A method for preparing triple helix plant polysaccharide by fermentation and application thereof in improving intestinal tract protection activity

Abstract

The application discloses a method for preparing triple helix plant polysaccharide through fermentation and application of the triple helix plant polysaccharide in improving intestinal tract protection activity, and the method is inoculating bacillus subtilis into a plant water extract, and then performing fermentation under specific conditions to obtain the triple helix plant polysaccharide, the triple helix plant polysaccharide prepared by the method can more effectively repair intestinal tract barrier damage and protect the inflamed intestinal tract, the method is simple and efficient, and preparation of the triple helix plant polysaccharide no longer depends on natural polysaccharide raw materials with triple helix structure, and has wide application prospects in the fields of triple helix polysaccharide production and functional food processing.

Description

Technical Field

[0001] This invention relates to the technical field of natural extracts, specifically to a method for preparing triple-helix plant polysaccharides through fermentation and its application in enhancing intestinal protective activity. Background Technology

[0002] Modification methods to enhance polysaccharide activity mainly include chemical modification, physical modification, and enzymatic hydrolysis. These modifications primarily focus on the monosaccharide composition, molecular weight regulation, and functional group modifications at the compound level, including two-dimensional modifications such as monosaccharide sequence and linkage patterns. In recent years, many researchers have concentrated their attention on the three-dimensional structure of polysaccharides, believing that it may be the most important factor determining polysaccharide activity. Reported three-dimensional structures of polysaccharides include chain-like, double-helix, triple-helix, worm-like, random coil, and rod-like structures. Among these structures, double-helix and triple-helix structures exhibit higher stiffness and stability, with natural polysaccharides possessing triple-helix structures reported to have better immunomodulatory activity.

[0003] Currently, the commercialization of such products is hindered by a lack of efficient research technologies for preparing high-performance triple-helix polysaccharides, mainly in the following aspects: First, the discovery of active triple-helix polysaccharide raw materials is relatively difficult. Triple-helix structures are scarce in natural plant polysaccharides; currently, only about 54 kinds of natural plant polysaccharides with triple-helix structures have been reported, such as licorice root, corn silk, Rosa laevigata, dandelion, black fungus, Dendrobium officinale, and shiitake mushroom. Second, the traditional preparation of triple-helix polysaccharides still heavily relies on natural raw materials with this structure, but the sources of these raw materials are limited, and the extraction and purification efficiency is not high. Therefore, providing a method for preparing triple-helix polysaccharides using plant raw materials that do not contain natural triple-helix polysaccharides has significant economic value. Summary of the Invention

[0004] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides a method for preparing triple-helix plant polysaccharides by fermentation and its application in improving intestinal protective activity.

[0005] The first objective of this invention is to provide the use of Bacillus subtilis as an additive in the preparation of triple-helix plant polysaccharides from plant raw materials that do not contain triple-helix polysaccharides themselves.

[0006] The second objective of this invention is to provide a method for preparing triple-helix plant polysaccharides.

[0007] A third objective of this invention is to provide a triple-helix plant polysaccharide prepared by the above-described preparation method.

[0008] A fourth objective of this invention is to provide the application of the above-mentioned triple-helix plant polysaccharide in the preparation of products that protect the intestinal barrier and / or repair inflammatory damage to the intestinal barrier.

[0009] A fifth objective of this invention is to provide the use of the above-mentioned triple-helix plant polysaccharide in the preparation of products for treating intestinal inflammatory damage.

[0010] The sixth object of the present invention is to provide a product for protecting the intestinal barrier.

[0011] Content of this invention:

[0012] The application of Bacillus subtilis as an additive in the preparation of triple-helix plant polysaccharides from plant raw materials that do not contain triple-helix polysaccharides themselves, wherein the plant raw materials include saponin rice, angelica, euryale seed, atractylodes macrocephala, asparagus, pomelo peel, fenugreek, or kudzu root.

[0013] Preferably, the Bacillus subtilis is a Bacillus subtilis that can secrete hemicellulase and / or mannanase and form an acidic environment after fermentation.

[0014] More preferably, the Bacillus subtilis is Bacillus subtilis CICC 10088.

[0015] A method for preparing a triple-helix plant polysaccharide involves inoculating a plant aqueous extract with Bacillus subtilis and then fermenting for 2.5–3.5 hours.

[0016] The plants mentioned include soapberry rice, angelica, foxnut, atractylodes macrocephala, asparagus, pomelo peel, fenugreek, or kudzu root.

[0017] The plant water extract contains a large amount of nutrients that Bacillus subtilis can utilize, which can provide the necessary nutritional conditions for the fermentation of Bacillus subtilis. In addition, the polysaccharides contained in the plant water extract are unmodified polysaccharides.

[0018] Preferably, in the plant aqueous extract, the mass-to-volume ratio of plant sample to water is 1 g: (45-55) mL.

[0019] More preferably, the mass-to-volume ratio of the plant sample to water is 1g:50mL.

[0020] Preferably, the Bacillus subtilis is a Bacillus subtilis that can secrete hemicellulase and / or mannanase and form an acidic environment after fermentation.

[0021] More preferably, the Bacillus subtilis is Bacillus subtilis CICC 10088.

[0022] Preferably, the inoculum amount of Bacillus subtilis is 1-5% of the volume of the plant water extract.

[0023] More preferably, the inoculum amount of Bacillus subtilis is 2% of the volume of the plant water extract.

[0024] Preferably, the fermentation time is 3 hours.

[0025] Preferably, the fermentation temperature is 28–32°C.

[0026] More preferably, the fermentation temperature is 30°C.

[0027] Preferably, after the fermentation is completed, the fermentation product is subjected to deproteinization, alcohol precipitation, and dialysis.

[0028] More preferably, the dialysis is performed using a 3000 Da dialysis bag.

[0029] Triple-helix plant polysaccharides prepared by any of the above preparation methods.

[0030] The monosaccharide composition of the triple-helix saponin rice polysaccharide includes arabinose, xylose, mannose, fructose, glucose, galactose and glucuronic acid.

[0031] The above-mentioned triple-helix plant polysaccharides are used in the preparation of products that protect the intestinal barrier and / or repair inflammatory damage to the intestinal barrier.

[0032] The above-mentioned triple-helix plant polysaccharide is used in the preparation of products for treating intestinal inflammatory damage.

[0033] As one possible approach, the products include, but are not limited to, pharmaceuticals and health foods, wherein the pharmaceuticals also include pharmaceutically acceptable excipients and the health foods also include food-acceptable additives.

[0034] A product for protecting the intestinal barrier, the product containing the aforementioned triple-helix plant polysaccharide.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention discloses a method for preparing triple-helix plant polysaccharides through fermentation and its application in enhancing intestinal protective activity. The method of this invention enables the preparation of triple-helix polysaccharides from plant raw materials that do not contain naturally occurring triple-helix polysaccharides, achieving the creation of triple-helix polysaccharides from scratch. The plant polysaccharides prepared by this method can more effectively repair intestinal barrier damage and protect inflamed intestines. This method is simple and efficient, and eliminates the reliance on naturally occurring triple-helix polysaccharide raw materials, thus showing broad application prospects in the production of triple-helix polysaccharides and the processing of functional foods. Attached Figure Description

[0037] Figure 1Figures show the intestinal leakage rate and triple helix structure analysis of crude polysaccharides after treatment under different fermentation conditions; A: Intestinal leakage rate of crude polysaccharides after fermentation by different strains; B: Triple helix structure analysis of crude polysaccharides after fermentation by different strains; C: Intestinal leakage rate of crude polysaccharides after fermentation with Bacillus subtilis for different times; D: Triple helix structure analysis of crude polysaccharides after fermentation with Bacillus subtilis for different times. In the figures, UF represents unfermented crude polysaccharide, BS represents crude polysaccharide fermented with Bacillus subtilis, LP represents crude polysaccharide fermented with Lactobacillus plantarum, and PC represents crude polysaccharide fermented with Bacillus coagulatingus.

[0038] Figure 2 The structure of the polysaccharide sample is shown below; A: HPGPC diagram of GSP-u and GSP-f, B: molecular weight of GSP-u-1 and GSP-u-2, C: molecular weight of GSP-f-1 and GSP-f-2.

[0039] Figure 3 The diagram shows the triple helix structure analysis of GSP-u, GSP-u-1, GSP-u-2, GSP-f, GSP-f-1, and GSP-f-2.

[0040] Figure 4 The effects of saponin rice polysaccharide before and after fermentation on the lifespan and intestinal barrier function of Drosophila enteritis; A: lifespan curve, B: mean lifespan, maximum lifespan and median lethal time (LT50), C: Drosophila intestinal leakage diagram, D: percentage of intestinal leakage.

[0041] Figure 5 The results of Congo red and intestinal leakage tests on nine plant polysaccharides before and after fermentation are shown below. A: Maximum absorption wavelength of Congo red in polysaccharides of Angelica sinensis (1), Mentha haplocalyx (2), and Euryale ferox (3) before and after fermentation under different concentrations of sodium hydroxide. B: Maximum absorption wavelength of Congo red in polysaccharides of Atractylodes macrocephala (4) and Cornus officinalis (5) before and after fermentation under different concentrations of sodium hydroxide. C: Maximum absorption wavelength of Congo red in polysaccharides of Asparagus cochinchinensis (7) and Citrus reticulata peel (8) before and after fermentation under different concentrations of sodium hydroxide. D: Maximum absorption wavelength of Congo red in polysaccharides of Fenugreek (9) and Pueraria lobata (10) before and after fermentation under different concentrations of sodium hydroxide. E: Intestinal leakage analysis of polysaccharides of Angelica sinensis (1), Euryale ferox (3), and Atractylodes macrocephala (4) before and after fermentation. F: Intestinal leakage analysis of polysaccharides of Asparagus cochinchinensis (7), Citrus reticulata peel (8), Fenugreek (9), and Pueraria lobata (10) before and after fermentation. In the figure, W represents polysaccharides before fermentation, and F represents polysaccharides after fermentation. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] Example 1: Effect of Fermentation Strains on the Triple Helix Structure of Saponin Rice Polysaccharide

[0045] I. Experimental Methods

[0046] 1. Strains and their activation

[0047] Lactobacillus plantarum ATCC 8014: purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number: B312299;

[0048] Bacillus subtilis CICC 10088: Purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number: BMZ137468;

[0049] Bacillus coagulans CICC 10358: purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number: BMZ142414;

[0050] Lactobacillus plantarum and Bacillus subtilis were cultured in MRS broth medium (Huankai Microbial Technology Co., Ltd.) at 30℃ for 24h and 48h respectively to obtain activated Lactobacillus plantarum and Bacillus subtilis; Bacillus decoagulation was cultured in TSA medium (Huankai Microbial Technology Co., Ltd.) at 30℃ for 48h to obtain activated Bacillus decoagulation.

[0051] 2. Preparation of water extract of soapberry rice

[0052] After processing the soapberry seeds and removing the seed coat, the translucent white endosperm was dried to obtain the experimental raw material, soapberry rice. The soapberry rice was ground into powder and passed through a 60-mesh sieve. 4g of soapberry rice powder was dissolved in 200mL of purified water and extracted for 12h. Then, it was pre-sterilized at 100℃ for 10min, and the pH was adjusted to 6.2 to obtain the soapberry rice water extract.

[0053] 3. Fermentation

[0054] The activated bacterial suspensions of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Bacillus coagulationus* were inoculated at a rate of 2% (1×10⁻⁶). 9 (CFU / mL) was added to the saponin rice water extract, stirred and mixed evenly under aseptic conditions, and fermented at 30℃ for 9 hours. An equal amount of sterile water was added as the unfermented group.

[0055] After fermentation, each group of solutions was inactivated at 100℃ for 15 min and centrifuged at 4000 rpm for 10 min. The supernatant was collected and deproteinized using the Savage method. Then, it was precipitated overnight at 4℃ with 3 volumes of anhydrous ethanol. The precipitate was dissolved in distilled water and placed in a 3000 Da dialysis bag. The water was changed every 3 hours, and the dialysis was performed for 24 hours. Then, it was freeze-dried to obtain crude polysaccharides, which were designated as crude polysaccharide from the unfermented group (UF), crude polysaccharide from the Lactobacillus plantarum fermentation group (LP), crude polysaccharide from the Bacillus subtilis fermentation group (BS), and crude polysaccharide from the Bacillus coagulans fermentation group (PC).

[0056] 4. Smurf Detection

[0057] (1) Fruit fly population, culture and treatment

[0058] Wild-type Canton-S strain fruit flies (Drosophila melanogaster, D. melanogaster) were obtained from the Shanghai Institute of Biochemistry and Cell Biology. The fruit flies were reared in basal medium (20g corn flour, 12.5g sucrose, 10g yeast extract, 2.5g agar powder, 1.2mL propionic acid, and 300mL distilled water) under the following conditions: 25℃, relative humidity (55%), and a 12h light / 12h dark cycle.

[0059] Newly emerged first-generation fruit flies were collected. Female fruit flies were selected and cultured for 3 days under CO2 anesthesia. They were then transferred to vials containing different filter papers and cultured for 48 hours, with the filter papers being replaced every 12 hours.

[0060] Control group: Filter paper was soaked in a 5% sucrose solution (w / v);

[0061] Induction group (SDS group): Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v);

[0062] Unfermented group (UF group): Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL crude polysaccharide solution from the unfermented group;

[0063] Lactobacillus plantarum fermentation group (LP group): Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL crude polysaccharide solution of Lactobacillus plantarum fermentation group;

[0064] Bacillus subtilis fermentation group (BS group): Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL Bacillus subtilis fermentation group crude polysaccharide solution;

[0065] Bacillus coagulation fermentation group (PC group): Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL crude polysaccharide solution of Bacillus coagulation fermentation group.

[0066] (2) The Smurfs Experiment

[0067] Brilliant Blue edible pigment was added to a 5% sucrose solution (w / v) to achieve a 2.5% (w / v) concentration, resulting in a sucrose solution containing Brilliant Blue. Female Drosophila melanogaster from each group, after 48 hours of culture, were transferred to empty tubes and starved for 2 hours, then fed 200 μL of the sucrose solution containing Brilliant Blue.

[0068] If the intestinal basal layer of the fruit fly is severely damaged, intestinal permeability will increase. The ingested brilliant blue will stain the mouthparts, digestive tract, abdominal cavity, thoracic cavity, head, and other parts, making the fruit fly appear blue all over. Twelve hours after feeding, the fruit flies were observed under a microscope, and the intestinal leakage rate was calculated based on the percentage of blue fruit flies in the total number of fruit flies.

[0069] 5. Congo Red Test

[0070] The presence of a triple helix structure in the crude polysaccharides of each group was determined using the Congo red assay. 2 mL of polysaccharide solution (2.5 mg / mL) was mixed with 2 mL of Congo red solution (80 μmol / L), followed by the addition of 1 mol / L NaOH solution. The concentration of NaOH in the mixture was increased sequentially from 0, 0.10, 0.20, 0.30, 0.40, and 0.50 mol / L. After standing at room temperature for 10 min, the characteristic absorption spectra in the wavelength range of 400–600 nm were scanned using a UV-Vis spectrophotometer and recorded.

[0071] II. Experimental Results

[0072] Smurf test results as follows Figure 1 As shown in Figure A, compared with the induction group, the intestinal leakage rate decreased by 7.4% in the UF group, 20.1% in the BS group, 12.9% in the LP group, and 15.3% in the PC group. This indicates that the saponin rice polysaccharides in each group have a protective effect on the Drosophila intestine, and the saponin rice polysaccharides fermented by Bacillus subtilis have the best protective effect.

[0073] The Congo red test results showed that the crude polysaccharides from the unfermented group, the fermented group of *Lactobacillus plantarum*, and the fermented group of *Bacillus cereus* did not exhibit a red shift, while the crude polysaccharides from the fermented group of *Bacillus subtilis* showed a slight red shift trend at 0.1 mol / L NaOH. Figure 1In section B), it is speculated that polysaccharides with a triple-helix structure form a stable complex with Congo red in a weakly alkaline solution, causing a red shift in its maximum absorption wavelength (λmax). This indicates that polysaccharides fermented by Bacillus subtilis have a tendency to produce triple-helix structures. Therefore, Bacillus subtilis was selected as the fermentation strain.

[0074] Example 2: Effect of fermentation time on the triple helix structure of saponin rice polysaccharide

[0075] I. Experimental Methods

[0076] The saponin rice water extract was prepared according to step 2 in Example 1. The activated Bacillus subtilis was inoculated at a rate of 2% (1×10⁻⁶). 9 (CFU / mL) was added to the saponin rice water extract, stirred and mixed evenly under aseptic conditions, and fermented at 30℃ for 3h, 6h, 9h, and 12h respectively. An equal volume of sterile water was added as the unfermented group.

[0077] After fermentation, each solution was inactivated at 100℃ for 15 min and centrifuged at 4000 rpm for 10 min. The supernatant was collected and deproteinized using the Savage method. Then, it was precipitated overnight at 4℃ with 3 volumes of anhydrous ethanol. The precipitate was dissolved in distilled water, placed in a 3000 Da dialysis bag, and the water was changed every 3 hours. Dialysis was performed for 24 hours, and then freeze-dried to obtain crude polysaccharide.

[0078] Perform Smurf detection and Congo Red testing according to steps 4 and 5 in Example 1.

[0079] II. Experimental Results

[0080] The results are as follows Figure 1 As shown in C and D, the crude polysaccharide from saponin rice fermented with Bacillus subtilis for 3 hours exhibited the best protective effect on the Drosophila gut. Furthermore, its maximum absorption wavelength increased from 400 nm at 0 mol / L NaOH to 406 nm at 0.1 mol / L NaOH, showing a significant red shift. This indicates the formation of a triple helix structure at this fermentation time point, while no triple helix structure was formed at other time points. Therefore, 3 hours was chosen as the fermentation time.

[0081] Example 3: Isolation, purification, molecular weight and yield determination of saponin rice polysaccharides before and after fermentation.

[0082] I. Experimental Methods

[0083] (1) Isolation and purification of saponin rice polysaccharide

[0084] The saponin rice water extract was prepared according to step 2 in Example 1. The activated Bacillus subtilis was inoculated at a rate of 2% (1×10⁻⁶). 9(CFU / mL) was added to the saponin rice water extract, stirred and mixed evenly under aseptic conditions, and fermented at 30℃ for 3 hours. An equal volume of sterile water was added as the unfermented group.

[0085] After fermentation, the solution was inactivated at 100℃ for 15 min and centrifuged at 4000 rpm for 10 min. The supernatant was collected and deproteinized using the Savage method. Precipitation was then carried out overnight at 4℃ with three volumes of anhydrous ethanol. The precipitate was dissolved in distilled water and placed in a 3000 Da dialysis bag. The water was changed every 3 hours, and the dialysis was repeated for 24 hours. The precipitate was then freeze-dried to obtain crude polysaccharides, designated as unfermented crude polysaccharide (GSP-u) and fermented crude polysaccharide (GSP-f), respectively. High-performance gel permeation chromatography (HPGPC) was used to analyze the unfermented and fermented crude polysaccharides.

[0086] Crude polysaccharides were purified using a Sephadex G-200 gel filtration column (1.6 cm × 60 cm) at a flow rate of 0.2 mL / min. Polysaccharide fractions with different retention times were collected, monitored at 220 nm using a refractive index detector, and concentrated at 50 °C. The concentrated products were then lyophilized to obtain pure polysaccharides GSP-u-1, GSP-u-2, GSP-f-1, and GSP-f-2, and the yields of each fraction were calculated.

[0087] (2) Molecular weight determination

[0088] Six dextran standards and six samples with relative molecular masses in the range of 13050 to 2000000 Da were dissolved in 500 μL of distilled aqueous solution (10 mg / mL), sterilized with a 0.22 μm filter membrane, and then analyzed by instrument.

[0089] A regression equation was established using the logarithm of the relative molecular mass of the dextran standard (log Mp) against the elution volume (V) to obtain a standard curve. The relative molecular mass of the sample polysaccharides was then calculated based on the standard curve.

[0090] Chromatographic conditions: TSK-GEL G-5000PW XL column (7.8mm×300mm) and TSK-GELG-3000PW XL column (7.8mm×300mm) were connected in series, with a differential refractive index detector, column temperature of 40℃, mobile phase of pure water, flow rate of 0.60mL / min, injection volume of 20μL, elution method of isogradient elution, and run time of 45min.

[0091] II. Experimental Results

[0092] Analysis of crude polysaccharides from saponin rice before and after fermentation using high-performance gel permeation chromatography (HPGPC) revealed two distinct peaks for both GSP-u and GSP-f. Figure 2 In the figure, A) represents GSP-u-1 (retention time of 17.8 min), GSP-u-2 (retention time of 21.4 min), GSP-f-1 (retention time of 19.0 min), and GSP-f-2 (retention time of 23.88 min), respectively.

[0093] Homogeneous fractions GSP-u-1, GSP-u-2, GSP-f-1, and GSP-f-2 of saponin rice polysaccharides before and after fermentation were successfully purified using a Sephadex G-200 gel filtration chromatography column, and their molecular weights were determined. The results are as follows: Figure 2 As shown in B and C, the molecular weight of GSP-u-1 after fermentation increased from 3.01 × 10⁻⁶. 6 g / mol decreased to 1.86 × 10 6 g / mol (GSP-f-1), the molecular weight of GSP-u-2 ranges from 6.05 × 10 g / mol (GSP-f-1). 5 g / mol decreased to 1.48 × 10 5 The change in g / mol (GSP-f-2) is likely due to the hydrolysis of polysaccharide molecular chains by hemicellulase and mannanase secreted by Bacillus subtilis during fermentation. This trend is similar to the fermentation results of carrot polysaccharide, longan polysaccharide, and rice bran polysaccharide, indicating that fermentation has a certain degradation effect on polysaccharides, leading to a decrease in Mw.

[0094] After fermentation, the dry yield of crude polysaccharides from saponin rice increased from 47.67±1.27% to 79.50±2.78%. Based on the dry weight of the two crude polysaccharide components obtained before and after fermentation, the purified yields were: GSP-u-1 6.58±0.89%, GSP-u-2 42.3±1.96%, GSP-f-1 1.97±2.35%, and GSP-f-2 63.2±3.16%. The yield of GSP-f-2 was approximately 20.9% higher than that of GSP-u-2. This is likely because fermentation converts a large amount of insoluble polysaccharides into soluble polysaccharides, thus effectively increasing the polysaccharide yield.

[0095] Since the yields of GSP-u-1 and GSP-f-1 after purification were both very low and their activities were poor, the second purified components GSP-u-2 and GSP-f-2 were retained.

[0096] Example 4: Monosaccharide composition analysis of saponin rice polysaccharides before and after fermentation

[0097] I. Experimental Methods

[0098] Take 0.01g of saponin rice polysaccharide samples (GSP-u, GSP-f, GSP-u-2, GSP-f-2) into a high-temperature resistant hydrolysis glass tube, add 0.3mL of 0.5M trifluoroacetic acid, hydrolyze at 80℃ for 16h, take 0.1mL of hydrolysate and blow dry with nitrogen to obtain saponin rice polysaccharide hydrolysis products.

[0099] Take 5 mg of monosaccharide standards (D-ribose, L-arabinose, D-galactose, mannose, D-xylose, D-galacturonic acid, rhamnose, fructose, glucose, and glucuronic acid) and 5 mg of saponin rice polysaccharide hydrolysate, dissolve them in 0.2 mL of pyridine, add 0.1 mL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide), and then place in a 50 °C oven for derivatization reaction for 1 h. After the reaction is complete, add 1 mL of water and 5 mL of n-hexane, vortex extract for 5 min, centrifuge at 4000 r / min for 5 min, and use the supernatant as a membrane buffer for gas chromatography-quadrupole / time-of-flight mass spectrometry (GC-O-TOF-MS) for analysis.

[0100] II. Experimental Results

[0101] After fermentation with Bacillus subtilis, the mannose content of GSP-f increased significantly, while the galactose content decreased significantly, and the proportions of other monosaccharides changed relatively little. As shown in Table 1, compared with GSP-u, the proportions of mannose, glucose, and arabinose in GSP-f increased by approximately 12.60%, 1.50%, and 0.66%, respectively, while the galactose content decreased by approximately 14.13%.

[0102] Compared with GSP-u-2, GSP-f-2 showed an increase in the proportions of mannose, glucose, and arabinose by approximately 10.23%, 0.41%, and 0.88%, respectively, while the proportion of galactose decreased by approximately 11.06%. These results indicate that the fermentation process significantly altered the monosaccharide composition of saponin rice polysaccharides.

[0103] Table 1 Monosaccharide composition of saponin rice polysaccharide

[0104] Monosaccharides GSP-u GSP-f GSP-u-2 GSP-f-2 Arabic sugar (Ara) 0.02±0.001c 0.66±0.02a 0.031±0.01bc 0.88±0.03a Xyl sugar 0.79±0.05a 0.63±0.09a 0.84±0.10a 0.69±0.03a Mannose 49.21±1.14c 61.81±1.78a 51.02±1.46bc 61.25±0.48a Fructose (Fructose) 1.13±0.85a 1.16±0.67a 0.85±0.26a 1.11±0.36a Glucose (Glc) 2.14±0.71bc 3.64±0.57a 2.11±0.09c 2.52±0.15abc Galactose 45.16±2.38bc 31.03±0.41a 44.13±1.79c 33.07±0.64ac Glucuronic acid (GlcA) 1.52±0.27a 0.97±0.04a 1.02±0.02a 0.67±0.08a

[0105] Note: Different lowercase letters (a, b, and c) indicate significant differences in GSP within the same row (p < 0.05).

[0106] Example 5: Monosaccharide composition analysis of saponin rice polysaccharides before and after fermentation

[0107] I. Experimental Methods

[0108] Take samples of saponin rice polysaccharides (GSP-u, GSP-f, GSP-u-2, GSP-f-2) and perform Congo red testing according to step 5 in Example 1.

[0109] II. Experimental Results

[0110] Experimental results are as follows Figure 3 As shown, the maximum absorption wavelength of GSP-f-2 in 0 mol / L NaOH solution is 494 nm. With increasing NaOH concentration to 0.1 mol / L, the maximum absorption wavelength red-shifts to 507 nm. When the NaOH concentration further increases to 0.2 mol / L, the maximum absorption wavelength decreases significantly. This red-shift phenomenon indicates that GSP-f-2 has a triple-helix conformation.

[0111] In contrast, neither the original crude polysaccharide (Control) nor the purified polysaccharide from GSP-u showed changes in the characteristic absorption wavelengths of the triple helix structure, indicating that the fermentation process induced the formation of a stable triple helix structure, which was maintained during purification.

[0112] Example 6: Effects of saponin rice polysaccharides before and after fermentation on fruit fly lifespan and intestinal barrier function.

[0113] I. Experimental Methods

[0114] (1) Fruit fly population, culture and treatment

[0115] Wild-type Canton-S strain fruit flies (Drosophila melanogaster, D. melanogaster) were obtained from the Shanghai Institute of Biochemistry and Cell Biology. The fruit flies were reared in basal medium (20g corn flour, 12.5g sucrose, 10g yeast extract, 2.5g agar powder, 1.2mL propionic acid, and 300mL distilled water) under the following conditions: 25℃, relative humidity (55%), and a 12h light / 12h dark cycle.

[0116] Newly emerged first-generation fruit flies were collected. Female fruit flies (5 tubes per group, 20 flies per tube, 100 flies per group) were selected under CO2 anesthesia and cultured for 3 days. Then, they were transferred to small bottles containing different filter papers and cultured for 48 hours. The filter papers were changed every 12 hours.

[0117] Control group: Filter paper was soaked in a 5% sucrose solution (w / v);

[0118] Induction group (SDS group): Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v);

[0119] GSP-u group: filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL GSP-u polysaccharide solution;

[0120] GSP-f group: Filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL GSP-f polysaccharide solution;

[0121] GSP-u-2 group: filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL GSP-u-2 polysaccharide solution;

[0122] GSP-f-2 group: filter paper was soaked in 0.6% SDS solution (w / v) + 5% sucrose solution (w / v) + 2 mg / mL GSP-f-2 polysaccharide solution.

[0123] (2) Fruit fly lifespan detection

[0124] Record the natural time of death for each individual fruit fly in each test tube until all fruit flies have died, and calculate the mean lifespan (Mean), maximum lifespan (Max), and median lethal time (LT50) for each experimental group.

[0125] (3) The Smurfs Experiment

[0126] The Smurf experiment was conducted according to step 4 in Example 1.

[0127] II. Experimental Results

[0128] The results of the fruit fly lifespan experiment showed that the lifespan of fruit flies fed with GSP-u, GSP-f, GSP-u-2, and GSP-f-2 groups was significantly longer than that of fruit flies fed with the induced group. Furthermore, the lifespan of fruit flies fed with fermented triple-helix polysaccharides was longer than that of fruit flies fed with unfermented polysaccharides. Figure 4 (A) in the middle.

[0129] Compared with the induction group, the mean lifespan, maximum lifespan and median lethal time of the GSP-u group increased by 10.07% (p<0.001), 8% (p<0.01) and 11.33% (p<0.001), respectively.

[0130] However, the GSP-f group showed increases of 19.9% ​​(p<0.0001), 15.1% (p<0.0001), and 15.67% (p<0.0001) compared to the GSP-u group, respectively.

[0131] The GSP-u-2 group showed increases of 8.6% (p<0.01), 10.9% (p<0.001), and 13.8% (p<0.0001) compared to the GSP-u group, respectively. However, there was no significant difference between the GSP-f-2 group and the GSP-f group. Figure 4 (B in the middle).

[0132] The above results indicate that the fermented triple-helix polysaccharides GSP-f and GSP-f-2 can more effectively reduce the impact of SDS-induced inflammatory damage on the lifespan of Drosophila melanogaster, suggesting that the triple-helix structure can effectively enhance the effect of saponin rice polysaccharide on improving the lifespan of Drosophila melanogaster.

[0133] The results of the Smurf experiment showed that very few fruit flies in the blank control group developed intestinal leakage, while significant intestinal leakage occurred after SDS induction. Figure 4 As shown in C and D, the intestinal leakage rate in the GSP-u group and the GSP-f group decreased by 17.3% (p<0.001) and 30.47% (p<0.0001) respectively compared with the induced group. The GSP-f group further reduced the leakage rate by 13.17% (p<0.001) compared with the GSP-u group. The GSP-u-2 group also improved the improvement effect by 8.1% compared with the GSP-u group (p<0.01), but it was still not as significant as the improvement effect of triple-helix saponin rice polysaccharide GSP-f. This indicates that the triple-helix structure can more effectively protect the integrity of the inflamed intestine in fruit flies.

[0134] Example 7: Effects of Bacillus subtilis fermentation on the triple helix structure of other plant polysaccharides

[0135] I. Experimental Methods

[0136] Angelica sinensis, peppermint, Euryale ferox, Atractylodes macrocephala, Cornus officinalis, Asparagus cochinchinensis, Pueraria lobata, Trigonella foenum-graecum, and pomelo peel were taken separately. The plant samples were ground into powder and passed through a 60-mesh sieve. 4g of the plant sample powder was dissolved in 200mL of purified water and extracted for 12h. Then, it was pre-sterilized at 100℃ for 10min and the pH value was adjusted to 6.2 to obtain the water extract.

[0137] The activated Bacillus subtilis was inoculated at a rate of 2% (1×10⁻⁶). 9 The extract (CFU / mL) was added to the water extract, stirred and mixed thoroughly under aseptic conditions, and fermented at 30°C for 3 hours. An equal volume of sterile water was added as the unfermented group.

[0138] After fermentation, each solution was inactivated at 100℃ for 15 min and centrifuged at 4000 rpm for 10 min. The supernatant was collected and deproteinized using the Savage method. Then, it was precipitated overnight at 4℃ with 3 volumes of anhydrous ethanol. The precipitate was dissolved in distilled water, placed in a 3000 Da dialysis bag, and the water was changed every 3 hours. Dialysis was performed for 24 hours, and then freeze-dried to obtain crude polysaccharide (W represents polysaccharide before fermentation, and F represents polysaccharide after fermentation).

[0139] Perform Smurf detection and Congo Red testing according to steps 4 and 5 in Example 1.

[0140] II. Experimental Results

[0141] The results are as follows Figure 5As shown in Figures A through D, the maximum absorption wavelengths of the polysaccharides from fermented Angelica sinensis (1F), Euryale ferox (3F), Atractylodes macrocephala (4F), Asparagus cochinchinensis (7F), Citrus reticulata peel (8F), Trigonella foenum-graecum (9F), and Pueraria lobata (10F) all exhibit a red shift, indicating the formation of a triple helix structure. However, the polysaccharides from Mentha haplocalyx and Cornus officinalis do not exhibit a triple helix structure. Figure 5 (A to D in the original text).

[0142] Polysaccharides from Angelica sinensis, Euryale ferox, Atractylodes macrocephala, Asparagus cochinchinensis, Pueraria lobata, Trigonella foenum-graecum, and Citrus reticulata peel, which produced triple-helix structures after fermentation, were used in a fruit fly larvae experiment to detect intestinal leakage. The results showed that the therapeutic effects of the fermented triple-helix plant polysaccharides were superior to those of the unfermented polysaccharides. Figure 5 (E~F in the middle).

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of Bacillus subtilis as an additive in the preparation of triple-helix plant polysaccharides from plant raw materials that do not contain triple-helix polysaccharides, characterized in that, The plant materials include saponin rice, angelica, fox nut, atractylodes macrocephala, asparagus, pomelo peel, fenugreek, or kudzu root.

2. The application according to claim 1, characterized in that, The Bacillus subtilis is a Bacillus subtilis that can secrete hemicellulase and / or mannanase and forms an acidic environment after fermentation.

3. The application according to claim 2, characterized in that, The Bacillus subtilis strain is Bacillus subtilis CICC10088.

4. A method for preparing a triple-helix plant polysaccharide, characterized in that, After inoculating the plant aqueous extract with Bacillus subtilis, ferment for 2.5–3.5 hours; The plants mentioned include soapberry rice, angelica, foxnut, atractylodes macrocephala, asparagus, pomelo peel, fenugreek, or kudzu root.

5. The preparation method according to claim 4, characterized in that, The Bacillus subtilis is a Bacillus subtilis that can secrete hemicellulase and / or mannanase and forms an acidic environment after fermentation.

6. The preparation method according to claim 5, characterized in that, The Bacillus subtilis is Bacillus subtilis CICC 10088.

7. The triple-helix plant polysaccharide prepared by any one of claims 4 to 6.

8. The use of the triple-helix plant polysaccharide according to claim 7 in the preparation of products that protect the intestinal barrier and / or repair inflammatory damage to the intestinal barrier.

9. The use of the triple-helix plant polysaccharide according to claim 7 in the preparation of products for treating intestinal inflammatory damage.

10. A product for protecting the intestinal barrier, characterized in that, The product contains the triple-helix plant polysaccharide as described in claim 7.

Images