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

Abstract

The application discloses application of kidney bean seed coat polysaccharide in preparation of products for improving intestinal flora and / or inhibiting intestinal inflammation of the elderly, and is characterized by the following steps: soaking and peeling white kidney beans, drying the seed coat in a hot air oven at 50-80 DEG C for 2-10 hours, grinding the seed coat with a high-speed pulverizer, and sieving to obtain white kidney bean seed coat fine powder, and extracting kidney bean seed coat polysaccharide from the white kidney bean seed coat fine powder by using citric acid, cellulase or ammonium oxalate, wherein the polysaccharide has the advantages of regulating the structure and metabolic function of intestinal microbial communities of the elderly, and especially has potential benefits in promoting the production of butyric acid and improving the intestinal health of the elderly.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of kidney bean polysaccharides, and particularly relates to application of kidney bean seed coat polysaccharides in preparation of products for improving intestinal flora and / or inhibiting intestinal inflammation of the elderly. BACKGROUND

[0002] Polysaccharides are an important class of biological macromolecules, have a wide range of biological activities and functional properties, kidney beans are a common legume crop, rich in various bioactive ingredients such as vitamins, minerals and amino acids, etc., especially suitable for special patients such as heart disease, hypokalemia, etc., also have the effects of preventing cancer, resisting cancer, etc. At present, the research on kidney bean polysaccharides is relatively common. For example, patent CN 110642962 B discloses a separation and purification method of mixed bean pectin polysaccharides, the experimental raw materials include red kidney beans, red beans and white beans, the preparation method quickly, efficiently and stably obtains pectin polysaccharides with high purity and good quality by using a series of physical methods to extract and purify pectin in the mixed beans after ultrafine grinding. Patent CN 118421726 A discloses a preparation method and application of kidney bean polysaccharides with enhanced immune function, which solves the problem of poor immune regulation effect of kidney bean polysaccharides obtained by using only alpha-amylase in the prior art. The preparation method is to grind the cooked kidney beans for compound enzymolysis and compound strain fermentation, then purify to obtain kidney bean polysaccharides, and then perform animal experiments to verify the immune regulation effect, which proves that the kidney bean polysaccharides can enhance the immune function through affecting the proliferation capacity of spleen lymphocytes and promoting antibody production in mice.

[0003] As an important by-product of beans, kidney bean seed coat has rich biological activity and nutritional value, and polysaccharides with excellent properties can be prepared through extraction process. Kidney bean seed coat polysaccharides have the effects of antioxidant, immune enhancement, anti-tumor, anti-virus, anti-radiation, blood lipid reduction, blood sugar reduction, anti-aging, etc., and can also regulate intestinal flora and promote intestinal health. At present, there is little research on kidney bean seed coat polysaccharides, and no relevant literature reports that polysaccharides extracted from kidney bean seed coat are used to improve the intestinal health of the elderly. SUMMARY

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

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

[0006] Furthermore, the polysaccharide from kidney bean seed coat was obtained by citric acid extraction. Its monosaccharide composition and molar ratio were: 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, in units of %.

[0007] Furthermore, the specific steps of the citric acid extraction method are as follows:

[0008] Step 1: Prepare fine powder of kidney bean seed coat: Soak white kidney beans and peel them. Dry the seed coat in a hot air oven at 50-80℃ for 2-10 hours, then grind it with a high-speed grinder and sieve it to obtain fine powder of white kidney bean seed coat.

[0009] Step 2: Citric acid extraction of kidney bean seed coat polysaccharide: Dissolve the white kidney bean seed coat powder obtained in Step 1 in 10-20 times its weight of deionized water, adjust the pH to 2 with 3M citric acid, extract by ultrasonic crushing, heat to 60-80℃, stir and incubate for extraction, filter and centrifuge after extraction, take the supernatant, precipitate with ethanol and centrifuge, take the precipitate for secondary alcohol precipitation, wash with anhydrous ethanol 2-3 times, and dry the obtained wet polysaccharide to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

[0010] Furthermore, the aforementioned kidney bean seed coat polysaccharide was obtained by cellulase extraction, and its monosaccharide composition and molar ratio were as follows: 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, in units of %.

[0011] Furthermore, the specific steps of the cellulase extraction method are as follows:

[0012] Step 1: Prepare fine powder of kidney bean seed coat: Soak white kidney beans and peel them. Dry the seed coat in a hot air oven at 50-80℃ for 2-10 hours, then grind it with a high-speed grinder and sieve it to obtain fine powder of white kidney bean seed coat.

[0013] Step 2: Extraction of kidney bean seed coat polysaccharide using cellulase: Dissolve the white kidney bean seed coat powder obtained in Step 1 in 10-20 times its weight of deionized water, adjust the pH to 5 with 3M citric acid, add 3wt% cellulase of the white kidney bean seed coat powder, heat to 50-60℃, stir and incubate for extraction, filter and centrifuge, take the supernatant, precipitate with ethanol and centrifuge, take the precipitate for secondary alcohol precipitation, wash with anhydrous ethanol 2-3 times, and dry the obtained wet polysaccharide to obtain kidney bean seed coat polysaccharide with high xylan and arabinose content.

[0014] Furthermore, the kidney bean seed coat polysaccharide was obtained by chelation extraction, and its monosaccharide composition and molar ratio were as follows: 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, and gluconic acid (GlcA) 0.38, in percentages.

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

[0016] Step 1: Prepare fine powder of kidney bean seed coat: Soak white kidney beans and peel them. Dry the seed coat in a hot air oven at 50-80℃ for 2-10 hours, then grind it with a high-speed grinder and sieve it to obtain fine powder of white kidney bean seed coat.

[0017] Step 2: Extraction of kidney bean seed coat polysaccharide with ammonium oxalate: Dissolve the white kidney bean seed coat powder obtained in Step 1 in 50mM ammonium oxalate solution at a mass ratio of 1:12-18, heat to 60-80℃, stir and incubate for extraction, filter and centrifuge, take the supernatant to precipitate with ethanol and centrifuge, take the precipitate for secondary alcohol precipitation, wash with anhydrous ethanol 2-3 times, and dry the obtained wet polysaccharide 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 as follows:

[0019] 1. This invention employs three different methods to extract polysaccharides from kidney bean seed coats. Each method has its own characteristics. For example, citric acid extraction helps improve the emulsifying properties of kidney bean polysaccharides; cellulase extraction helps preserve the natural structure and function of polysaccharides; and ammonium oxalate extraction helps dissolve high molecular weight polysaccharides.

[0020] 2. This invention uses the prepared polysaccharide to conduct in vitro fecal fermentation experiments and cell experiments on elderly people, and measures the changes in pH of the fermentation broth, changes in intestinal flora, dynamic changes in monosaccharide composition, dynamic changes in SCFA production, and the determination of pro-inflammatory and anti-inflammatory factors. This demonstrates that polysaccharide has a regulatory effect on the structure and metabolic function of the intestinal microbiota in the elderly, especially its potential benefits in promoting butyrate production and improving intestinal health in the elderly. It is expected to provide a safe and effective intervention for improving intestinal function in the elderly, and shows unique advantages in regulating the intestinal flora in the elderly. Attached Figure Description

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

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

[0023] Figure 3 Scanning electron microscope (SEM) images of three polysaccharides: AEP, EEP, and CEP, where A1 represents the magnification of AEP.

[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 This refers to the pH changes during in vitro fermentation.

[0027] Figure 5 Venn plot showing the number of specific OTUs in different polysaccharide samples at different fermentation time points;

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

[0029] Figure 7 PCoA score plots for different polysaccharide samples;

[0030] Figure 8 The relative abundance (%) of the gut microbiota at the phylum level in different groups;

[0031] Figure 9 Heatmap showing the relative abundance changes of gut microbiota in different groups;

[0032] Figure 10The relative abundance (%) of the gut microbiota at the genus level in different groups;

[0033] Figure 11 Heatmap showing the relative abundance changes of gut microbiota in different groups;

[0034] Figure 12 The graph shows the changes in short-chain fatty acid (SCFA) content during fermentation in different groups. The letters ac represent significant differences between them.

[0035] Figure 13 To detect the gene expression level of tight junction protein in HT-29 cells in an intestinal model with inflammation and injury using Western blotting, and to determine the effect of each sample group on the expression of tight junction protein in HT-29 cells in the intestinal model with inflammation and injury, the following values ​​were used: #: 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 effect of each polysaccharide group on the release of inflammatory factors from HT-29 cells in an intestinal model of inflammation injury was investigated. The values ​​were as follows: #: p < 0.05 compared to the Control group; ##: p < 0.01 compared to the Control group; *: p < 0.05 compared to the LPS group; **: p < 0.01 compared to the LPS group; ***: p < 0.001 compared to the LPS group; ns: no significant difference compared to the LPS group. Detailed Implementation

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

[0038] Specific Implementation Example 1: Preparation of Polysaccharides from Kidney Bean Seed Coat

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

[0040] Step 1: Preparation of kidney bean seed coat powder: White kidney beans are produced in Chaoyang City, Liaoning Province, China. Soak and peel the white kidney beans, dry the seed coat in a hot air oven at 60℃ for 2-10 hours, grind the dried kidney bean seed coat with a high-speed grinder, and sieve it 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: Dissolve the white kidney bean seed coat powder obtained in Step 1 in 15 times its weight of deionized water, adjust the pH to 2 with 3M citric acid, ultrasonically crush and extract for 30 min, heat to 70℃, stir and incubate for 3 h. During the experiment, cover the beaker with plastic wrap to prevent solvent evaporation. After extraction, filter and centrifuge, take the supernatant and rotary evaporate to 1 / 3 of the original volume, add 70% ethanol, store at 4℃ for 12 h, centrifuge at 10,000 rpm for 10 min, take the precipitate and perform a second alcohol precipitation for 12 h, wash with anhydrous ethanol 2-3 times to remove impurities, and then dry the obtained wet polysaccharide in a hot air oven at 55℃ to obtain polysaccharide AEP.

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

[0043] Step 1: Same as in Example 1 above;

[0044] Step 2: Cellulase extraction of polysaccharides from kidney bean seed coats: Dissolve fine powder of white kidney bean seed coats in 15 times its weight of deionized water, adjust the pH to 5 with 3M citric acid, add 3wt% cellulase of the white kidney bean seed coat powder, heat to 55℃, stir and incubate for 3 hours. During the experiment, cover the beaker with plastic wrap to prevent solvent evaporation. After extraction, filter and centrifuge. Take the supernatant and rotary evaporate to 1 / 3 of the original volume, then add 70% ethanol. Store at 4℃ for 12 hours, then centrifuge at 10,000 rpm for 10 minutes. Take the precipitate and perform a second alcohol precipitation for 12 hours. Wash with anhydrous ethanol 2-3 times to remove impurities. Then dry the obtained wet polysaccharide in a hot air oven at 55℃ to obtain polysaccharide EEP.

[0045] Example 3: Extraction of polysaccharides from kidney bean seed coat using a chelating agent extraction method, including the following steps.

[0046] Step 1: Same as in Example 1 above;

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

[0048] Specific Example 2: Physicochemical Characterization of Kidney Bean Seed Coat Polysaccharides

[0049] 1. Monosaccharide composition and content analysis

[0050] Take 10 mg of each of the three types of kidney bean seed coat polysaccharide samples prepared in Example 1 and 4 mL of 2M trifluoroacetic acid in a hydrolysis tube. After purging with nitrogen for 1 min to remove air, seal the tube and hydrolyze at 120℃ for 1 h. Dry the mixture with nitrogen to remove excess acid, dilute, and filter through a 0.2 μm filter membrane. Use a Dionex ICS-3000 analyzer from Dionex Corporation (USA), with an analytical column of CarboPac™ PA20 3×150 mm, column temperature 35℃; eluent of 250 mM NaOH and 1 M NaAC, flow rate 0.5 mL / min; and injection volume of 10 μL to analyze the monosaccharide composition of the polysaccharide samples. The results are shown in Table 1. As can be seen from the table, the monosaccharide composition of AEP, EEP, and CEP mainly contains xylose, arabinose, galactose, galacturonic acid (GalA), rhamnose, glucose (Glc), gluconic acid (GlcA), and trehalose (Fuc). The molar ratios of these monosaccharides vary. AEP and CEP have relatively high molar ratios of Xyl and Ara, suggesting that Xyl and Ara may be the main components that build the backbone of AEP and CEP. This may be because the glycosidic bonds formed by Xyl and Ara are very sensitive to acid hydrolysis, resulting in the release of more of them. EEP, on the other hand, has the highest Gla content.

[0051] Table 1. Monosaccharide molar ratio (%)

[0052]

[0053] 2. Molecular weight analysis

[0054] The molecular weight of 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 Shodex 806, the mobile phase was 0.1 M sodium chloride solution, the flow rate was 0.5 mL / min, and the injection volume was 100 μL. Data collection and analysis were performed 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 widest molecular weight distribution.

[0055] Table 2 Molecular weight and dispersity index of polysaccharides

[0056]

[0057] Specific Implementation Example 3: Structural Characteristics of Kidney Bean Seed Coat Polysaccharide.

[0058] 1. Fourier transform infrared spectroscopy analysis

[0059] The three types of kidney bean seed coat polysaccharide samples prepared in Specific Example 1 were thoroughly dried in an oven at 105℃, sieved through a 200-mesh sieve, and then uniformly ground with pure KBr at a ratio of 1:100. After tableting, they were placed in a Fourier transform infrared spectroscopy scanner at 4000-400cm. -1 Thirty-two scans were performed within the area. Data collection and processing were performed using offline OMMIC software; the results are shown below. Figure 1 The visible wavelength range of the infrared spectrum is 3279-3355 cm⁻¹. -1 The peak value and 2926-2946cm -1 The peak value is a characteristic absorption peak of polysaccharides.

[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 substances. 80 μL of the supernatant was collected, air bubbles were removed, and the sample was placed in a SAXA instrument at 25℃. The distance from the sample to the detector was 540.74 mm. Data points were acquired using 3 × 300 seconds of exposure. Data analysis was performed using GIFT software. The results are shown below. Figure 2 .Depend on Figure 2 The results from the AC study showed the effect of different extraction methods on the polysaccharide structure, and 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, and imaged using SEM at an accelerating potential of 5 kV, with magnifications of 5000× and 10000×. The results are shown in [Figure number missing]. Figure 3 The figure shows the differences in chemical structure of different polysaccharides. Ultrasonic treatment may make the surfaces of the three polysaccharides rougher. Among them, the surface of CEP is the most porous and exhibits 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 water adsorption and binding.

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

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

[0066] 1. Experimental Methods

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

[0068] (2) Preparation of basic nutrient culture medium: Dissolve 2.0g peptone, 2.0g yeast, 0.1g NaCl, 40.0mg K2HPO4, 40.0mg KH2PO4, 10.0mg MgSO4, 10.0mg CaCl2, 2.0g NaHCO3, 0.5g cysteine-HCl, 0.5g bile salts, 2.0mL Tween-80, 5.0μL vitamin K1 and 20.0mg hemin in deionized water and bring the volume to 1L. Then adjust the pH to 7.4 with 0.1mol / 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 culture medium. Under the same conditions, basal nutrient culture medium without any carbon source was used as a blank control (CON), and fructooligosaccharide (FOS) was used as a positive control. All fermentation experiments were performed in triplicate. Fermentation was carried out in an incubator shaker at 37℃ and 120 rpm. Fermentation samples were collected at 0, 12, 24, and 36 hours after the start of fermentation, with three samples collected from each group.

[0070] (5) Methods for measuring indicators:

[0071] A. Fermentation broth pH: Centrifuge the fermentation broth at 9000g for 20min, collect the supernatant, and measure the pH of each sample using a pH meter.

[0072] B. DNA Extraction and 16S rRNA Sequencing: One sample from each group was centrifuged at 3000 rpm for 10 min, rapidly 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. The DNA concentration was determined using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer, and its quality was assessed by 1% agarose gel electrophoresis. Full-length 16S rRNA gene amplification of genomic DNA was performed using primers 27F:AGRGTTTGATYNTGGCTCAG and 1492R:TASGGHTACCTTGTTASGACTT. The amplified products were then subjected to multiplex sequencing using forward and reverse primers with sample-specific PacBio barcodes. Sequencing data were analyzed using the UCHIME algorithm (v8.1) to remove chimeric sequences, obtaining clean reads. Subsequently, sequences with ≥97% similarity were clustered into Operational Taxonomic Units (OTUs) using USEARCH (v10.0). Finally, based on the Naive Bayes classifier in QIIME2 and combined with the SILVA database, representative readings were annotated, and the confidence threshold was set to 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 a Dionex ICS-3000 ion chromatograph from Dionex Corporation, USA.

[0074] D. Short-chain fatty acids: Take 1 mL of sample and add it to a 20 mL glass bottle. Add 104 μL of 6 M hydrochloric acid and 105 μL of 500 ppm 2-ethylbutyric acid. Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid at 10-1000 ppm are used to prepare external standard solutions. Short-chain fatty acids were determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HSP-GC-MS). The solid-phase extraction head adsorbed the sample for 30 min at 40 °C, followed by thermal desorption at the injection port for 3 min. The desorbed substances were separated using a DB-WAXUI column with helium as the carrier gas at a flow rate of 1 mL / min. The inlet and ion source temperatures were 250 °C. The gas chromatograph oven temperatures were set as follows: 0-10 min, 50-100 °C, 10-14 min, 100-120 °C, 5 °C / min, 14-39 min, 120-150 °C, 2 °C / min, 39-56 min, 150-220 °C, 10 °C / min. The ionization voltage was 70 eV, and the emission current was 35 μA. The content of short-chain fatty acids was expressed as mmol / 10 mL of fermentation broth.

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

[0076] 2. Analysis of Experimental Results

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

[0078] pH changes during in vitro fermentation are shown in the figure. Figure 4 .Depend on Figure 4 The results showed that polysaccharides with different structures had significantly different effects on pH. The pH of all groups decreased throughout the fermentation process, with the AEP and EEP groups exhibiting a particularly sharp decrease, indicating that the production of more organic acids during fermentation may promote gut microbial activity. In contrast, the CO and CY groups showed a slower rate of pH decrease, suggesting lower acid production capacity during fermentation. These results indicate that polysaccharides with different structures have varying effects on regulating the gut microenvironment during fermentation. The accumulation of acidic products helps inhibit the growth of harmful bacteria and maintain gut health, particularly having a potentially positive impact on the gut microecological balance in the elderly.

[0079] (2) Diversity analysis of gut microbiota

[0080] like Figure 5 As shown, the initial gut bacteria in the CO and CY groups contained 158 and 201 specific OTUs, respectively. With prolonged fermentation, the number of specific OTUs in the CY group decreased significantly, while the change in the CO group was more gradual. When CEP was used as the main carbon source, the number of specific OTUs decreased to 51 after 24 hours of fermentation, indicating a significant decrease in interspecies abundance, and this decrease was greater than in other groups. These results demonstrate that different carbon sources have different effects on gut microbiota structure.

[0081] Figure 6 The study showed that the gut microbiota exhibited significant diversity under the intervention of polysaccharides with different structural features. With prolonged fermentation time, the richness, diversity, evenness, and genetic diversity of the microbiota in each group decreased to varying degrees. Figure 6 It can be seen that, compared with other groups, AEP significantly reduced microbial α-diversity. PCoA analysis of the known data yielded... Figure 7 ,like Figure 7 The gut microbiota composition of the unfermented CO group and the CY group is different. The gut microbiota of each group is relatively different in the early stage of fermentation, indicating that there are significant differences in the fermentation effect of different polysaccharides. However, the gut microbiota composition of each group is similar in the later stage of fermentation.

[0082] (3) Regulation of the gut microbiota at the phylum level by polysaccharides

[0083] Figure 8 , 9 This study demonstrates the changes in the composition and relative abundance of the gut microbiota at the phylum level under different polysaccharide treatment groups. Figure 8 Stacked bar charts were used to illustrate the relative abundance distribution of different microbial phyla in each treatment group. In the early stages of fermentation, the gut microbiota of the elderly was dominated by four bacterial phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidota, with the total relative abundance of these phyla exceeding 90%. With prolonged fermentation, the microbial community structure changed significantly, particularly in the polysaccharide-free control fermentation, where the abundance of Actinobacteria decreased significantly, while the abundance of Proteobacteria increased. Figure 9 By using heatmaps combined with cluster analysis, the similarities and differences in the gut microbiota among different treatment groups were visually presented. The results indicate that polysaccharide fermentation in the gut environment not only affects the proliferation of specific bacterial communities but also leads to significant changes in the composition of the gut microbiota. These data further reveal the regulatory role of polysaccharides and their derivatives in the gut microbiota, contributing to a deeper understanding of the functions and mechanisms of polysaccharides in the gut health of the elderly.

[0084] (4) Regulation of the gut microbiota by polysaccharides at the genera level

[0085] Figure 10 , 11 This study demonstrates the changes in the composition and relative abundance of the gut microbiota at the genus level under different polysaccharide treatment groups. Figure 10 Stacked bar charts were used to illustrate the relative abundance distribution of different microbial genera in each treatment group. At the initial stage of fermentation, the genus composition of the gut microbiota differed between elderly and young individuals. For example, the relative abundance of *E. coli* was 0.16% and *Eubacterium* was 11.08% in the gut microbiota of elderly individuals, while the relative abundance of *E. coli* was 2.15% and *Eubacterium* reached 18.52% in the gut of young individuals. During fermentation, the gut microbiota structure underwent significant changes. The relative abundance of *E. coli* and *Bifidobacterium* in the gut of both elderly and young individuals changed over time. *E. coli* was dominant under carbon-free conditions, while *Bifidobacterium* increased under specific polysaccharide conditions. AEP and EEP promoted the growth of *Bifidobacterium* and inhibited *E. coli*. In the CEP group, *Clostridium* increased, affecting the abundance of *Bifidobacterium*. These changes reflect the regulatory role of polysaccharides in the gut microbiota. Figure 11The heatmap further illustrates the relative abundance changes of *Escherichia coli*, gradually shifting from dark blue in the early stages of fermentation to light red in the later stages, indicating that *E. coli* abundance gradually increases or decreases over time. Overall, these data highlight the significant regulatory role of different polysaccharides in the gut microbiota of older adults, particularly the dynamic balance changes between *Bifidobacterium* and *Escherichia coli*.

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

[0087] Table 3 shows the changes in monosaccharides during polysaccharide fermentation in different groups. With prolonged fermentation time, the contents of Ara, Gal, Glc, and GalA gradually decreased, indicating that gut microbiota effectively utilized these monosaccharides. In the SHP group, microorganisms significantly preferentially consumed Glc and GalA. The utilization rate of Ara exceeded 60% in all groups, while the utilization rates of Gal and Glc also exceeded 50%, promoting the metabolic activity of gut microbiota. GalA utilization ranged from 20% to 72%, demonstrating the promoting effect of polysaccharide structure on microbial fermentation metabolism and its ability to promote intestinal metabolic function in the elderly.

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

[0089]

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

[0091] like Figure 12 As shown, Figure 12 This study illustrates the changes in short-chain fatty acid (SCFA) content during fermentation in different polysaccharide treatment groups. With prolonged fermentation time, various SCFAs significantly increased, indicating efficient utilization of carbohydrates by the gut microbiota. Acetic acid, propionic acid, and butyric acid, among other SCFAs, are the main products of dietary fiber fermentation and possess metabolic and immunomodulatory functions. The AEP group showed a significant increase in acetic acid and butyric acid content, possibly related to the increased relative abundance of *Bifidobacterium* and *Clostridium fibrillii*. Propionic acid and isobutyric acid content showed relatively small changes, indicating little difference between groups. Valeric acid and isovaleric acid content were low, mainly originating from protein fermentation. The increase in SCFAs reflects the prebiotic effect of polysaccharides, with the AEP group exhibiting 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 other groups, indicating that the gut microbiota gradually transitioned from carbohydrate fermentation to partial protein metabolism, beginning 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 investigating 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, with 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 of polysaccharide products on TNF-α release, tight junction protein expression, and inflammatory factor release in HT-29 cells were analyzed. This study aims to reveal the potential protective mechanism of white kidney bean polysaccharides on intestinal health in the elderly at the cellular level, providing theoretical support and practical basis for improving intestinal health in the elderly and developing functional foods for them.

[0094] 1. Experimental Methods

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

[0096] (2) Immunoblot detection of protein expression: Cells from each group were collected, lysed with an appropriate amount of RIPA lysis buffer for 30 min, and then centrifuged at 1200 r / min for 15 min at 4℃. The supernatant was collected. The total protein concentration of cells was measured using the BCA method. Proteins were separated using 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked in 5% BSA solution at room temperature for 1 h. After blocking, the PVDF membranes were washed with TBST buffer and primary antibodies were added: Claudin-1 (1:1000), Claudin-3 (1:1000), Occludin (1:2000), Zonulin-1 (1:1000), and Zonulin-3 (1:1000). The membranes were incubated overnight at 4℃. The PVDF membranes were removed from the primary antibodies, washed with TBST buffer, and then incubated in anti-rabbit IgG (1:20000) secondary antibody at room temperature in the dark for 1 h. After washing again, the membrane was developed using an Odyssey dual-color infrared fluorescence imaging system. GAPDH was used as an internal reference protein, and the data were analyzed using ImageJ software.

[0097] (3) Inflammatory factor assay: Cells were incubated according to the above grouping. The supernatant was collected and the contents of pro-inflammatory factors TNF-α, IL-6, IL-8, 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 plotted after being analyzed and processed by GraphPad Prism software. The variance analysis (ANOVA) was performed using SPSS 17.0 software at the 5% significance level.

[0099] 2. Analysis of Experimental Results

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

[0101] Figure 13 The effects of each polysaccharide sample on LPS-induced tight junction proteins in HT-29 cells were demonstrated. Figure 13 It was found that, 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 significantly reduced (p<0.05). Subsequent treatment with polysaccharide products from different stages of intestinal fermentation was also observed. Furthermore, the expression of tight junction proteins Claudin-3, Claudin-1, Occludin, ZO-3, and ZO-1 showed varying degrees of increase in all groups (p<0.05), especially in the AEP group, where the expression levels of all five proteins were significantly increased (p<0.05), and the effect was more pronounced in the fermentation endpoint (T36-AEP) group compared to the fermentation initiation point (T0-AEP). Therefore, the polysaccharide products from intestinal 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 the integrity of the intestinal barrier in the elderly.

[0102] (2) Effects of gut microbiota fermentation polysaccharide fermentation products on the release of inflammatory factors in HT-29 cells

[0103] Figure 14 The anti-inflammatory effects of each polysaccharide sample on LPS-induced HT-29 cell inflammation model were demonstrated. Figure 14Compared with the Control group, LPS significantly increased the levels of pro-inflammatory factors TNF-α, IL-6, IL-8, and IL-12 in HT-29 cells (p<0.05). After treatment with fermentation products for 24 h, the CO group showed no significant change in the release of TNF-α, IL-6, IL-8, and IL-12 (p>0.05), while the groups with added polysaccharides showed varying degrees of decrease in the levels of pro-inflammatory factors TNF-α, IL-6, IL-8, and IL-12. This suggests that the release of TNF-α and IL-6 may be reduced by inhibiting NF-κB activation. The AEP group showed better inhibition of IL-8, with T36-AEP showing better downregulation than T0-AEP. This suggests that the secretion of IL-8 may be reduced by inhibiting the MAPK pathway. The SCP group showed the most significant inhibitory effect on IL-12, suggesting that the level of IL-12 may be reduced 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 mainly inhibit the release of pro-inflammatory factors by inhibiting pathways such as NF-κB and JAK / STAT. 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, with the upregulation of both inflammatory factors being significantly better at T36-AEP than at T0-AEP. Furthermore, SCP also promoted IL-10 release. Overall, fermentation products at different stages inhibited LPS-induced inflammatory responses and demonstrated a protective effect on the intestinal barrier in the elderly.

[0105] In summary, the regulatory effect of kidney bean seed coat polysaccharide on the gut microbiota was evaluated using an in vitro anaerobic fermentation model in the elderly. The study 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 butyric acid, demonstrating its potential as a prebiotic. This could help enhance intestinal barrier function and improve gut health in the elderly.

[0106] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. The application of a kidney bean seed coat polysaccharide in the preparation of products that improve the intestinal flora and / or inhibit intestinal inflammation in the elderly, characterized in that, The aforementioned kidney bean seed coat polysaccharide was extracted using a citric acid extraction method, with the specific steps as follows: Step 1: Prepare fine powder of kidney bean seed coat: Soak white kidney beans and peel them. Dry the seed coat in a hot air oven at 50-80℃ for 2-10 hours, then grind it with a high-speed grinder and sieve it to obtain fine powder of white kidney bean seed coat. Step 2: Citric acid extraction of kidney bean seed coat polysaccharide: Dissolve the white kidney bean seed coat powder obtained in Step 1 in 10-20 times its weight of deionized water, adjust the pH to 2 with 3 M citric acid, extract by ultrasonic crushing, heat to 60-80°C, stir and incubate for extraction, filter and centrifuge after extraction, take the supernatant, precipitate with ethanol and centrifuge, take the precipitate for secondary alcohol precipitation, wash with anhydrous ethanol 2-3 times, and dry the obtained wet polysaccharide to obtain kidney bean seed coat polysaccharide.

2. The application according to claim 1, characterized in that: The aforementioned kidney bean seed coat polysaccharide was obtained by citric acid extraction. Its monosaccharide composition and molar ratio are as follows: trehalose 0.36, rhamnose 3.10, arabinose 18.94, galactose 9.31, glucose 1.52, xylose 63.47, galacturonic acid 2.84, gluconic acid 0.46, in units of .

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