Application of kidney bean seed coat polysaccharide in preparation of product for improving intestinal flora of old people and / or inhibiting intestinal inflammation

Abstract

The invention discloses an application of kidney bean seed coat polysaccharide in preparation of a product for improving intestinal flora of old people and / or inhibiting intestinal inflammation, which is characterized in that the kidney bean seed coat polysaccharide is extracted by a citric acid extraction method, a cellulase extraction method and a chelating agent extraction method, and comprises the following steps: soaking and peeling white kidney beans; the method comprises the following steps: drying the seed coats in a hot air oven at 50-80 DEG C for 2-10 hours, grinding the dried seed coats with a high-speed grinder, sieving the ground seed coats to obtain white kidney bean seed coat fine powder, and extracting the kidney bean seed coat polysaccharide from the white kidney bean seed coat fine powder through citric acid, cellulase or ammonium oxalate. Particularly, the invention has potential benefits in the aspects of promoting butyric acid production and improving intestinal health of old people.

Description

Technical Field

[0001] The present invention belongs to the technical field of kidney bean polysaccharides, and specifically relates to the use of kidney bean seed coat polysaccharide in the preparation of a product for improving the intestinal flora of the elderly and / or inhibiting intestinal inflammation. Background Art

[0002] Polysaccharides are an important class of biomacromolecules with a wide range of biological activities and functional properties. Kidney beans are a common legume crop, rich in various bioactive components, such as vitamins, minerals, and amino acids. They are particularly suitable for patients with special conditions such as heart disease and hypokalemia, and also have anti-cancer and anti-cancer effects. Currently, research on kidney bean polysaccharides is quite common. For example, patent CN 110642962 B discloses a method for the isolation and purification of pectin polysaccharides from legumes. The experimental raw materials include red kidney beans, adzuki beans, and white lentils. This preparation method ultrafinely grinds the beans and then uses a series of physical methods to extract and purify the pectin, resulting in a rapid, efficient, and stable production of high-purity, high-quality pectin polysaccharides. Patent CN 118421726A discloses a method for the preparation and application of kidney bean polysaccharides with immune-enhancing properties. This preparation method addresses the problem of poor immune-regulating effects of kidney bean polysaccharides obtained using only α-amylase enzymatic hydrolysis, which is a common technique in existing technologies. The preparation method comprises the following steps: grinding the cooked kidney beans into pulp, subjecting them to composite enzymatic hydrolysis and composite strain fermentation, and then purifying them to obtain kidney bean polysaccharides. Subsequently, animal experiments are conducted to verify the immunomodulatory effect, which proves that the kidney bean polysaccharides can enhance immunity by affecting the proliferation capacity of spleen lymphocytes, promoting the production of mouse antibodies, and other pathways.

[0003] As an important byproduct of legumes, kidney bean seed coats possess rich biological activity and nutritional value. Polysaccharides with excellent properties can be extracted from these products. These polysaccharides have antioxidant, immune-enhancing, anti-tumor, anti-viral, anti-radiation, lipid-lowering, blood sugar-lowering, and anti-aging properties. They can also regulate intestinal flora and promote intestinal health. Currently, research on kidney bean seed coat polysaccharides is limited, and no literature has reported the extraction of polysaccharides from kidney bean seed coats for improving intestinal health in the elderly. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an application of kidney bean seed coat polysaccharide in the preparation of a product for improving the intestinal flora of the elderly and / or inhibiting intestinal inflammation.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: use of kidney bean seed coat polysaccharide in the preparation of products for improving the intestinal flora of the elderly and / or inhibiting intestinal inflammation.

[0006] Furthermore, the kidney bean seed coat polysaccharide is obtained by citric acid extraction, and its monosaccharide composition and molar ratio are trehalose (Fuc) 0.36, rhamnose (Rha) 3.10, arabinose (Ara) 18.94, galactose (Gal) 9.31, glucose (Glc) 1.52, xylose (Xyl) 63.47, galacturonic acid (GalA) 2.84, and gluconic acid (GlcA) 0.46, and the unit is %.

[0007] Furthermore, the citric acid extraction method comprises the following specific steps:

[0008] Step 1, preparing kidney bean seed coat fine powder: soaking and peeling white kidney beans, drying the seed coats in a hot air oven at 50-80°C for 2-10 hours, grinding with a high-speed grinder, and sieving to obtain white kidney bean seed coat fine powder;

[0009] Step 2, extracting kidney bean seed coat polysaccharide with citric acid: dissolving the white kidney bean seed coat fine powder obtained in step 1 in deionized water 10-20 times its weight, adjusting the pH value to 2 with 3M citric acid, performing ultrasonic crushing and extraction, heating to 60-80°C, stirring and incubating for extraction, filtering and centrifuging after the extraction is completed, taking the supernatant for ethanol precipitation and then centrifuging, taking the precipitate for secondary alcohol precipitation, washing with anhydrous ethanol 2-3 times, and drying the obtained wet polysaccharide to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

[0010] Furthermore, the kidney bean seed coat polysaccharide is obtained by cellulase extraction, and its monosaccharide composition and molar ratio are trehalose (Fuc) 0.4, rhamnose (Rha) 2.83, arabinose (Ara) 9.74, galactose (Gal) 11.13, glucose (Glc) 51.92, xylose (Xyl) 20.77, galacturonic acid (GalA) 2.83, and gluconic acid (GlcA) 0.38, and the unit is %.

[0011] Furthermore, the cellulase extraction method comprises the following specific steps:

[0012] Step 1, preparing kidney bean seed coat fine powder: soaking and peeling white kidney beans, drying the seed coats in a hot air oven at 50-80°C for 2-10 hours, grinding with a high-speed grinder, and sieving to obtain white kidney bean seed coat fine powder;

[0013] Step 2, cellulase extraction of kidney bean seed coat polysaccharide: the white kidney bean seed coat fine powder prepared in step 1 is dissolved in deionized water 10-20 times its weight, the pH is adjusted to 5 with 3M citric acid, 3wt% of cellulase by weight of the white kidney bean seed coat fine powder is added, the mixture is heated to 50-60°C, stirred and incubated for extraction, and then filtered and centrifuged. The supernatant is ethanol precipitated and centrifuged, the precipitate is subjected to secondary alcohol precipitation, washed with anhydrous ethanol 2-3 times, and the obtained wet polysaccharide is dried to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

[0014] Furthermore, the kidney bean seed coat polysaccharide is obtained by chelating agent extraction method, and its monosaccharide composition and molar ratio are rhamnose (Rha) 1.32, arabinose (Ara) 15.57, galactose (Gal) 5.58, glucose (Glc) 1.45, xylose (Xyl) 73.44, galacturonic acid (GalA) 2.26, gluconic acid (GlcA) 0.38, and the unit is %.

[0015] Furthermore, the specific steps of the chelating agent extraction method are as follows:

[0016] Step 1, preparing kidney bean seed coat fine powder: soaking and peeling white kidney beans, drying the seed coats in a hot air oven at 50-80°C for 2-10 hours, grinding with a high-speed grinder, and sieving to obtain white kidney bean seed coat fine powder;

[0017] Step 2, ammonium oxalate extraction of kidney bean seed coat polysaccharide: the white kidney bean seed coat fine powder obtained in step 1 is dissolved in 50mM ammonium oxalate solution at a mass ratio of 1:12-18, heated to 60-80°C, stirred and incubated for extraction, filtered and centrifuged, the supernatant is ethanol precipitated and then centrifuged, the precipitate is subjected to secondary alcohol precipitation, washed with anhydrous ethanol 2-3 times, and the obtained wet polysaccharide is dried to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] 1. The present invention adopts three different methods to extract kidney bean seed coat polysaccharides, each method has its own characteristics, such as citric acid extraction helps to improve the emulsification properties of kidney bean polysaccharides; cellulase extraction helps to retain the natural structure and function of polysaccharides; ammonium oxalate extraction helps to dissolve high molecular weight polysaccharides.

[0020] 2. The prepared polysaccharides of the present invention are used to conduct in vitro fecal fermentation experiments and cell experiments on the elderly, and to measure the changes in the pH of the fermentation liquid, changes in the intestinal flora, dynamic changes in monosaccharide composition, dynamic changes in SCFA production, and determination of pro-inflammatory and anti-inflammatory factors, thereby proving that the polysaccharide has a regulatory effect on the intestinal microbial community structure and metabolic function of the elderly, especially its potential benefits in promoting butyrate production and improving the intestinal health of the elderly. It is expected to provide a safe and effective intervention method for improving the intestinal function of the elderly, and show unique advantages in regulating the intestinal flora of the elderly. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Fourier transform infrared spectra of three polysaccharides: AEP, EEP and CEP;

[0022] Figure 2 SAXS scattering patterns (A), radial distribution function (RDF) patterns (B) and Kratky plots (C) of three polysaccharides: AEP, EEP and CEP;

[0023] Figure 3 Scanning electron microscope images of three polysaccharides: AEP, EEP, and CEP, where A1: AEP magnification

[0024] ×5000, A2: AEP magnification ×10,000, B1: EEP magnification ×5000, B2: EEP magnification

[0025] ×10,000, C1: CEP magnification ×5000, C2: CEP magnification ×10,000;

[0026] Figure 4 is the change of pH during in vitro fermentation;

[0027] Figure 5 The Venn diagram of the number of special OTUs in different polysaccharide samples at different fermentation time points;

[0028] Figure 6 is the α-diversity index of different polysaccharide samples at different fermentation time points;

[0029] Figure 7 PCoA score diagram of different polysaccharide samples;

[0030] Figure 8 is the relative abundance of intestinal microbial communities at the phylum level in different groups (%);

[0031] Figure 9 Heat map of the relative abundance changes of intestinal microbial communities in different groups;

[0032] Figure 10is the relative abundance of intestinal microbial communities at the genus level in different groups (%);

[0033] Figure 11 Heat map of the relative abundance changes of intestinal microbial communities in different groups;

[0034] Figure 12 The changes in the content of short-chain fatty acids (SCFAs) in different groups during fermentation are shown in the figure (ac). Different letters indicate significant differences between them.

[0035] Figure 13 Western blotting was used to detect the gene expression levels of tight junction proteins in HT-29 cells in the inflammatory intestinal injury model and the effects of each group of samples on the expression of tight junction proteins in HT-29 cells in the inflammatory intestinal injury model, where #: p < 0.05 compared with the Control group; ##: p < 0.01 compared with the Control group; *: p < 0.05 compared with the LPS group; **: p < 0.01 compared with the LPS group; ***: p < 0.001 compared with the LPS group; ns: no significant difference compared with the LPS group.

[0036] Figure 14 The figures show the effects of each group of polysaccharides on the release of inflammatory factors by HT-29 cells in the inflammatory damage intestinal model, where #: p < 0.05 compared with the Control group; ##: p < 0.01 compared with the Control group; *: p < 0.05 compared with the LPS group; **: p < 0.01 compared with the LPS group; ***: p < 0.001 compared with the LPS group; ns: no significant difference compared with the LPS group. DETAILED DESCRIPTION

[0037] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0038] Specific Example 1: Preparation of kidney bean seed coat polysaccharide.

[0039] Example 1: Preparation of kidney bean seed coat polysaccharide by citric acid extraction method, comprising the following steps:

[0040] Step 1. Preparation of kidney bean seed coat powder: White kidney beans are produced in Chaoyang City, Liaoning Province, China. The white kidney beans are soaked and peeled, and the seed coats are dried in a hot air oven at 60°C for 2-10 hours. The dried kidney bean seed coats are ground using a high-speed grinder and sieved through an 80-mesh sieve to obtain fine white kidney bean seed coat powder.

[0041] Step 2, citric acid extraction of kidney bean seed coat polysaccharide: the white kidney bean seed coat fine powder prepared in step 1 is dissolved in deionized water 15 times its weight, the pH value is adjusted to 2 with 3M citric acid, ultrasonically crushed and extracted for 30 minutes, heated to 70°C, stirred and incubated for extraction for 3 hours. During the experiment, the beaker is covered with plastic wrap to prevent solvent evaporation; after the extraction is completed, it is filtered and centrifuged, and the supernatant is evaporated to 1 / 3 of the original volume and then added with 70% ethanol. After storing at 4°C for 12 hours, it is centrifuged at 10,000 rpm for 10 minutes, and the precipitate is subjected to secondary alcohol precipitation for 12 hours, and then washed with anhydrous ethanol 2-3 times to remove impurities. The resulting wet polysaccharide is then dried in a hot air oven at 55°C to obtain polysaccharide AEP.

[0042] Example 2: Preparation of kidney bean seed coat polysaccharide by cellulase extraction, comprising the following steps:

[0043] Step 1, same as in above embodiment 1;

[0044] Step 2: Cellulase extraction of kidney bean seed coat polysaccharide: white kidney bean seed coat powder was dissolved in deionized water 15 times its weight, the pH was adjusted to 5 with 3M citric acid, 3 wt% of cellulase by weight of white kidney bean seed coat powder was added, the mixture was heated to 55°C, stirred and incubated for extraction for 3 hours, and the beaker was covered with plastic wrap during the experiment to prevent solvent evaporation. After the extraction was completed, it was filtered and centrifuged, and the supernatant was evaporated to 1 / 3 of the original volume and 70% ethanol was added. After storage at 4°C for 12 hours, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was subjected to secondary alcohol precipitation for 12 hours. It was then washed 2-3 times with anhydrous ethanol to remove impurities, and the resulting wet polysaccharide was dried in a hot air oven at 55°C to obtain polysaccharide EEP.

[0045] Example 3: Extraction of kidney bean seed coat polysaccharide by chelating agent extraction method, comprising the following steps:

[0046] Step 1, same as in above embodiment 1;

[0047] Step 2, chelating agent extraction of kidney bean seed coat polysaccharide: the white kidney bean seed coat fine powder obtained in step 1 is dissolved in 50mM ammonium oxalate solution at a mass ratio of 1:15, heated to 70°C, stirred and incubated for extraction for 3 hours. During the experiment, the beaker is covered with plastic wrap to prevent solvent evaporation; after the extraction is completed, it is filtered and centrifuged, and the supernatant is evaporated to 1 / 3 of the original volume and then added with 70% ethanol. After storing at 4°C for 12 hours, it is centrifuged at 10,000 rpm for 10 minutes, and the precipitate is subjected to secondary alcohol precipitation for 12 hours, washed with anhydrous ethanol 2-3 times to remove impurities, and then the obtained wet polysaccharide is dried in a hot air oven at 55°C to obtain polysaccharide CEP.

[0048] Specific Example 2: Physical and Chemical Characterization of Kidney Bean Seed Coat Polysaccharide

[0049] 1. Monosaccharide composition and content analysis

[0050] 10 mg of the three kidney bean seed coat polysaccharide samples prepared in Specific Example 1 and 4 mL of 2M trifluoroacetic acid were placed in a hydrolysis tube. The tube was filled with nitrogen for 1 minute to expel air, then sealed and hydrolyzed at 120°C for 1 hour. The mixture was dried with nitrogen to remove excess acid, diluted, and filtered through a 0.2 μm filter. The monosaccharide composition of the polysaccharide samples was analyzed using a DIONEX ICS-3000 (USA) with a CarboPac™ PA20 3×150 mm analytical column, a column temperature of 35°C, 250 mM NaOH and 1 M NaAC as eluents, a flow rate of 0.5 mL / min, and a 10 μL injection volume. The results are shown in Table 1. As can be seen from the table, the monosaccharide compositions of AEP, EEP, and CEP mainly include xylose (Xyl), arabinose (Ara), galactose (Gal), galacturonic acid (GalA), rhamnose (Rha), glucose (Glc), gluconic acid (GlcA), and trehalose (Fuc). The molar ratios of these monosaccharides are different. The molar ratios of Xyl and Ara are higher in AEP and CEP, indicating that Xyl and Ara may be the main components of the AEP and CEP skeletons. This may be because the glycosidic bond formed by Xyl and Ara is very sensitive to acidic hydrolysis, resulting in more release; while the Gla content is the highest in EEP.

[0051] Table 1 Monosaccharide molar ratio (%)

[0052]

[0053] 2. Molecular weight analysis

[0054] The molecular weight of kidney bean seed coat polysaccharides was determined using SEC-MALLS analysis on an Agilent 1260 Series HPLC system with an Optilab rEX refractive index detector. The chromatographic column was a Shodex 806 column, the mobile phase consisted of 0.1 M sodium chloride solution, at a flow rate of 0.5 mL / min, and the injection volume was 100 μL. Data were collected and analyzed using online Astra software. The results are shown in Table 2. The polydispersity indices of AEP, EEP, and CEP were 1.65, 1.32, and 1.04, respectively, indicating that AEP had the broadest molecular weight distribution.

[0055] Table 2 Molecular weight and dispersion coefficient of polysaccharides

[0056]

[0057] Specific Example 3: Structural characteristics of kidney bean seed coat polysaccharide.

[0058] 1. Fourier transform infrared spectroscopy

[0059] The three kidney bean seed coat polysaccharide samples prepared in the specific embodiment 1 were fully dried in an oven at 105°C, sieved through 200 mesh, and then evenly ground with pure KBr at a ratio of 1:100. After tableting, the tablets were placed on a Fourier transform infrared spectrometer scanner at 4000-400 cm -1 32 scans were performed within the range. Data were collected and processed using offline OMMIC software. Figure 1 , the visible wavelength range of infrared spectrum is 3279-3355cm -1 The peaks at 2926-2946 cm -1 The peak is the characteristic absorption peak of polysaccharide.

[0060] 2. Small-angle X-ray scattering

[0061] The kidney bean seed coat polysaccharide sample was dissolved and centrifuged at 10,000 rpm for 30 min to remove insoluble matter. 80 μL of the supernatant was taken to remove bubbles and placed on the SAXA instrument at 25°C and a sample-to-detector distance of 540.74 mm. Data points were acquired using 3×300 s exposures and analyzed using GIFT software. Figure 2 .Depend on Figure 2 Middle AC shows the effect of different extraction methods on the polysaccharide structure, and it was found that the CEP polysaccharide had the most compact conformation.

[0062] 3. Scanning electron microscope

[0063] The kidney bean seed coat polysaccharide sample was fixed on the sample stage, coated with gold powder under vacuum conditions, and photographed using a SEM at an accelerating potential of 5kV with magnifications of 5000× and 10,000×. Figure 3 The figure shows the differences in the chemical structures of different polysaccharides. Ultrasonic treatment may make the surfaces of the three polysaccharides rougher. Among them, the surface of CEP is the loosest and presents wavy wrinkles, which increases its surface area and exposes more polar groups such as hydroxyl and carboxyl groups, as well as other water binding sites, which is conducive to the adsorption and binding of water.

[0064] Specific Example 4: Effects of kidney bean seed coat polysaccharide on intestinal health and intestinal microbial composition in the elderly.

[0065] The three polysaccharides AEP, EEP, and CEP prepared in Specific Example 1 and two commercially available polysaccharides SLP and SHP (purchased from Sigma-Aldrich) were subjected to an in vitro fecal fermentation experiment. Two groups of elderly people (CO) and young people (CY) were set up as blank control groups, and kidney bean seed coat was used to verify the effects of polysaccharides on the intestinal health and intestinal microbial composition of the elderly.

[0066] 1. Experimental methods

[0067] (1) Preparation of fecal inoculum: Fecal samples were collected from three healthy elderly individuals who had not received antibiotic treatment for at least 3 months. Feces were collected in plastic bags, air-deprived, and sealed. The feces were homogenized with sterile phosphate-buffered saline (pH 7.4) to obtain a 10% w / v fecal suspension. The feces were magnetically stirred for 15 minutes, and then filtered through gauze. All operations were performed under an anaerobic workbench (gas phase: 80% N2, 10% CO2, 10% H2). Three replicate fecal inocula were obtained from three donors.

[0068] (2) Preparation of basal nutrient medium: 2.0 g peptone, 2.0 g yeast, 0.1 g NaCl, 40.0 mg K2HPO4, 40.0 mg KH2PO4, 10.0 mg MgSO4, 10.0 mg CaCl2, 2.0 g NaHCO3, 0.5 g cysteine-HCl, 0.5 g bile salt, 2.0 mL Tween-80, 5.0 μL vitamin K1, and 20.0 mg hemin were dissolved in deionized water to a volume of 1 L, and then the pH was adjusted to 7.4 with 0.1 mol / L HCl solution.

[0069] (3) In vitro fecal fermentation: 50 mg of kidney bean seed coat polysaccharide sample and 1 mL of fecal inoculum were accurately added to 9 mL of basal nutrient medium. Under the same conditions, basal nutrient medium without any carbon source was used as a blank control (CON), and oligofructose (FOS) was used as a positive control. All fermentation experiments were performed in triplicate. Fermentation was carried out in an incubator shaker at 37°C and 120 rpm. Fermentation samples were collected 0, 12, 24, and 36 hours after the start, with three samples collected for each group.

[0070] (5) Index determination method:

[0071] A. Fermentation broth pH: The fermentation broth was centrifuged at 9000 g for 20 min, the supernatant was collected, and the pH of each sample was measured using a pH meter.

[0072] B. DNA Extraction and 16S rRNA Gene Sequencing: One sample from each group was centrifuged at 3000 rpm for 10 min, snap-frozen in liquid nitrogen, and stored at −80°C for microbial analysis. DNA was extracted from the samples using the Omega Bio-Tek DNA Extraction Kit. DNA concentration was determined using the Qubit dsDNA HS Assay Kit and a Qubit 4.0 Fluorometer, and quality was assessed by 1% agarose gel electrophoresis. Full-length 16S rRNA genes were amplified from genomic DNA using primers 27F: AGRGTTTGATYNTGGCTCAG and 1492R: TASGGHTACCTTGTTASGACTT. The amplified products were sequenced in multiplex using forward and reverse primers with sample-specific PacBio barcodes. Sequencing data were detected and chimeric sequences removed using the UCHIME algorithm (v8.1) to obtain clean reads. Subsequently, sequences with a similarity ≥97% were clustered into operational taxonomic units (OTUs) using USEARCH (v10.0). Finally, representative reads were annotated based on the Naive Bayes classifier in QIIME2 combined with the SILVA database, and the confidence threshold was set at 70%.

[0073] C. Carbohydrate degradation: One sample was taken from each group, and the degree of carbohydrate degradation in different fermentation groups was determined using an ion chromatograph DIONEX ICS-3000 from Dionex, USA.

[0074] D. Short-chain fatty acids: Place 1 mL of sample into a 20 mL glass vial, add 104 μL of 6 M hydrochloric acid and 105 μL of 500 ppm 2-ethylbutyric acid. Prepare an external standard solution with 10-1000 ppm of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid. Short-chain fatty acids were determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry. The sample was adsorbed on a solid-phase extraction head at 40°C for 30 minutes, followed by thermal desorption at the inlet for 3 minutes. The desorbed substances were separated on a DB-WAXUI column using helium as the carrier gas at a flow rate of 1 mL / min. The inlet and ion source temperatures were 250°C, and the gas chromatograph oven temperature was set as follows: 0-10 minutes, 50-100°C, 10-14 minutes, 100-120°C, 5°C / min, 14-39 minutes, 120-150°C, 2°C / min, 39-56 minutes, and 150-220°C, 10°C / min. The ionization voltage was 70 eV, and the emission current was 35 μA. Short-chain fatty acid content is expressed as mmol / 10 mL of fermentation broth.

[0075] (4) Statistical methods: The statistical differences between the data were analyzed by analysis of variance (ANOVA) and Tukey test at a significance level of 5% using SPSS 21.0 software, and the graphics were drawn using Origin 9.0 software.

[0076] 2. Analysis of experimental results

[0077] (1) pH changes during in vitro fermentation

[0078] The pH changes during in vitro fermentation are shown in Figure 4 .Depend on Figure 4 The effects of polysaccharides with different structures on pH values showed significant differences. The pH value of each group showed a downward trend throughout the fermentation process, with the AEP and EEP groups experiencing a particularly sharp decline. This suggests that the production of more organic acids during fermentation may promote intestinal microbial activity. In contrast, the pH value of the CO and CY groups decreased more slowly, indicating lower acid production during fermentation. These results suggest that polysaccharides with different structures have different regulatory effects on the intestinal microenvironment during fermentation. The accumulation of acidic products helps inhibit the growth of harmful bacteria and maintain intestinal health, with potential positive effects on the intestinal microecological balance in the elderly.

[0079] (2) Analysis of intestinal flora diversity

[0080] like Figure 5 As shown, the CO and CY groups initially contained 158 and 201 unique OTUs, respectively. As fermentation time increased, the number of unique OTUs in the CY group decreased significantly, while the CO group changed more slowly. When CEP was used as the primary carbon source, the number of unique OTUs decreased to 51 after 24 hours of fermentation, indicating a significant decrease in species abundance, with a greater magnitude than in the other groups. These results demonstrate that different carbon sources have different effects on the structure of the intestinal microbiota.

[0081] Figure 6 It shows that the intestinal microbial community has formed obvious diversity under the intervention of polysaccharides with different structural characteristics. With the extension of fermentation time, the richness, diversity, uniformity and genetic diversity of each group of microbial communities have decreased to varying degrees. Figure 6 It can be seen that AEP significantly reduced the α-diversity of microorganisms compared with other groups. PCoA analysis of known data yielded Figure 7 ,like Figure 7 As shown, the intestinal flora compositions of the non-fermented CO group and the CY group were different. The intestinal flora of each group was relatively different in the early stage of fermentation, indicating that there were obvious differences in the fermentation effects of different polysaccharides, while the intestinal flora compositions of each group were similar in the late stage of fermentation.

[0082] (3) Regulation of polysaccharides on the intestinal microbiome at the phylum level

[0083] Figure 8 、 9 The changes in the composition and relative abundance of intestinal microbial communities at the phylum level under different polysaccharide treatment groups are shown. Figure 8 The relative abundance distribution of different microbial phyla in each treatment group is shown in a stacked bar chart. During the initial fermentation phase, the gut microbial community of elderly individuals was dominated by four bacterial phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidota. The combined relative abundance of these phyla exceeded 90%. As fermentation progressed, the microbial community structure shifted significantly, particularly in the polysaccharide-free control fermentation, where the abundance of Actinobacteria decreased significantly while that of Proteobacteria increased. Figure 9 Heatmaps, combined with cluster analysis, visually visualized the similarities and differences in microbial communities across treatment groups. The results demonstrate that polysaccharide fermentation in the intestinal environment not only influences the proliferation of specific bacterial communities but also leads to significant changes in the composition of the intestinal microbiome. These data further reveal the regulatory effects of polysaccharides and their derivatives on intestinal microbial communities, contributing to a deeper understanding of the functions and mechanisms of polysaccharides in intestinal health in the elderly.

[0084] (4) Regulation of polysaccharides on the intestinal microbiome at the genus level

[0085] Figure 10 、 11 The changes in the composition and relative abundance of the intestinal microbial community at the genus level under different polysaccharide treatment groups are shown. Figure 10 The relative abundance distribution of different microbial genera in each treatment group is presented in a stacked bar chart. During the initial fermentation phase, differences in the genus composition of the microbiota between the elderly and young individuals were observed. For example, the relative abundance of Escherichia coli in the intestinal microbiota of the elderly was 0.16%, and that of Eubacterium spp. was 11.08%. In the young individuals, the relative abundance of Escherichia coli was 2.15%, and that of Eubacterium spp. reached 18.52%. During the fermentation process, the structure of the intestinal microbial community changed significantly. The relative abundance of Escherichia coli and Bifidobacterium spp. in the intestines of both elderly and young individuals varied over time. E. coli dominated under carbon-free conditions, while Bifidobacterium spp. increased under specific polysaccharide conditions. AEP and EEP promoted the growth of Bifidobacterium spp. and inhibited E. coli. In the CEP group, Clostridium spp. increased, affecting the abundance of Bifidobacterium spp. These changes reflect the regulatory effects of polysaccharides on the intestinal microbial community. Figure 11The heat map further illustrates the relative abundance of E. coli, which gradually shifts from dark blue in the early stages of fermentation to light red in the later stages, indicating that the abundance of E. coli increases or decreases over time. Overall, these data highlight the significant regulatory effects of different polysaccharides on the gut microbiome in the elderly, particularly regarding the dynamic balance between Bifidobacterium and E. coli.

[0086] (5) Dynamic changes in monosaccharide composition during fermentation

[0087] Table 3 shows the changes in monosaccharide content during polysaccharide fermentation in different groups. Ara, Gal, Glc, and GalA levels gradually decreased with fermentation time, indicating that intestinal microorganisms effectively utilized these monosaccharides. Microorganisms in the SHP group significantly prioritized the consumption of Glc and GalA. Ara utilization exceeded 60% in all groups, while utilization rates for Gal and Glc also exceeded 50%, promoting intestinal microbial metabolic activity. GalA utilization ranged from 20% to 72%, demonstrating the polysaccharide structure's facilitation of microbial fermentation and metabolism, potentially boosting intestinal metabolic function in the elderly.

[0088] Table 3 Dynamic changes in the contents of SLP, SHP, AEP, EEP and CEP monosaccharides during in vitro fermentation

[0089]

[0090] (6) Dynamic changes in SCFA production during fermentation

[0091] like Figure 12 As shown, Figure 12 Figure 2 shows changes in short-chain fatty acid (SCFA) content during fermentation in different polysaccharide-treated groups. With prolonged fermentation, the levels of various SCFAs increased significantly, indicating efficient carbohydrate utilization by the gut microbiota. SCFAs, such as acetate, propionate, and butyrate, are the primary products of dietary fiber fermentation and have metabolic and immunomodulatory functions. Acetate and butyrate levels increased significantly in the AEP group, likely related to increased relative abundance of Bifidobacterium and Clostridium freundii. Propionate and isobutyrate levels changed less, indicating limited differences between groups. Valeric and isovaleric acid levels were low, primarily derived from protein fermentation. The increase in SCFAs reflects the prebiotic effect of polysaccharides, with the AEP group containing the highest total SCFA content. After 36 hours of fermentation, the SCFAs / BCFAs ratio in the AEP and CEP groups was significantly higher than in the other groups, indicating that the gut microbiota gradually transitioned from carbohydrate fermentation to partial protein metabolism and began to utilize more protein as a substrate. This was particularly evident in specific microbial community structures and fermentation environments.

[0092] Specific Example 5: Effects of kidney bean seed coat polysaccharide on intestinal related cells in the elderly.

[0093] By exploring the effects of LPS-induced HT-29 cell viability and TNF-α release, a cell model suitable for studying intestinal inflammation in the elderly was established. The polysaccharide AEP, which performed best in in vitro fermentation experiments, and the polysaccharide SCP, which had a larger molecular weight, were selected for the experiment. The elderly (CO) group served as the control group and the control group served as the blank group. The effects of intestinal flora fermentation polysaccharide products on TNF-α release, tight junction protein expression, and inflammatory factor release in HT-29 cells were analyzed, revealing the potential protective mechanism of white kidney bean polysaccharides on intestinal health in the elderly at the cellular level, in order to provide theoretical support and practical basis for improving intestinal health in the elderly and developing functional foods for the elderly.

[0094] 1. Experimental methods

[0095] (1) Cell culture: HT-29 cells were cultured in a medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in a humidified incubator at 37°C with 5% CO2. When the cells reached 80% to 90% confluency, they were subcultured for subsequent experiments.

[0096] (2) Immunoblotting to detect protein expression: Collect cells from each group and add appropriate amount of RIPA lysis buffer to lyse for 30 minutes, then centrifuge at 4°C and 1200 r / min for 15 minutes to collect the supernatant. The total cell protein concentration was measured by BCA method, and proteins were separated using 12% SDS-PAGE gel. The proteins were transferred to PVDF membrane and blocked in 5% BSA solution at room temperature for 1 hour. After blocking, the PVDF membrane was washed with TBST buffer and the primary antibodies: Claudin-1 (1:1000), Claudin-3 (1:1000), Occludin (1:2000), Zonulin-1 (1:1000), Zonulin-3 (1:1000) were added and incubated at 4°C overnight. The PVDF membrane was removed from the primary antibody, washed with TBST buffer, and incubated in anti-rabbit IgG (1:20000) secondary antibody at room temperature in the dark for 1 hour. After washing again, the membrane was placed in the Odyssey two-color infrared fluorescence imaging system for development, and GAPDH was used as the internal reference protein, and the data were analyzed using Image J software.

[0097] (3) Inflammatory factor determination: Cells were incubated in the same groups as above. The supernatant was collected and the levels of pro-inflammatory factors TNF-α, IL-6, IL-8, and IL-12 and anti-inflammatory factors IL-10 and TGF-β were quantified by ELISA according to the manufacturer's instructions.

[0098] (4) Statistical methods: The experimental data were analyzed and processed by GraphPad Prism software and then plotted. The differences were analyzed by analysis of variance (ANOVA) using SPSS 17.0 software at a significance level of 5%.

[0099] 2. Analysis of experimental results

[0100] (1) Effects of intestinal microbiota polysaccharide fermentation products on the expression of tight junction proteins in HT-29 cells

[0101] Figure 13 The figure shows the effect of each group of polysaccharide samples on the tight junction proteins of HT-29 cells induced by LPS. Figure 13 Compared with the control group, the gene expression levels of key tight junction proteins such as Claudin-3, Claudin-1, Occludin, ZO-3, and ZO-1 in the LPS model group were generally significantly decreased (p < 0.05). Subsequently, the mice were treated with polysaccharide products produced by microbial fermentation at different stages. Furthermore, the expression of tight junction proteins Claudin-3, Claudin-1, Occludin, ZO-3, and ZO-1 in each group showed varying degrees of increase (p < 0.05). In the AEP group, the expression levels of these five proteins were significantly increased (p < 0.05), and the effect was more pronounced at the end of fermentation (T36-AEP) compared to the start of fermentation (T0-AEP). Therefore, polysaccharide products produced by microbial fermentation can protect against LPS-induced intestinal barrier damage by upregulating the expression of tight junction proteins between intestinal epithelial cells, which is of great significance for regulating the intestinal immune system and protecting intestinal barrier integrity in the elderly.

[0102] (2) Effects of intestinal flora polysaccharide fermentation products on the release of inflammatory factors from HT-29 cells

[0103] Figure 14 The results show that each group of polysaccharide samples has anti-inflammatory effects on the LPS-induced HT-29 cell inflammation model. Figure 14It can be seen that compared with the Control group, LPS caused a significant increase in the levels of proinflammatory factors TNF-α, IL-6, IL-8 and IL-12 in HT-29 cells (p<0.05). Subsequently, after 24 hours of treatment with fermentation products in each group, the release of TNF-α, IL-6, IL-8 and IL-12 in the CO group did not change significantly (p>0.05), while the levels of proinflammatory factors TNF-α, IL-6, IL-8 and IL-12 in the groups with added polysaccharides decreased to varying degrees. The study found that this may reduce the release of TNF-α and IL-6 by inhibiting the activation of NF-κB; the AEP group performed better in inhibiting IL-8, and the downregulation effect at T36-AEP was better than that at T0-AEP. The study found that this may reduce the secretion of IL-8 by inhibiting the MAPK pathway; the SCP group had the most significant inhibitory effect on IL-12. The study found that this may reduce the level of IL-12 by inhibiting the JAK / STAT pathway.

[0104] In addition, the levels of anti-inflammatory factors IL-10 and TGF-β were also measured. IL-10 and TGF-β are key anti-inflammatory factors that inhibit the release of pro-inflammatory factors mainly by inhibiting NF-κB and JAK / STAT pathways, such as Figure 14 LPS stimulation was found to upregulate IL-10 and TGF-β levels, leading to a compensatory increase in anti-inflammatory factors to suppress excessive inflammation. The AEP group showed a significant increase in IL-10 and TGF-β levels, and the upregulation of these two inflammatory factors was significantly greater at T36-AEP than at T0-AEP. Furthermore, SCP also promoted the release of IL-10. Overall, fermentation products at different stages have an inhibitory effect on LPS-induced inflammatory responses and a protective effect on the intestinal barrier in the elderly.

[0105] In summary, the regulatory effect of kidney bean seed coat polysaccharide on intestinal microbiota was evaluated through an in vitro anaerobic fermentation model in the elderly. It was found that it can promote the growth of beneficial bacteria such as Bifidobacterium, inhibit the proliferation of Escherichia coli, and increase the production of short-chain fatty acids such as butyrate, showing its potential as a prebiotic, which can help enhance the intestinal barrier function and improve the intestinal health of the elderly.

[0106] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. Use of kidney bean seed coat polysaccharide in the preparation of a product for improving the intestinal flora and / or inhibiting intestinal inflammation in the elderly.

2. The use according to claim 1, characterized in that: The kidney bean seed coat polysaccharide is obtained by citric acid extraction, and its monosaccharide composition and molar ratio are trehalose 0.36, rhamnose 3.10, arabinose 18.94, galactose 9.31, glucose 1.52, xylose 63.47, galacturonic acid 2.84, and gluconic acid 0.46, and the unit is %.

3. The use according to claim 2, characterized in that The citric acid extraction method comprises the following steps: Step 1, preparing kidney bean seed coat fine powder: soaking and peeling white kidney beans, drying the seed coats in a hot air oven at 50-80°C for 2-10 hours, grinding with a high-speed grinder, and sieving to obtain white kidney bean seed coat fine powder; Step 2, extracting kidney bean seed coat polysaccharide with citric acid: dissolving the white kidney bean seed coat fine powder obtained in step 1 in deionized water 10-20 times its weight, adjusting the pH value to 2 with 3M citric acid, performing ultrasonic crushing and extraction, heating to 60-80°C, stirring and incubating for extraction, filtering and centrifuging after the extraction is completed, taking the supernatant for ethanol precipitation and then centrifuging, taking the precipitate for secondary alcohol precipitation, washing with anhydrous ethanol 2-3 times, and drying the obtained wet polysaccharide to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

4. The use according to claim 1, characterized in that: The kidney bean seed coat polysaccharide is obtained by extracting through a cellulase extraction method, and its monosaccharide composition and molar ratio are trehalose 0.4, rhamnose 2.83, arabinose 9.74, galactose 11.13, glucose 51.92, xylose 20.77, galacturonic acid 2.83, and gluconic acid 0.38, and the unit is %.

5. The use according to claim 4, characterized in that The specific steps of the cellulase extraction method are as follows: Step 1, preparing kidney bean seed coat fine powder: soaking and peeling white kidney beans, drying the seed coats in a hot air oven at 50-80°C for 2-10 hours, grinding with a high-speed grinder, and sieving to obtain white kidney bean seed coat fine powder; Step 2, cellulase extraction of kidney bean seed coat polysaccharide: the white kidney bean seed coat fine powder prepared in step 1 is dissolved in deionized water 10-20 times its weight, the pH is adjusted to 5 with 3M citric acid, 3wt% of cellulase by weight of the white kidney bean seed coat fine powder is added, the mixture is heated to 50-60°C, stirred and incubated for extraction, and then filtered and centrifuged. The supernatant is ethanol precipitated and centrifuged, the precipitate is subjected to secondary alcohol precipitation, washed with anhydrous ethanol 2-3 times, and the obtained wet polysaccharide is dried to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

6. The use according to claim 1, characterized in that: The kidney bean seed coat polysaccharide is extracted by a chelating agent extraction method, and the monosaccharide composition and molar ratio thereof are rhamnose 1.32, arabinose 15.57, galactose 5.58, glucose 1.45, xylose 73.44, galacturonic acid 2.26, and gluconic acid 0.38, and the unit is %.

7. The use according to claim 6, characterized in that The specific steps of the chelating agent extraction method are as follows: Step 1, preparing kidney bean seed coat fine powder: soaking and peeling white kidney beans, drying the seed coats in a hot air oven at 50-80°C for 2-10 hours, grinding with a high-speed grinder, and sieving to obtain white kidney bean seed coat fine powder; Step 2, ammonium oxalate extraction of kidney bean seed coat polysaccharide: the white kidney bean seed coat fine powder obtained in step 1 is dissolved in 50mM ammonium oxalate solution at a mass ratio of 1:12-18, heated to 60-80°C, stirred and incubated for extraction, filtered and centrifuged, the supernatant is ethanol precipitated and then centrifuged, the precipitate is subjected to secondary alcohol precipitation, washed with anhydrous ethanol 2-3 times, and the obtained wet polysaccharide is dried to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

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