value at 490nm and x represents the D-glucose concentration; then, the absorbance value of the polysaccharide solution corresponding to each grade of precipitate at 490nm is tested, and the total sugar content of each grade of precipitate after freeze-drying obtained in step (3) is calculated based on the standard curve.

8. The application according to claim 1, wherein, In step (5), the precipitate with the highest total sugar content after freeze-drying is dissolved in water, and the concentration of the resulting polysaccharide solution is 1-3 mg / mL.

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