Method for preparing robbia polysaccharide with triple helix structure by fermenting bacillus velezensis and application thereof

Abstract

The application provides a method for preparing apocynum venetum polysaccharide with a triple helix structure through fermentation of bacillus velezensis and application thereof. The apocynum venetum is subjected to fermentation treatment by bacillus velezensis, and through synergistic effect of various extracellular enzymes secreted by the strain, efficient degradation of the cell wall of the apocynum venetum and sufficient release of the polysaccharide are realized. Experiments show that the polysaccharide obtained by the application has a significant triple helix structure feature, and the polysaccharide obtained by ultrasonic cellulase complex method and cellulase method does not have the structure. It is found for the first time that fermentation of bacillus velezensis can induce apocynum venetum polysaccharide to form a stable triple helix structure, and directional construction of the higher structure of the apocynum venetum polysaccharide is realized. The apocynum venetum polysaccharide with the triple helix structure can be used as a probiotic preparation for promoting generation of short-chain fatty acids in the intestinal tract and improving intestinal microecology, and can be applied to the fields of functional food and intestinal health.

Description

Technical Field

[0001] This invention belongs to the field of natural active polysaccharide development and utilization technology, and relates to a technical method for regulating the higher-order structure of plant polysaccharides through microbial fermentation. Specifically, it relates to a method and application of *Bacillus belye* fermentation to prepare *Apocynum venetum* polysaccharide with a triple-helix structure. Particularly, it relates to its application in promoting the production of short-chain fatty acids, improving intestinal microecology, and preparing prebiotic preparations. Background Technology

[0002] Apocynum venetum ( Apocynum venetum Apocynum venetum (L.) is an important plant used for both food and medicine, widely distributed in arid and semi-arid regions of my country, such as Xinjiang. It is rich in polysaccharides, flavonoids, phenolic acids, and other bioactive components, possessing various physiological functions including lowering blood pressure, anti-oxidation, and immune regulation. In recent years, with the deepening research on natural active polysaccharides in functional foods and gut health, Apocynum venetum polysaccharides have received widespread attention due to their safe source, abundant resources, and potential prebiotic functions.

[0003] Studies have shown that the bioactivity of polysaccharides is related not only to their monosaccharide composition and molecular weight, but also closely related to their higher-order structure. Among these, the triple helix structure, as an important spatial conformation of natural bioactive polysaccharides, can significantly affect the stability, bioavailability, and interaction with gut microbiota, and is one of the key structural factors determining the functional activity of polysaccharides. However, the formation and maintenance of the polysaccharide triple helix structure are significantly affected by the extraction process; traditional extraction methods often fail to yield bioactive polysaccharides with intact higher-order structures.

[0004] Currently, the extraction of Apocynum venetum polysaccharides mainly employs hot water extraction, acid-base extraction, enzymatic hydrolysis, and ultrasound-assisted extraction. While hot water extraction is simple to operate, it involves high extraction temperatures and energy consumption, and is prone to polysaccharide molecule degradation and conformational disruption. Acid-base extraction, although improving extraction efficiency, is susceptible to glycosidic bond breakage and damage to higher-order structures under strong acid and alkaline conditions. Enzymatic hydrolysis offers good selectivity, but suffers from high enzyme costs and sensitivity to reaction conditions. Ultrasound-assisted extraction, while improving efficiency, suffers from strong equipment dependence and difficulty in controlling structural stability during industrial scale-up. Therefore, existing extraction technologies still have significant limitations in obtaining Apocynum venetum polysaccharides with specific higher-order structures, particularly the triple-helix structure.

[0005] In recent years, microbial fermentation technology has gradually become an important technical route for the preparation of natural polysaccharides due to its advantages such as mild reaction conditions, high selectivity, and the ability to achieve targeted degradation of plant cell walls. During fermentation, various enzyme systems produced by microorganisms, such as cellulase, hemicellulase, and pectinase, can effectively disrupt plant cell walls, improve polysaccharide release efficiency, and potentially regulate the molecular structure of polysaccharides. However, there are currently few research reports on the fermentation preparation of Apocynum venetum polysaccharides. Existing studies mainly focus on improving polysaccharide yield, while in-depth research is lacking on the formation mechanism of higher-order polysaccharide structures and changes in functional activity during fermentation.

[0006] Bacillus belesiensis ( Bacillus velezensis As a microorganism with strong extracellular enzyme secretion and plant cell wall degradation capabilities, *Bacillus belyss* has been reported to be used for the synthesis of microbial polysaccharides or the degradation of fungal polysaccharides. However, current research has not revealed the effect of *Bacillus belyss* fermentation on the formation of the triple helix structure of *Apocynum venetum* polysaccharides, nor has it reported the application value of *Apocynum venetum* polysaccharides obtained through this process in regulating intestinal microbial metabolism and promoting short-chain fatty acid production. Furthermore, existing research on *Bacillus belyss* polysaccharides largely focuses on the extracellular polysaccharides produced by the strain itself, rather than utilizing the strain to ferment plant materials to regulate the higher-order structure of plant polysaccharides.

[0007] Therefore, developing a method for preparing Apocynum venetum polysaccharide with a triple helix structure using Bacillus belye fermentation, clarifying its structural characteristics and its function in promoting the production of short-chain fatty acids in the intestine, is of great significance for enriching the preparation technology system of Apocynum venetum polysaccharide, revealing its structure-function relationship, and promoting its high-value utilization in functional foods and intestinal health. Summary of the Invention

[0008] Purpose of the invention The purpose of this invention is to overcome the problems of low extraction efficiency, easy destruction of polysaccharide higher structure, unstable biological activity and insufficient functional properties in existing Apocynum venetum polysaccharide extraction technology, and to provide an Apocynum venetum polysaccharide with a triple helix structure prepared by Bacillus belye fermentation and its application.

[0009] Technical solution To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing Apocynum venetum polysaccharide.

[0010] This invention discloses a method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belye, comprising the following steps: (1) Activation of strains and preparation of seed culture Bacillus berberis (Bacillus velezensis) After activation, the culture medium was inoculated into a liquid culture medium and cultured to obtain a seed culture. (2) Processing of Apocynum venetum raw materials After washing and drying, the leaves of Apocynum venetum are pulverized and sieved. The resulting powder is then subjected to petroleum ether degreasing and ethanol reflux decolorization treatment in sequence. After drying, pretreated Apocynum venetum powder is obtained. The pretreated Apocynum venetum powder is added to distilled water at a material-to-liquid ratio of 1:10 to 1:30 (g / mL), sterilized, and cooled to obtain Apocynum venetum aqueous solution. (3) Fermentation culture The seed liquid was inoculated into the Apocynum venetum aqueous solution obtained in step (2) at a volume fraction of 5% to 15%, and cultured by shaking fermentation at 25 to 37°C and 50 to 150 r / min for 12 to 48 h. (4) Solid-liquid separation and concentration After fermentation, the supernatant was inactivated, cooled to room temperature, collected by centrifugation, filtered, and concentrated under reduced pressure to obtain the concentrated solution. (5) Deproteinization Sevag reagent was added to the concentrate to remove proteins, and the polysaccharide aqueous phase was collected. (6) Alcohol precipitation and drying Ethanol was added to the aqueous phase of the polysaccharide for alcohol precipitation. The precipitate was collected, reconstituted, and then freeze-dried to obtain Apocynum venetum polysaccharide with a triple helix structure.

[0011] Further, the activation medium for Bacillus belyssus in step (1) is NA solid medium; the activation culture conditions are 25-37℃ for 24-48 h; the liquid medium is NB liquid medium; and the seed culture conditions are 25-37℃, 100-200 r / min shaking culture for 24-48 h.

[0012] Further, the sieving in step (2) is sieving through a 60-100 mesh sieve; the petroleum ether defatting is performed by adding petroleum ether at a material-to-liquid ratio of 1:4 to 1:8 (g / mL) and stirring for 0.5 to 2 h, repeating 1 to 3 times; the ethanol reflux decolorization is performed by adding 95% ethanol at a material-to-liquid ratio of 1:4 to 1:8 (g / mL) and refluxing at 80 to 100°C for 1 to 3 h, repeating until the extract is nearly colorless.

[0013] Furthermore, the fermentation culture conditions described in step (3) are 28℃, 90 r / min shaking culture for 24 h, and the seed liquid inoculation amount is 10% by volume.

[0014] Further, in step (4), the inactivation is performed by heating in a water bath at 80-100°C for 5-15 minutes; the centrifugation is performed by centrifugation at 3000-5000 r / min for 5-15 minutes; and the vacuum concentration is performed by vacuum concentration at 45-70°C to 1 / 5-1 / 2 of the original volume.

[0015] Further, the Sevag reagent mentioned in step (5) is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1; the deproteinization treatment is to add 1 / 5 to 1 / 3 volume of Sevag reagent to the concentrate, shake vigorously and centrifuge, and then take off the upper polysaccharide aqueous phase. This operation is repeated 2 to 5 times.

[0016] Further, the ethanol mentioned in step (6) is pre-cooled 95% ethanol, and the amount added is 3 to 5 times the volume of the polysaccharide aqueous phase; the alcohol precipitation conditions are standing at 4°C for 12 to 16 h; the freeze drying is pre-freezing at -80°C overnight, and then drying for 24 to 48 h under the conditions of cold trap temperature ≤ -50°C and vacuum degree ≤ 10Pa.

[0017] Secondly, the present invention provides a polysaccharide of Apocynum venetum with a triple helix structure.

[0018] The polysaccharide prepared by the above method has the following structural characteristics: (1) The weight-average molecular weight is 10-50 kDa, the number-average molecular weight is 10-50 kDa, and the polydispersity coefficient is close to 1, which is a highly homogeneous polysaccharide. (2) It is composed of rhamnose, arabinose, galactose, glucose and galacturonic acid (GalA), of which the content of galacturonic acid is 30% to 55%; (3) It has a stable triple helix structure. In the Congo red experiment, the maximum absorption wavelength of the polysaccharide-Congo red complex showed a significant red shift as the NaOH concentration increased. (4) The microstructure is an irregular sheet structure with thick and complete sheets, smooth and rounded edges, flat and dense surface, few pores, and no obvious fractures or collapses.

[0019] Thirdly, the present invention also provides the following applications of the above-mentioned Apocynum venetum polysaccharide having a triple helix structure: (1) Use in the preparation of formulations for promoting the formation of short-chain fatty acids, wherein the short-chain fatty acids include propionic acid and / or butyric acid; (2) Application in the preparation of prebiotic formulations for improving gut microbiota, wherein the prebiotic formulations are used to enrich beneficial bacteria of the Bacteroidota phylum in the gut and / or inhibit potentially harmful bacteria of the Bacillota phylum. (3) A prebiotic preparation comprising the above-mentioned Apocynum venetum polysaccharide having a triple helix structure and acceptable excipients.

[0020] Fourthly, the present invention provides the aforementioned Bacillus belesiensis ( Bacillus velezensis Application in the fermentation preparation of Apocynum venetum polysaccharide with a triple helix structure.

[0021] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to use Bacillus belye to ferment Apocynum venetum. Through the synergistic action of multiple extracellular enzymes secreted by the strain, the cell wall of Apocynum venetum is efficiently degraded and polysaccharides are fully released. Experiments show that the yield of Apocynum venetum polysaccharides obtained by the method of this invention can reach 10.01%, which is significantly higher than that of the ultrasonic cellulase complex method (8.10%) and the cellulase method (7.82%), thus improving the extraction efficiency and resource utilization rate of Apocynum venetum polysaccharides.

[0022] (2) This invention uses a mild bio-fermentation method to replace the traditional high-temperature and acid-base extraction process, avoiding excessive degradation and damage to the polysaccharide molecular structure, which is beneficial to maintaining the natural conformation and biological activity of the polysaccharide. The obtained polysaccharide has a neutral sugar content of 71.14% and a protein content of only 2.44%, with a purity significantly higher than the control method.

[0023] (3) This invention is the first to discover that Bacillus belye fermentation can induce the formation of a stable triple helix structure in Apocynum venetum polysaccharide, thus realizing the directional construction of the higher-order structure of Apocynum venetum polysaccharide. Congo red experiments show that the polysaccharide obtained by this invention has significant triple helix structure characteristics, while the polysaccharides obtained by the ultrasonic cellulase complex method and the cellulase method do not have this structure.

[0024] (4) The molecular weight of the polysaccharide obtained in this invention is significantly reduced (Mw 17.51 ​​kDa), which is about 23.7% to 27.1% of that of the control method, while maintaining high homogeneity (Mw / Mn=1.01). The low molecular weight characteristic gives the polysaccharide better water solubility and higher bioavailability.

[0025] (5) The triple-helix structure of Apocynum venetum polysaccharide obtained in this invention has good prebiotic activity and can be effectively utilized by beneficial intestinal bacteria during in vitro intestinal flora fermentation. After 24 h of fermentation, the propionic acid concentration in the BAC group reached 0.772 mg / mL and the butyric acid concentration reached 0.484 mg / mL, both of which were significantly higher than those in the control method.

[0026] (6) This invention reveals the structure-function linkage mechanism of “Bacillus belye fermentation-polysaccharide triple helix structure formation-promoting short chain fatty acid generation”, providing a new technical approach for the high-value utilization of Apocynum venetum polysaccharide, and also providing new research ideas for the structural modification and functional enhancement of natural plant polysaccharides.

[0027] (7) The process conditions of this invention are mild, the operation is simple and the safety is high. It is suitable for industrial scale-up production and has good application prospects and industrialization value. Attached Figure Description

[0028] Figure 1This is a schematic diagram illustrating the preparation of Apocynum venetum polysaccharide and the determination of its indicators using the Bacillus venetum fermentation method of this invention.

[0029] Figure 2 The results show the monosaccharide composition of polysaccharides prepared by the ultrasonic cellulase complex method (CEL-U), the cellulase method (CEL), and the method of the present invention (BAC).

[0030] Figure 3 Scanning electron microscopy results of polysaccharides prepared by the ultrasonic cellulase complex method (CEL-U), the cellulase method (CEL), and the method of the present invention (BAC).

[0031] Figure 4 The triple helix structure of polysaccharides prepared by the ultrasonic cellulase complex method (CEL-U), the cellulase method (CEL), and the method of the present invention (BAC) was determined.

[0032] Figure 5 The method of this invention (BAC) is used for the determination of pH and short-chain fatty acids in in vitro fermentation using the ultrasonic cellulase complex method (CEL-U), the cellulase method (CEL), and the method of this invention (BAC).

[0033] Figure 6 Metagenomic data on in vitro fermentation of polysaccharides prepared by the ultrasonic cellulase complex method (CEL-U), the cellulase method (CEL), and the method of this invention (BAC). Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments and accompanying drawings. This description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0035] Example 1: Preparation of Apocynum venetum polysaccharide (1) Activation of strains and preparation of seed culture Bacillus berberis ( Bacillus velezensis, The culture was streaked onto NA solid medium (beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar powder 15.0–20.0 g / L, pH 7.2–7.4) at the China General Microbiological Culture Collection Center (CGMCC, accession number: CGMCC No. 24640) and incubated at 28°C for 36 h. Single colonies were picked and inoculated onto NB liquid medium (beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, pH 7.2–7.4) and incubated at 28°C with shaking at 150 r / min for 36 h to obtain the seed culture.

[0036] (2) Processing of Apocynum venetum raw materials Wash the leaves of Apocynum venetum, dry them at 40℃, and then pulverize them through an 80-mesh sieve. Add petroleum ether at a ratio of 1:5 (g / mL) and stir for 1 h. After standing and separating, discard the petroleum ether layer. Repeat the degreasing process 3 times and evaporate the residual petroleum ether. Then add 95% ethanol at a ratio of 1:5 (g / mL) and reflux at 90℃ for 2 h. After filtration, discard the filtrate and repeat the decolorization process until the ethanol extract is nearly colorless. Air dry. Add the pretreated Apocynum venetum powder to distilled water at a ratio of 1:20 (g / mL) and sterilize at 121℃ for 20 min. Cool and set aside for later use.

[0037] (3) Fermentation culture Inoculate the seed culture into the above Apocynum venetum aqueous solution at a volume fraction of 10%, and culture at 28℃ and 90 r / min for 24 h with shaking. After fermentation, inactivate the culture by heating in a water bath at 100℃ for 10 min, and then cool to room temperature.

[0038] (4) Solid-liquid separation and concentration The fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. After vacuum filtration, the supernatant was concentrated under reduced pressure at 50-65℃ to 1 / 3 of its original volume to obtain the concentrated liquid.

[0039] (5) Deproteinization Mix the concentrate with Sevag reagent (chloroform: n-butanol = 4:1, v / v) at a volume ratio of 4:1, shake vigorously, and centrifuge at 4000 r / min for 10 min. Collect the upper aqueous phase. Repeat the above operation 3 to 4 times until there is no obvious protein layer after centrifugation.

[0040] (6) Alcohol precipitation and drying Slowly add 4 times the volume of pre-cooled 95% ethanol to the deproteinized polysaccharide solution, let it stand at 4℃ for 12-16 h for alcohol precipitation; centrifuge at 4000 r / min for 10 min to collect the precipitate, dissolve it in distilled water to remove residual ethanol, pre-freeze at -80℃ overnight, and then freeze-dry under vacuum (cold trap temperature ≤ -50℃, vacuum degree ≤ 10Pa, 24-48 h) to obtain the Apocynum venetum polysaccharide product.

[0041] Example 2: Preparation of Apocynum venetum polysaccharide (1) Activation of strains and preparation of seed culture Bacillus berberis ( Bacillus velezensisThe culture was streaked onto NA solid medium (3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, 15.0–20.0 g / L agar powder, pH 7.2–7.4) and incubated at 28°C for 36 h. Single colonies were picked and inoculated onto NB liquid medium (3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, pH 7.2–7.4) and incubated at 28°C with shaking at 150 rpm for 36 h to obtain the seed culture.

[0042] (2) Processing of Apocynum venetum raw materials Wash the leaves of Apocynum venetum, dry them at 30℃, and then pulverize them through a 60-mesh sieve. Add petroleum ether at a ratio of 1:8 (g / mL) and stir for 2 hours. After standing and separating, discard the petroleum ether layer. Repeat the degreasing process 3 times and evaporate the residual petroleum ether. Then add 95% ethanol at a ratio of 1:8 (g / mL) and reflux at 80℃ for 3 hours. After filtration, discard the filtrate and repeat the decolorization process until the ethanol extract is nearly colorless. Air dry. Add the pretreated Apocynum venetum powder to distilled water at a ratio of 1:10 (g / mL) and sterilize at 121℃ for 20 minutes. Cool and set aside for later use.

[0043] (3) Fermentation culture Inoculate the seed culture into the above Apocynum venetum aqueous solution at a volume fraction of 15%, and culture at 28℃ and 90 r / min for 24 h with shaking. After fermentation, inactivate the culture by heating in a water bath at 100℃ for 10 min, and then cool to room temperature.

[0044] (4) Solid-liquid separation and concentration The fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. After vacuum filtration, the supernatant was concentrated under reduced pressure at 50-65℃ to 1 / 3 of its original volume to obtain the concentrated liquid.

[0045] (5) Deproteinization Mix the concentrate with Sevag reagent (chloroform: n-butanol = 4:1, v / v) at a volume ratio of 4:1, shake vigorously, and centrifuge at 4000 r / min for 10 min. Collect the upper aqueous phase. Repeat the above operation 3 to 4 times until there is no obvious protein layer after centrifugation.

[0046] (6) Alcohol precipitation and drying Slowly add 4 times the volume of pre-cooled 95% ethanol to the deproteinized polysaccharide solution, let it stand at 4℃ for 12-16 h for alcohol precipitation; centrifuge at 4000 r / min for 10 min to collect the precipitate, dissolve it in distilled water to remove residual ethanol, pre-freeze at -80℃ overnight, and then freeze-dry under vacuum (cold trap temperature ≤ -50℃, vacuum degree ≤ 10Pa, 24-48 h) to obtain the Apocynum venetum polysaccharide product.

[0047] Example 3: Preparation of Apocynum venetum polysaccharide (1) Activation of strains and preparation of seed culture Bacillus berberis ( Bacillus velezensis The culture was streaked onto NA solid medium (3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, 15.0–20.0 g / L agar powder, pH 7.2–7.4) and incubated at 28°C for 36 h. Single colonies were picked and inoculated onto NB liquid medium (3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, pH 7.2–7.4) and incubated at 28°C with shaking at 150 rpm for 36 h to obtain the seed culture.

[0048] (2) Processing of Apocynum venetum raw materials Wash the leaves of Apocynum venetum, dry them at 40℃, and then pulverize them through a 100-mesh sieve. Add petroleum ether at a ratio of 1:3 (g / mL) and stir for 1 h. After standing and separating, discard the petroleum ether layer. Repeat the degreasing process 3 times and evaporate the residual petroleum ether. Then add 95% ethanol at a ratio of 1:4 (g / mL) and reflux at 95℃ for 3 h. After filtration, discard the filtrate and repeat the decolorization process until the ethanol extract is nearly colorless. Air dry. Add the pretreated Apocynum venetum powder to distilled water at a ratio of 1:20 (g / mL) and sterilize at 121℃ for 20 min. Cool and set aside for later use.

[0049] (3) Fermentation culture Inoculate the seed culture into the above Apocynum venetum aqueous solution at a volume fraction of 5%, and culture at 28℃ and 90 r / min for 24 h with shaking. After fermentation, inactivate the culture by heating in a water bath at 100℃ for 10 min, and then cool to room temperature.

[0050] (4) Solid-liquid separation and concentration The fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was collected. After vacuum filtration, the supernatant was concentrated under reduced pressure at 50-65℃ to 1 / 3 of its original volume to obtain the concentrated liquid.

[0051] (5) Deproteinization Mix the concentrate with Sevag reagent (chloroform: n-butanol = 4:1, v / v) at a volume ratio of 4:1, shake vigorously, and centrifuge at 4000 r / min for 10 min. Collect the upper aqueous phase. Repeat the above operation 3 to 4 times until there is no obvious protein layer after centrifugation.

[0052] (6) Alcohol precipitation and drying Slowly add 4 times the volume of pre-cooled 95% ethanol to the deproteinized polysaccharide solution, let it stand at 4℃ for 12-16 h for alcohol precipitation; centrifuge at 4000 r / min for 10 min to collect the precipitate, dissolve it in distilled water to remove residual ethanol, pre-freeze at -80℃ overnight, and then freeze-dry under vacuum (cold trap temperature ≤ -50℃, vacuum degree ≤ 10Pa, 24-48 h) to obtain the Apocynum venetum polysaccharide product.

[0053] Example 4: Determination of Chemical Composition (1) Determination of neutral sugar content The neutral sugar content was determined using anhydrous glucose as a standard by the phenol-sulfuric acid method.

[0054] (2) Determination of uronic acid content The uronic acid content was determined using the 3-phenylphenol method with galacturonic acid as the reference standard.

[0055] (3) Protein content determination The protein content was determined using the Bradford method with bovine serum albumin as the standard.

[0056] The results are shown in Table 1. The results indicate that the polysaccharide prepared by the method of this invention (BAC) has a neutral sugar content of 71.14%, a uronic acid content of 27.27%, and a protein content of 2.44%. Compared with the ultrasonic cellulase complex method (CEL-U) and the cellulase method (CEL), the polysaccharide obtained by the method of this invention has the highest sugar content and a significantly lower protein content, indicating higher polysaccharide purity. This result is attributed to the fact that the enzyme system secreted by Bacillus belyssus during fermentation can effectively degrade the cell wall, while the mild conditions help protect the polysaccharide structure, thereby retaining more active ingredients, significantly reducing protein impurities, and significantly increasing uronic acid content.

[0057] Table 1 shows the yield, chemical composition, and molecular weight of polysaccharides prepared by the ultrasonic cellulase complex method (CEL-U), the cellulase method (CEL), and the method BAC. Yield % 8.10 7.82 10.01 Neutral sugar content % 65.95 63.28 71.14 Glucuronic acid % 16.90 10.85 27.27 protein% 2.53 2.91 2.44 Mw (kDa) 73.74 64.52 17.51 Mn (kDa) 72.07 63.74 17.28 Mp (kDa) 74.65 62.35 17.71 Mw / Mn 1.02 1.01 1.01 Example 5: Structural Characterization (1) Molecular weight determination 5 mg of polysaccharide sample was dissolved in 1 mL of 0.05 M NaCl solution to prepare a 5 mg / mL sample solution. After vortexing, the solution was centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm microporous membrane and transferred to a sample vial. Analysis was performed using a high-performance liquid chromatography (HPLC) system with a differential detector. The chromatographic column was a tandem gel column (8 × 300 mm, BRT 105-103-101), the mobile phase was 0.2 M NaCl, the flow rate was 0.7 mL / min, the column temperature was 40℃, and the injection volume was 50 μL.

[0058] The molecular weight determination results are shown in Table 1. The weight-average molecular weight (Mw) of the Apocynum venetum polysaccharide prepared by the method of this invention (BAC) was 17.51 ​​kDa, and the number-average molecular weight (Mn) was 17.28 kDa, which were approximately 23.7% and 27.1% of the CEL-U group and the CEL group, respectively, indicating a significant reduction in molecular weight. Simultaneously, the polydispersity index (Mw / Mn) of the BAC group was 1.01, comparable to that of the CEL group (1.01) and the CEL-U group (1.02), both very close to 1. This indicates that while effectively reducing the molecular weight of the polysaccharide, the fermentation process maintained a highly concentrated molecular weight distribution and excellent uniformity, resulting in a homogeneous polysaccharide. Its low molecular weight characteristic suggests that Bacillus belesiensis, during fermentation, secreted specific glycoside hydrolases to moderately and uniformly degrade the Apocynum venetum cell wall polysaccharide, breaking down the large polysaccharide into smaller, more structurally regular fragments.

[0059] Compared with ultrasonic cellulase complex method (CEL-U) and cellulase method (CEL), the low molecular weight polysaccharide obtained by the method of the present invention has better water solubility and higher bioavailability, and its active groups are more easily exposed, which is beneficial to enhancing antioxidant, immunomodulatory and other biological activities.

[0060] (2) Monosaccharide composition analysis 5 mg of polysaccharide sample was mixed with 2 mL of 3 M trifluoroacetic acid and hydrolyzed at 120 °C. After hydrolysis, the acid solution was evaporated to dryness under a nitrogen stream, and 5 mL of water was added and vortexed. 50 μL of the mixture was added to 950 μL of deionized water, centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm microporous membrane before analysis. High-performance anion exchange chromatography (HPLC) was used. The column was a Dionex Carbopac™ PA20 (3 × 150 mm). Mobile phase A was H2O, mobile phase B was 15 mM NaOH, and mobile phase C was a mixture of 15 mM NaOH and 100 mM NaOAc. The flow rate was 0.3 mL / min, the injection volume was 25 μL, and the column temperature was 30 °C.

[0061] Monosaccharide composition Figure 2Analysis showed that the polysaccharides obtained by the three methods were all composed of rhamnose, arabinose, galactose, glucose, and galacturonic acid (GalA). The GalA content in the BAC group was as high as 41.00%, significantly higher than that in the CEL-U and CEL groups; and the uronic acid content in the BAC group reached 27.27%, approximately 2.5 times that of the CEL group and 1.6 times that of the CEL-U group. Both GalA and uronic acid are characteristic indicators of pectin-type acidic polysaccharides, and their correlation indicates that *Bacillus belye* fermentation can directionally release and enrich bound pectin polysaccharides in the cell walls of *Apocynum venetum*. High uronic acid content endows the product with better water solubility, stronger metal ion chelating ability, and more significant biological activity.

[0062] (3) Scanning electron microscopy (SEM) observation The dried polysaccharide sample was fixed on the sample stage and observed using a scanning electron microscope at an accelerating voltage of 5.00 kV. The microstructure of the sample surface was recorded at an appropriate magnification.

[0063] Scanning electron microscopy observation results as follows Figure 3 As shown, the CEL-U group of polysaccharides exhibits a highly fragmented, entangled network structure with severe depolymerization of fiber bundles, a high degree of fragmentation, a rough surface, and a dense distribution of pores and cracks. This morphology indicates that the mechanical shearing force generated during ultrasonic treatment, combined with the action of cellulase, caused significant physicochemical damage to the polysaccharide backbone. The CEL group showed numerous discrete fiber fragments, poor lamellar continuity, localized irregular collapses, and a significantly insufficient structural regularity. The BAC group exhibited an irregular lamellar structure with thick, intact lamellars, smooth and rounded edges, a flat and dense surface with few pores, and no obvious fractures or collapses. These morphological characteristics indicate that the fermentation extraction method of this invention causes minimal damage to the polysaccharide structure and can better preserve its natural original morphology.

[0064] The microstructure of polysaccharides is closely related to their physicochemical properties such as solubility and viscosity. These structural features are beneficial to maintaining the microbial availability of polysaccharides during in vitro fermentation, thereby affecting their prebiotic potential.

[0065] (4) Congo Red Experiment (Determination of Triple Helix Structure) The triple helix structure of polysaccharides was detected using the Congo red method. An equal volume of polysaccharide solution (1 mg / mL) and Congo red solution (100 μM) were mixed, and NaOH solution was added dropwise to achieve final concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 M, respectively. After standing at room temperature for 10 min, the solution was scanned in the wavelength range of 400–600 nm, and the maximum absorption wavelength was recorded.

[0066] The results of the Congo Red experiment are as follows Figure 4As shown. The polysaccharide of Apocynum venetum prepared by the method of the present invention was measured by the Congo red assay. The results showed that, compared with the blank control group, the maximum absorption wavelength of the polysaccharide-Congo red complex in the BAC group showed a significant red shift with increasing NaOH concentration, indicating that the polysaccharide in the BAC group has a stable triple helix structure; while no obvious red shift was observed in the CEL-U group and the CEL group, indicating that they do not have a triple helix structure.

[0067] Existing techniques such as traditional hot water extraction or acid-base extraction, used to prepare Apocynum venetum polysaccharides, did not exhibit a significant triple-helix structure under the same conditions. The triple-helix structure is a crucial higher-order conformational basis for polysaccharides to exert various biological activities, including immunomodulation, antitumor activity, and antioxidant activity, directly influencing their prebiotic potential and medicinal value. The method of this invention, through a mild microbial fermentation process, effectively avoids the destruction of the higher-order structure of the polysaccharide, preserving its triple-helix conformation—a significant advantage over existing extraction methods. Polysaccharides with a triple-helix structure can specifically recognize pattern recognition receptors on the surface of immune cells, effectively activating downstream immune signaling pathways, thereby significantly enhancing immunomodulatory, antioxidant, and other biological activities. Simultaneously, the triple-helix structure endows the polysaccharide with superior water solubility and spatial conformational stability, which is beneficial for maintaining the integrity of the active structure during processing and storage, and improving its water-holding capacity and textural properties. In contrast, the mechanical shearing force generated by ultrasonic cavitation in the CEL-U group and the intense action of single enzymatic hydrolysis in the CEL group both disrupted the hydrogen bond network and helical conformation between polysaccharide chains, leading to the disintegration of the triple helix structure. This not only reduced the potential for biological activity but also affected the physicochemical stability of the product. Therefore, the triple helix structure characteristics of the BAC group further demonstrate that the Bacillus belyss fermentation method can completely preserve the natural higher conformation of polysaccharides under mild conditions, and the resulting product has significant advantages in terms of biological activity, stability, and functional applications.

[0068] Example 6: In vitro fermentation experiment Fecal samples were collected from healthy volunteers, mixed in equal volumes, diluted to 25% (w / v) with sterile PBS, homogenized, filtered through gauze, and the filtrate was collected as inoculum. The basal fermentation medium (containing peptone, yeast extract, inorganic salts, L-cysteine ​​hydrochloride, bile salts, Tween 80, heme, etc.) was sterilized at 121℃ for 15 min and then mixed with the inoculum at a volume ratio of 1:4. A blank control group (without polysaccharide) and an experimental group (with Apocynum venetum polysaccharide) were included. Anaerobic fermentation was performed at 37℃ for 24 h, with samples taken at 0, 6, 12, and 24 h. pH was directly measured using a pH meter; short-chain fatty acids were acidified, extracted with ether, and analyzed by gas chromatography. DNA was extracted from fermentation samples and analyzed for changes in bacterial community composition using high-throughput sequencing.

[0069] (1) pH value changes and determination of short-chain fatty acids pass Figure 5It can be seen that the pH value of the fermentation broth gradually decreased during in vitro fermentation with the accumulation of short-chain fatty acids. Experimental results showed that the pH value of each experimental group continued to decrease with prolonged fermentation time, with the BAC group showing a significantly faster decrease than the CEL-U, CEL, and blank control groups. The pH value at the fermentation endpoint was even lower, which is closely related to the large-scale production of short-chain fatty acids. Short-chain fatty acid determination results showed that the concentration of each group increased with prolonged fermentation time. The total acid production of the BAC group was significantly higher than that of the CEL-U, CEL, and blank control groups, indicating that intestinal microorganisms can continuously and efficiently utilize the polysaccharides from the BAC group as metabolic substrates. Among these, the BAC group showed particularly outstanding promoting effects on propionic and butyric acids. At 24 h of fermentation, the propionic acid concentration reached 0.772 mg / mL, and the butyric acid concentration reached 0.484 mg / mL, both significantly higher than those of the CEL-U and CEL groups.

[0070] The aforementioned acid-producing advantages are directly related to the structural characteristics of the BAC group polysaccharides: First, the BAC group has the lowest molecular weight (Mw 17.51 ​​kDa), making it easier for intestinal flora to recognize, degrade, and utilize the polysaccharides. Second, the BAC group has the highest galacturonic acid content (41.00%), indicating that acidic polysaccharides rich in galacturonic acid have a higher compatibility with the carbohydrate-active enzyme system of acid-producing bacteria. Third, the BAC group has a complete triple helix structure, which helps maintain the spatial stability of the active site and promotes the specific recognition and metabolism of the polysaccharides by the flora. The increased production of propionic and butyric acids is consistent with the significant increase in the abundance of acid-producing bacteria such as Segatella, Bacteroides, and Prevotella in the BAC group, indicating that the polysaccharides of this invention effectively promote the enrichment and metabolic activity of beneficial acid-producing bacteria through their unique molecular structural characteristics.

[0071] (2) Metagenomics analysis Metagenomic analysis after in vitro fermentation showed that ( Figure 6The fermentation microbial community structure in the BAC group was significantly remodeled. At the phylum level, Bacteroidota became the absolutely dominant phylum (relative abundance ~70%), while Bacillota abundance decreased accordingly. At the genus level, Segatella was the dominant genus, with beneficial genera such as Bacteroides, Phocaeicola, and Prevotella being enriched simultaneously, and Clostridium having the lowest abundance. The heatmap showed clear clustering among the treatment groups. The BAC group was highly enriched in Segatella and Leyella (both belonging to Bacteroidetes), which carry abundant glycoside hydrolases and polysaccharide lysins, enabling efficient degradation of plant cell wall polysaccharides. In summary, this fermentation polysaccharide can directionally enrich beneficial Bacteroidetes with degradation functions and inhibit potentially harmful bacteria. The high degree of biological repeatability within the BAC group indicates that the regulatory effect is stable and reliable. CAZy gene family functional analysis showed that the BLANK control group was generally biased towards basal metabolism and cell wall remodeling (mainly enriched in the GH77, GT4 / 2, and GH23 enzyme families), while the experimental groups were directionally enriched in plant cell wall degrading enzyme systems. Among them, the BAC group was significantly enriched with glycoside hydrolases GH43, GH5, GH10, GH26, GH53, polysaccharide lyases PL1 and CE6 gene families. These enzymes, through functional complementarity, constitute a complete extracellular degradation system for cellulose, hemicellulose, and pectin. While the CEL and CEL-U groups possessed certain degradation potential, their community characteristics differed significantly. The BAC group exhibited a unique microbial community structure and was highly enriched with key beneficial bacteria genera such as Segatella and Leyella. Furthermore, its dominant bacteria carried abundant GH43, GH5, and PL1 polysaccharide degrading enzyme genes, forming a functionally complementary synergistic metabolic network with the Segatella symbiotic bacteria, greatly promoting the release of bound polysaccharides from *Apocynum venetum*. This method was significantly superior to the CEL and CEL-U groups in optimizing the microbial community ecology.

[0072] In summary, the fermented polysaccharide obtained in this invention can directionally enrich beneficial Bacteroidetes with degradation functions and inhibit potentially harmful bacteria, and the biological repeatability within the BAC group is highly consistent, indicating that the regulatory effect is stable and reliable, and it is an efficient and stable fermentation strategy.

[0073] Example 7: Preparation of prebiotic formulations The triple-helix polysaccharide prepared in Example 1 was used as the active ingredient and, with acceptable excipients, was prepared into a prebiotic formulation using conventional methods. The formulation may be in the form of powder, tablets, capsules, or oral liquid. Excipients include, but are not limited to, fillers, binders, lubricants, disintegrants, and flavoring agents.

[0074] Comparative Example 1: Preparation of Apocynum venetum polysaccharide by ultrasonic cellulase complex method (CEL-U) Using Apocynum venetum leaves as raw material, after defatting and decolorization pretreatment, distilled water was added at a material-to-liquid ratio of 1:20, cellulase was added, and enzymatic extraction was carried out under ultrasonic-assisted conditions. Subsequent steps included centrifugation, concentration, deproteinization, alcohol precipitation, and freeze-drying to obtain Apocynum venetum polysaccharide. The polysaccharide yield was 8.10%, with a Mw of 73.74 kDa, and no triple helix structure was observed.

[0075] Comparative Example 2: Preparation of Apocynum venetum polysaccharide by cellulase method (CEL) Using Apocynum venetum leaves as raw material, after defatting and decolorization pretreatment, distilled water was added at a material-to-liquid ratio of 1:20, and cellulase was added for enzymatic extraction. Subsequent steps including centrifugation, concentration, deproteinization, alcohol precipitation, and freeze-drying yielded Apocynum venetum polysaccharide. The polysaccharide yield was 7.82%, with a molecular weight of 64.52 kDa, and no triple helix structure was observed.

[0076] In summary, the method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation with Bacillus venetum provided in this invention features mild process conditions, simple operation, and high safety. The raw material, Apocynum venetum, is abundant and widely available, and the fermentation strain, Bacillus venetum, is easy to cultivate and scale up. This method is suitable for industrial-scale production and has good application prospects and industrialization value. The obtained Apocynum venetum polysaccharide with a triple-helix structure can be used as a prebiotic preparation to promote the production of short-chain fatty acids in the intestine and improve the intestinal microecology, showing broad application prospects in the fields of functional foods and intestinal health.

Claims

1. A method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belye, characterized in that, Includes the following steps: (1) Activation of strains and preparation of seed culture After activating Bacillus belye, it was inoculated into liquid culture medium and cultured to obtain seed culture. (2) Processing of Apocynum venetum raw materials After washing and drying, the leaves of Apocynum venetum are pulverized and sieved. The resulting powder is then subjected to petroleum ether degreasing and ethanol reflux decolorization treatment in sequence. After drying, pretreated Apocynum venetum powder is obtained. The pretreated Apocynum venetum powder is added to distilled water at a material-to-liquid ratio of 1:10 to 1:30 (g / mL), sterilized, and cooled to obtain Apocynum venetum aqueous solution. (3) Fermentation culture The seed liquid was inoculated into the Apocynum venetum aqueous solution obtained in step (2) at a volume fraction of 5% to 15%, and cultured by shaking fermentation at 25 to 37°C and 50 to 150 r / min for 12 to 48 h. (4) Solid-liquid separation and concentration After fermentation, the supernatant was inactivated, cooled to room temperature, collected by centrifugation, filtered, and concentrated under reduced pressure to obtain the concentrated solution. (5) Deproteinization Sevag reagent was added to the concentrate to remove proteins, and the polysaccharide aqueous phase was collected. (6) Alcohol precipitation and drying Ethanol was added to the aqueous phase of the polysaccharide for alcohol precipitation. The precipitate was collected, reconstituted, and then freeze-dried to obtain Apocynum venetum polysaccharide with a triple helix structure.

2. The method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belye according to claim 1, characterized in that, The sieving in step (2) refers to passing through a 60-100 mesh sieve; the petroleum ether defatting is carried out by adding petroleum ether at a material-to-liquid ratio of 1:4 to 1:8 (g / mL) and stirring for 0.5 to 2 hours, repeating 1 to 3 times; the ethanol reflux decolorization is carried out by adding 95% ethanol at a material-to-liquid ratio of 1:4 to 1:8 (g / mL) and refluxing at 80 to 100°C for 1 to 3 hours, repeating until the extract is nearly colorless.

3. The method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belye according to claim 1, characterized in that, The fermentation culture conditions described in step (3) are 28℃, 90 r / min shaking culture for 24 h, and the seed liquid inoculation amount is 10% by volume.

4. The method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belye according to claim 1, characterized in that, The Sevag reagent mentioned in step (5) is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1; the deproteinization treatment involves adding 1 / 5 to 1 / 3 volume of Sevag reagent to the concentrate, shaking vigorously, centrifuging, and aspirating the upper polysaccharide aqueous phase. This process is repeated 2 to 5 times.

5. The method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belye according to claim 1, characterized in that, The ethanol mentioned in step (6) is pre-cooled 95% ethanol, and the amount added is 3 to 5 times the volume of the polysaccharide aqueous phase; the alcohol precipitation conditions are standing at 4°C for 12 to 16 h; the freeze drying is pre-freezing at -80°C overnight, and then drying for 24 to 48 h under the conditions of cold trap temperature ≤ -50°C and vacuum degree ≤ 10 Pa.

6. The method for preparing Apocynum venetum polysaccharide with a triple-helix structure by fermentation of Bacillus belyi according to any one of claims 1 to 5, characterized in that, The Apocynum venetum polysaccharide has at least one of the following structural features: (a) Weight-average molecular weight is 10–50 kDa; (b) It is composed of rhamnose, arabinose, galactose, glucose and galacturonic acid, of which the content of galacturonic acid is 30% to 55%; (c) It has a triple helix structure, and in the Congo red experiment, the maximum absorption wavelength of the polysaccharide-Congo red complex showed a significant red shift as the NaOH concentration increased; (d) The microstructure is an irregular sheet-like structure with thick and complete sheets, smooth and rounded edges, and a flat and dense surface.

7. The use of the triple-helix structured Apocynum venetum polysaccharide according to claim 6 in the preparation of formulations for promoting the production of short-chain fatty acids; wherein the short-chain fatty acids include propionic acid and / or butyric acid.

8. The application of the triple-helix structured Apocynum venetum polysaccharide according to claim 6 in the preparation of a prebiotic formulation for improving intestinal microecology; wherein the prebiotic formulation is used to enrich beneficial Bacteroidetes in the gut and / or inhibit potentially harmful Bacillota bacteria.

9. A prebiotic preparation, characterized in that, It comprises the triple-helix structured Apocynum venetum polysaccharide as described in claim 6 and acceptable excipients.

10. The application of *Bacillus belye* in the fermentation preparation of *Apocynum venetum* polysaccharide with a triple helix structure, characterized in that... The Bacillus belye is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 24640.

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