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11 February 2026

20 Pages

Extraction-Dependent Structural Diversity of White Kidney Bean Cotyledon Polysaccharides Determines Their Effects on Ex Vivo Fermentation of the Elderly Gut Microbiota

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1
School of Food and Health, Beijing Technology & Business University, Beijing 100048, China
2
Key Laboratory of Geriatric Nutrition and Health (Beijing Technology & Business University), Ministry of Education, Beijing 100048, China
3
Centre for Healthy Eating & Food Innovation (HEFI), Maastricht University—Campus Venlo, Villa Floraweg 1, 5928 SZ Venlo, The Netherlands
*
Author to whom correspondence should be addressed.
Nutrients2026, 18(4), 591;https://doi.org/10.3390/nu18040591
This article belongs to the Section Phytochemicals and Human Health
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Abstract

Background/Objectives: Aging is accompanied by profound alterations in gut microbiota composition, reduced microbial diversity, and impaired metabolic resilience. Dietary polysaccharides with different structures exhibit varying efficacy in alleviating these age-related disorders. In this study, we aimed to extract and characterize polysaccharides from white kidney bean (Phaseolus vulgaris L.) cotyledon using different methods and to evaluate their fermentation behavior by the elderly gut microbiota. Methods: White kidney bean cotyledon polysaccharides were extracted using ultrasound-assisted acid (WKBC-P1), alkaline (WKBC-P2), and chelator (WKBC-P3) methods. Ex vivo fermentation of WKBC-P1, WKBC-P2, and WKBC-P3 was performed using pooled fecal microbiota from the elderly aged 65–70 years old. Results: Monosaccharides profiling revealed that WKBC-P1 and WKBC-P3 were rich in arabinose, whereas WKBC-P2 contained high levels of glucose. Molecular weight analysis indicated that acidic extraction led to smaller, more fragmented polysaccharides, while chelating extraction produced the highest molecular weight and most uniform fractions. Ex vivo fecal fermentation showed that WKBC-P1 and WKBC-P2 significantly increased the relative abundance of Bifidobacterium and Bacteroides_H while suppressing potentially harmful Proteobacteria. Correspondingly, both fractions induced robust production of short-chain fatty acids (SCFAs), while reducing branched short-chain fatty acids (bSCFAs), indicating a metabolic shift toward carbohydrate fermentation rather than protein catabolism. Conclusions: Overall, these findings demonstrate that acidic and alkaline extractions yield fractions that most effectively support beneficial microbial growth and SCFAs generation in aging microbiota. This knowledge might indicate that white kidney bean cotyledon polysaccharides represent promising prebiotic candidates for maintaining gut homeostasis and metabolic health in elderly populations.

1. Introduction

White kidney beans (Phaseolus vulgaris L.) are among the most widely consumed legumes worldwide and represent a key element of the Mediterranean dietary pattern. Beyond their nutritional value, they contain bioactive components such as α-amylase inhibitors, resistant starch, and polyphenols that have been associated with beneficial effects on metabolic health, weight regulation, and glycemic control. These characteristics have led to increasing recognition of white kidney beans as a promising natural source of nutraceutical compounds [1,2]. Accumulating evidence indicates that white kidney beans and their bioactive components also strengthen and regulate gut barrier integrity by promoting tight junction function and supporting mucosal protection. In addition, they modulate gut microbiota composition, reduce inflammatory responses, and contribute to the prevention of obesity and diabetes [3,4,5]. Most existing studies have investigated white kidney beans as whole foods or crude extracts, demonstrating broad health-promoting properties. However, the specific contributions and mechanistic actions of individual polysaccharide fractions in regulating gut barrier function and overall host physiology remain insufficiently understood and warrant further detailed investigation.
Maintaining a balanced gut microbiota is essential for sustaining overall health, particularly in the elderly, where microbial diversity and metabolic activity decline markedly with age [6]. Age-related dysbiosis often coincides with impaired gut barrier integrity and chronic low-grade inflammation [7]. Dietary polysaccharides, such as those present in white kidney beans, can counteract these processes by promoting beneficial microbial populations, reinforcing intestinal barrier function, and attenuating age-associated inflammatory responses, thereby contributing to healthier aging and improved intestinal resilience [8]. For instance, ex vivo batch fecal fermentation of water extractable polysaccharides from red kidney bean (Phaseolus vulgaris) stimulated growth of beneficial gut microbiota, especially Bifidobacterium spp. and Lactobacillus spp. [9]. Similarly, polysaccharides extracted from Portulaca oleracea have been shown to beneficially modulate the gut microbiota of aged rats by lowering the Firmicutes/Bacteroidetes ratio and reducing the abundance of Fusobacteria [10].
The extent and specificity of these gut regulatory effects of polysaccharides are strongly determined by the monosaccharide composition, glycosidic linkages, three-dimensional conformation, molecular weight, and distribution. These structural characteristics are profoundly influenced by the extraction methods employed. Research by Ding et al. revealed that mung bean episperm polysaccharides extracted using methods such as hot water, acid, alkali, and enzymes exhibit distinct monosaccharide compositions and molecular weights. Among these, alkali-extracted polysaccharides demonstrated the highest antioxidant activity, while hot water-extracted polysaccharides exhibited significant anti-inflammatory effects [11]. This underscores the importance of utilizing standardized and well-characterized polysaccharide preparations when evaluating the biological activity of polysaccharide formulations and their potential health benefits for the elderly population [12,13]. Currently, extraction methods of polysaccharides include hot water extraction, acid-base extraction, chelating agent extraction, enzymatic extraction, ultrasonic-assisted extraction, and microwave-assisted extraction. Among these methods, hot water extraction is frequently employed for laboratory-scale polysaccharide extraction due to its simplicity and low cost [14]. However, long processing time and relatively low yield hinder its broad application. In contrast, alternative methods such as acid-base extraction could significantly reduce extraction duration while improving the yield [15]. Chelating agent extraction also provides a higher yield compared to hot water extraction, and the obtained polysaccharides exhibited superior biological activity, including enhanced antioxidant activity and fermentation performance [16,17].
Aging is increasingly recognized as a major global health and economic challenge, closely linked to physiological decline and gut microbiota dysbiosis. These alterations contribute to intestinal barrier dysfunction, chronic inflammation, and metabolic disorders observed in the elderly. Insufficient intake of dietary polysaccharides, which is estimated to be below optimal levels in older populations, may further exacerbate these disturbances. To address this, novel and functionally characterized polysaccharide sources are needed. Therefore, the present study aimed to extract and characterize polysaccharides from white kidney bean cotyledons using different methods and to examine how their structural features affect anaerobic fermentation by the elderly gut microbiota, focusing on short-chain fatty acid production, microbial modulation, and their potential to support gut barrier integrity and metabolic health during aging.

2. Materials and Methods

2.1. Materials

White kidney bean was produced in Liaoning Province, China. Commercial apple polysaccharides (CAPs) were purchased from Sigma Aldrich (St. Louis, MO, USA). Monosaccharide standards of fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), galacturonic acid (GalA), and glucuronic acid (GlcA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

2.2. Extraction of Kidney Bean Cotyledon Polysaccharides

White kidney beans were first soaked in water at 60 °C for 2 h to soften the seed coat, after which the cotyledons were manually separated and dried in a hot-air oven at 60 °C until a constant weight was achieved. The dried cotyledons were then finely ground using a high-speed grinder, and the resulting powder was passed through an 80-mesh sieve to obtain a uniform particle size suitable for extraction. To enhance both the purity and yield of kidney bean cotyledon polysaccharides, three extraction procedures, including acidic (WKBC-P1), alkaline (WKBC-P2), and chelating agent (WKBC-P3), were systematically optimized.
Polysaccharide extraction was performed following the method of Gutohrlein et al., with modifications [18]. For the preparation of WKBC-P1, a defined amount of dried kidney bean cotyledon powder was suspended in deionized water at a ratio of 1:15 (w/v). The pH was adjusted to 2.0 using citric acid, after which the suspension was subjected to ultrasonic treatment for 30 min to facilitate cell wall disruption and improve extraction efficiency. The mixture was then heated to 70 °C, continuously stirred, and incubated for 3 h. Following extraction, the solution was filtered and centrifuged to remove insoluble residues. The resulting supernatant was concentrated to one-third of its original volume using a rotary evaporator, and polysaccharides were precipitated twice with 70% ethanol at 4 °C for 12 h. The precipitates were recovered by centrifugation (6000× g, 20 min), washed 2–3 times with anhydrous ethanol to eliminate impurities, and subsequently dried at 55 °C until a constant weight was achieved.
The extraction protocols for WKBC-P2 and WKBC-P3 followed the same procedure with the following modifications: for WKBC-P2, the pH was adjusted to 12.0 using sodium hydroxide instead of citric acid to achieve alkaline extraction conditions; for WKBC-P3, the cotyledon powder was suspended in a 50 mM ammonium oxalate solution (1:15, w/v) to facilitate polysaccharide release through metal ion chelation.

2.3. Monosaccharides Composition

The monosaccharide composition of kidney bean cotyledon polysaccharides was determined as described by Zhu et al. with an ion chromatograph (DIONEX ICS-3000 USA, Dionex Corporation, Sunnyvale, CA, USA) [19]. In brief, 10 mg of the sample or the lyophilized fermentation supernatant was hydrolyzed with trifluoroacetic acid (TFA, 2 mol/L) at 120 °C for 3 h. After hydrolysis, trifluoroacetic acid (TFA) was completely removed under a gentle stream of nitrogen gas, and the resulting hydrolysate was dissolved in ultrapure water. The solution was then filtered through a 0.2 μm membrane prior to chromatographic analysis. Monosaccharide composition was analyzed using an ion chromatograph (DIONEX ICS-3000, Dionex Corporation, Sunnyvale, CA, USA) equipped with a CarboPac™ PA20 analytical column (3 × 150 mm) and a pulsed amperometric detector (PAD). The column was maintained at 35 °C, with a flow rate of 0.5 mL/min and an injection volume of 10 μL.
The same analytical procedure was also employed to assess polysaccharide degradation during ex vivo fermentation, allowing comparison of structural changes and monosaccharide release over time. The utilization rate of monosaccharides was determined as follows:
Degradation rate (%) = (C0 − C36)/C0 × 100
where C0 and C36 were the concentration of monosaccharides at the beginning (T0) and the end (T36) of fermentation.

Molecular Weight

The weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw/Mn) were analyzed using size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS; Wyatt Technology, USA) following the method described by Liu et al. [20]. The analysis was performed using multi-angle laser scattering (Wyatt Technology, Santa Barbara, CA, USA), a refractive index (RI) detector, an L-2400 UV detector (Wyatt Technology, CA, USA), high-performance size exclusion chromatography (Dawn Heleos II, Wyatt Technology, CA, USA), and a Shodex SB-803 HQ chromatographic column (8 × 300 mm, Showa Denko K.K., Tokyo, Japan). Polysaccharide samples (3 mg) were dissolved in 1 mL of ultra-pure water and filtered through a 0.22 μm membrane filter prior to injection. The conditions were set as follows: mobile phase, ultra-pure water; flow rate, 0.5 mL/min; injection volume, 20 μL; and sample concentration, 1 mg/mL. Data collection and analysis were conducted using Astra 6.1 software (Wyatt Technologies, CA, USA).

2.4. Fourier Transform Infrared Spectroscopy (FT-IR)

Fourier-transform infrared (FT-IR) spectra of the different kidney bean cotyledon polysaccharides were acquired using a PerkinElmer Spectrum 400 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA) [21]. The dried samples were homogenized with spectroscopic-grade potassium bromide (KBr) at a ratio of 1:100 (w/w), finely ground to achieve uniform mixing, and pressed into transparent pellets using a hydraulic press. FT-IR spectra were recorded in the range of 4000–400 cm−1, with 24 consecutive scans performed for each sample to enhance signal quality. Data acquisition and spectral processing were carried out using OMNIC 8.2 software (Thermo Nicolet, Waltham, MA, USA).

2.5. Small Angle X-Ray Scattering (SAXS)

Small-angle X-ray scattering (SAXS) procedure followed the method of Yu et al. with minor modifications [22]. The polysaccharide sample (10 mg/mL) was dissolved in water overnight to ensure complete dissolution, after which approximately 80 μL of the supernatant was sealed in a sample vial. The sample was then degassed under vacuum to minimize background scattering. The test temperature was set to 25 °C, and the probe distance was 540.74 mm. Data points were acquired using three 300-s exposures. Data analysis was performed using a SAXS instrument (Anton Paar, Graz, Austria) and Generalized Indirect Fourier Transformation (GIFT).

2.6. Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) was employed to observe the surface morphology and microstructural differences between commercially available kidney bean cotyledon polysaccharides and those obtained through different extraction methods. Dried polysaccharide powders were mounted onto aluminum stubs using double-sided carbon adhesive tape and subsequently sputter-coated with a thin layer of gold under vacuum to enhance conductivity. Imaging was performed at an acceleration voltage of 5 kV, and micrographs were captured at magnifications of 5000× and 10,000× to visualize surface texture, particle arrangement, and structural integrity of the samples.

2.7. Preparation of Fecal Inoculum

Fecal samples were collected from three healthy elderly individuals (two males and one female, aged 65–70 years old) and three healthy adults (one male and two females, aged 25–26 years old) who had not received antibiotics, dietary supplements including probiotics, prebiotics, laxatives, and anti-inflammatory drugs, or medical treatment for at least 3 months. The feces of the young were used solely as a positive microbial community reference. The studies involving human participants were reviewed and approved by the Ethical Committee for Scientific Research, School of Food and Health, Beijing Technology and Business University (NO. 253 of 2025). All participants provided written informed consent, adhering to relevant laws and institutional guidelines. Samples were collected in plastic bags, sealed after air removal, and homogenized with sterile phosphate-buffered saline (pH 7.4) to prepare a 10% (w/v) fecal suspension, which was then magnetically stirred for 15 min. The suspension was made by pooling fecal samples equally from each of the three donors to avoid donor-donor variation. The pooled samples were filtered through three layers of gauze, with all operations performed in an anaerobic workbench (gas phase: 80% N2, 10% CO2, 10% H2). Three replicate fecal inocula were prepared from each of the suspensions. All subsequent processing was completed within 1 h of collection.

2.8. Ex Vivo Fermentation of Kidney Bean Cotyledon Polysaccharides

The preparation of basal nutrient medium was based on the method described by Gu et al., with minor modifications [23]. The medium contained 1.0 L of solution with 2.0 g peptone, 2.0 g yeast extract, 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 salts, 2.0 mL Tween 80, 5.0 μL vitamin K1, and 20.0 mg hemin (previously dissolved in 6 mol/L NaOH) in deionized water. The pH of the medium was adjusted to 7.4 using 0.1 mol/L HCl. For each fermentation assay, 9 mL of the prepared basal medium was supplemented with 50 mg of the polysaccharide sample and 1 mL of freshly prepared fecal inoculum to make the final concentration of the polysaccharide at 5 mg/mL [24]. A control group containing basal medium without any added carbon source was included to serve as a blank reference. All fermentation experiments were conducted in triplicate under anaerobic conditions at 37 °C and 30 rpm using a shaking incubator.
Fermentation samples were collected at 0, 12, 24, and 36 h post-incubation. Three replicates were collected per group: one was centrifuged at 6000× g for 10 min to separate the supernatant and precipitate, then rapidly frozen in liquid nitrogen and stored at −80 °C for microbial analysis. The remaining two were collected in safe-lock microcentrifuge tubes for determining carbohydrate degradation and short-chain fatty acid (SCFA) levels. These two samples were heat-inactivated in a 100 °C water bath for 10 min to halt bacterial enzyme activity, then stored at −80 °C.

2.9. Short Chain Fatty Acids (SCFAs)

Short-chain fatty acids (SCFAs) were quantified using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS), following the analytical procedure described by Chen et al. [25]. This technique allows sensitive and accurate detection of volatile fatty acids generated during ex vivo fermentation, providing detailed insights into the metabolic activity of the gut microbiota. For SCFAs analysis, 1 mL of each fermentation sample was transferred into a 20 mL glass vial, followed by the addition of 104 μL of 6 M hydrochloric acid and 105 μL of an internal standard solution (2-ethylbutyric acid, 500 ppm). Separation was then performed using a DB-WAXUI column (30 cm × 0.25 mm × 0.25 μm). The content of SCFAs was quantified based on the standard curve, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid. The program settings were as follows: 50–120 °C from 0 to 7 min at 10 °C/min, 120–150 °C from 7 to 32 min at 2 °C/min, and 150–220 °C from 32 to 49 min at 10 °C/min. The inlet and ion source temperatures were both maintained at 250 °C.

2.10. DNA Extraction and 16S rRNA Gene Sequencing of Gut Microbial Analysis

DNA was extracted from samples using a DNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. The DNA concentration of the samples was determined using the Qubit dsDNA HS Assay Kit and Qubit 4.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, San Diego, CA, USA), and the quality was assessed via 1% agarose gel electrophoresis. The full-length 16S rRNA gene from genomic DNA was amplified using universal primers 27F (AGRGTTTGATYNTGGCTCAG) and 1492R (TASGGHTACCTTGTTASGACTT). Multiplex sequencing was performed using forward and reverse 16S primers, with primer tails containing PacBio barcode sequences specific to each sample. Chimeric sequences were detected and removed using the UCHIME algorithm (v8.1) to obtain clean reads. Sequences with ≥97% similarity were clustered into the same operational taxonomic unit (OTU) using USEARCH (v10.0). All selected representative reads were annotated using the Naive Bayes classifier in QIIME2 with the SILVA database, at a 70% confidence threshold.

2.11. Statistical Analysis

All experiments were performed in triplicate, with data reported as mean ± standard deviation (SD). Statistical differences were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s test in SPSS 21 (IBM, Armonk, NY, USA) at a 5% significance level. Figures were generated using Origin 9.0 (OriginLab Inc., Northampton, MA, USA).

3. Results

3.1. Monosaccharides Composition and Molecular Weight Analysis

To evaluate how different extraction methods affect the chemical structure and composition of white kidney bean cotyledon polysaccharides (WKBC-P), three extraction approaches were applied: acidic (WKBC-P1), alkaline (WKBC-P2), and chelating agent-based (WKBC-P3). These methods were chosen because each targets distinct molecular interactions within the plant cell wall. Acidic extraction primarily disrupts non-covalent bonds, alkaline extraction breaks hydrogen and ester linkages, and chelating extraction releases polysaccharides bound to metal ions. A commercial apple pectin polysaccharide (CAP) was included as a reference. As summarized in Table 1, all polysaccharide samples, including CAP, WKBC-P1, WKBC-P2, and WKBC-P3, contained galacturonic acid (GalA), arabinose (Ara), glucose (Glc), galactose (Gal), xylose (Xyl), rhamnose (Rha), fucose (Fuc), and glucuronic acid (GlcA) as their main monosaccharide constituents. CAP exhibited a higher proportion of acidic sugars, particularly GalA and Rha, followed by Ara and Gal, whereas the levels of Xyl, Glc, Fuc, and GlcA were comparatively low. The extraction methods had distinct effects on the molar ratios of individual monosaccharides. Both WKBC-P1 and WKBC-P3 displayed similar monosaccharide compositions, characterized by a notably high proportion of Ara.
Table 1. The physicochemical properties, monosaccharide composition (molar proportion, %), and molecular parameters of CAP, WKBC-P1, WKBC-P2, and WKBC-P3.
The average molecular weight and distribution characteristics of the polysaccharides varied depending on the extraction method used. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (Mw/Mn) were determined for WKBC-P1, WKBC-P2, and WKBC-P3. As presented in Table 1, the Mw and Mn values of WKBC-P1 were 66.3 kDa and 46.9 kDa, respectively, which were significantly lower than those of WKBC-P2 (88.9 kDa and 65.3 kDa) and WKBC-P3 (99.1 kDa and 68.4 kDa) (p < 0.05). The polydispersity index of all samples ranged from 1.36 to 1.83, indicating relatively narrow and homogeneous molecular weight distributions. This suggests that the extracted polysaccharides were structurally uniform, with a consistent molecular size distribution across preparations [26].

3.2. Fourier Transform-Infrared Spectroscopy (FT-IR)

FT-IR was applied to further analyze the atomic vibration and functional group characteristics of the polysaccharides. FT-IR spectra of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 were in the range of 400–4000 cm−1 as shown in Figure 1A. All four samples showed similar absorption bands, indicating consistency in functional group composition. In FT-IR spectra, a broad and intense absorption peak was observed in the range of 3200–3400 cm−1, corresponding to the stretching vibration of hydroxyl (-OH) groups, which is one of the typical characteristic peaks of polysaccharide molecules. The absorption peak near 2935 cm−1 was likely associated with the stretching vibration of C-H bonds [27], further confirming the carbon–hydrogen backbone in the polysaccharides. The absorption peak near 1230 cm−1 further revealed sulfated groups (SO3-) in the polysaccharide structure, corresponding to asymmetric stretching vibration of sulfur-oxygen bond (S=O) [28]. Multiple absorption bands between 1000 and 1200 cm−1 may arise from the stretching vibrations of C-O-H side groups, rings, and C-O-C glycosidic bonds, which indicate pyranose monomers [28]. The intense absorption peaks near 1150 cm−1 and 1045 cm−1 further confirmed the presence of pyran rings [29]. In particular, the asymmetric C-O-C stretching vibration peak at 1150 cm−1 indicated the presence of -OCH3 groups. Moreover, the absorption peak near 857 cm−1 is a typical feature of α-glycosidic bonds [30]. FT-IR analysis illustrated that CAP, WKBC-P1, WKBC-P2, and WKBC-P3 had similar chemical compositions, all containing hydroxyl groups, glycosidic bonds, pyranose monomers, and other typical groups of polysaccharides.
Figure 1. Structural characterization of CAP, WKBC-P1, WKBC-P2, and WKBC-P3. (A) FT-IR spectra. (B) SEM images at 10,000× magnification. (C) SAXS intensity curves. (D) Kratky plots of SAXS scattering profiles. (E) Normalized P(r) curves. CAP, commercial apple polysaccharides.

3.3. Small-Angle X-Ray Scattering (SAXS)

SAXS is a sensitive and non-destructive technique, which is widely used to investigate structural features of polysaccharides, including ordering, disordering, and intermolecular interactions. As shown in Figure 1C, the SAXS plot clearly exhibited scattering features of all polysaccharides. Overall shapes of the scattering curves for all samples remained consistent, suggesting negligible intermolecular interactions among these polysaccharides [22,31]. WKBC-P2 showed the highest scattering intensity, followed by WKBC-P3, WKBC-P1, and CAP.
To further investigate the molecular morphology of the polysaccharides, Kratky curves were plotted for the high q region, as shown in Figure 1D. CAP, WKBC-P1, WKBC-P2, and WKBC-P3 displayed small peaks at 0.027 nm−1, 0.082 nm−1, 0.061 nm−1, and 0.041 nm−1, respectively. These characteristic peaks further revealed certain folded conformation and flexibility of the polysaccharide chain in solution [32].
In addition, the particle size of each sample was further evaluated by fitting the distance distribution function P(r) to determine the maximum size of the molecules (Dmax). Figure 1E showed that the particle sizes of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 were 45.90 nm, 29.93 nm, 39.60 nm, and 43.23 nm, respectively, with corresponding Dmax values of 128.25 nm, 85.50 nm, 114.60 nm, and 124.80 nm. It is worth mentioning that WKBC-P1 had the smallest Dmax, indicating a more compact molecular conformation, which might be associated with the degree of molecular entanglement and the specific extraction methods.

3.4. Scanning Electron Microscopy (SEM)

Distinct micro-structural characteristics were observed among CAP and WKBC polysaccharides prepared with different extraction methods (Figure 1B). Specifically, CAP exhibited a complete, ordered layer structure with a smooth surface and tightly packed arrangement. Whereas WKBC-P1 showed irregular porous particles with a rough surface and loosely arranged molecules. The relatively loose network structure of WKBC-P1 might be attributed to its lower molecular weight, which usually results in a less dense network and higher porosity [33]. Differently, WKBC-P2 displayed irregular stacking of the layered structure, with numerous surface wrinkles and fine fragment formation. Consistent with previous research, the fragmented structure of WKBC-P2 may arise from polysaccharide depolymerization and degradation induced by alkaline extraction [34]. Whereas WKBC-P3 was primarily composed of fragments with varied sizes, consisting of numerous small particles and irregular pores, showing an overall fragmented and complex structure. Such a porous structure might be attributed to the weak acidic environment of the chelating agent solution, with the ability to cause polysaccharides depolymerization and degradation [35].

3.5. Regulation of Gut Microbiota at the Genus Level

The composition of gut microbiota undergoes substantial changes with advancing age, and such alterations have been linked to an increased risk of physical decline and metabolic dysfunction. Dietary polysaccharides are known to help restore microbial balance, although the efficacy strongly depends on their structural characteristics. Therefore, this study examined the effects of WKBC-P1, WKBC-P2, and WKBC-P3 on gut microbiota composition of the elderly individuals using an ex vivo fermentation model. Commercial apple pectin polysaccharide (CAP) was included as a reference sample for comparison due to its high popularity as a mature heteropolysaccharide and application in ex vivo fermentation [36]. To gain a more detailed understanding of the microbial shifts induced by different polysaccharide extraction, the composition of the gut microbiota was further analyzed at the genus level (Figure 2A). At the beginning of fermentation (T0), the dominant genera across all groups included Blautia_A, Gemmiger_A, Faecalibacterium, Bifidobacterium, Escherichia, and Romboutsia_B, which are commonly found in the human intestinal ecosystem. The relative abundance of Faecalibacterium reached 16.69% and 27.04% (p < 0.05), respectively, and Bifidobacteria accounted for 11.01% and 13% in the CY and CO groups. Additionally, the CO group contained 4.08% Romboutsia_B, which was not detected in the CY group. The findings from Bai et al. indicated a positive correlation between Romboutsia_B and inflammatory markers [37]. The Blautia_A abundance in the CO group (6.10%) also showed a decrease compared to the CY group (9.67%) (p < 0.05). This was in line with the declining trend among Parkinson’s disease patients [38]. However, as fermentation progressed, the relative abundance of Faecalibacterium and Bifidobacterium decreased significantly, while Escherichia and Clostridium_T gradually became the dominant microbial communities. In the CO group, the relative abundance of Escherichia remained stable from 66.7% to 65.7%, while Clostridium_T increased by 12.71-fold compared to T0 (from 0.99% to 13.57%, p < 0.05). The corresponding heatmap in Figure 2B visually illustrates these temporal changes.
Figure 2. Relative abundance of gut microbiota at the genus level by fermentation of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 at T0, T12, T24, and T36 with pooled fecal samples of the elderly over 65 years old. (A) Relative abundance of each genus by fermentation of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 at T0, T12, T24, and T36. (B) Heatmap of the relative abundance distribution of the genus by fermentation of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 at T0, T12, T24, and T36. CY represents the microbial community structure of the young, and CO was set as a sample control with pooled fecal samples of the elderly without any carbohydrate supplement. CAP, commercial apple polysaccharides.
Supplementation with polysaccharides produced distinct modulatory effects on the gut microbiota composition of the elderly. As shown in Figure 2A, Escherichia levels in the polysaccharide fermentation groups gradually decreased as fermentation progressed, with WKBC-P2 and WKBC-P3 reducing them by 65.42% and 68.14%, respectively (p < 0.05). Concurrently, the CO group exhibited a significant increase in Clostridium_T, whereas WKBC-P1, WKBC-P2, and WKBC-P3 significantly inhibited their growth (from 13.57% to 1.21%, 1.75%, and 6.50%, respectively) (p < 0.05). This indicates that white kidney bean cotyledon polysaccharides can reduce the number of harmful bacteria to a certain extent. For each polysaccharide fermentation group, fermentation with CAP induced only modest microbial changes, resulting in a moderate increase in Bifidobacterium by 43.95% at T24 and 49.19% at T36 compared to T12, without affecting the abundance of Escherichia. In contrast, fermentation with white kidney bean cotyledon polysaccharides produced a much stronger response across all extraction types. WKBC-P1, in particular, markedly enriched Bifidobacterium populations from 1.16% at T12 to 5.51% and 12.2% at T24 and T36 (p < 0.05), showing a 3.75-fold increase at T24 and a 9.52-fold increase at T36 relative to T12. WKBC-P2- and WKBC-P3-derived polysaccharides also stimulated the growth of Bifidobacterium during fermentation, showing 3.67-fold and 5.39-fold increases at T36 compared to T12, respectively. The relative abundance of Bifidobacterium in CO only reached 1.73% at T36, while this was significantly enhanced to 3.7%, 12.2%, 5.09%, and 7.03% (p < 0.05) with CAP, WKBC-P1, WKBC-P2, and WKBC-P3 supplements, respectively. The heatmap presented in Figure 2B clearly visualizes these changes, with color variations corresponding to the increased abundance of Bifidobacterium over time.
It is noteworthy that fermentation with WKBC-P2, but not CAP, WKBC-P1, and WKBC-P3, led to a pronounced enrichment of Bacteroides_H, and reached the highest relative abundance among all groups at 14.61% at T36, which was significantly higher than the level of 3.49% at T36 in CO (p < 0.05). The heatmap clearly reflects this increase, showing an intensified red coloration toward the end of fermentation (T36). In addition, we also observed that WKBC-P2 promoted the growth and proliferation of Parabacteroides_B. Compared to the CO group at T36, the WKBC-P2 supplement significantly increased the relative abundance of Parabacteroides_B from 4.27% to 6.00%, with an elevation by 40.52%. Furthermore, white kidney bean cotyledon polysaccharides significantly reduced the relative abundance of Clostridium_T in the gut microbiota, particularly WKBC-P1 and WKBC-P2. By the end of fermentation at T36, the relative abundance of Clostridium_T was decreased to 1.21% and 1.75% of WKBC-P1 and WKBC-P2, respectively, which were significantly lower than the level of 13.57% in the CO group (p < 0.05).

3.6. Dynamic Changes in Monosaccharide Composition During Fermentation

The monosaccharide components of polysaccharides serve as essential nutrient sources for gut microbial fermentation and play a crucial role in shaping both the composition of gut microbiota and the spectrum of metabolites produced throughout the fermentation process [39]. Overall, the concentrations of Ara, Glc, and Gal gradually declined across all samples during fermentation, indicating that these monosaccharides served as preferred carbon sources for the gut microbiota of the elderly (Table 2). By the end of fermentation (T36), Ara, Glc, and Gal in the CAP sample were utilized by 70.84%, 76.82%, and 63.22%, respectively, while Rha showed a utilization rate of 48.77% at T36. In contrast, the most abundant monosaccharide, GalA, was scarcely degraded.
Table 2. Monosaccharide degradation of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 by fermentation with gut microbiota of the elderly over 65 years old.
Monosaccharide utilization of WKBC-P1, WKBC-P2, and WKBC-P3 by gut microbiota of the elderly varied with fermentation. WKBC-P1 was more readily degraded, as Ara, Glc, and Gal of WKBC-P1 were already utilized at T12, and kept decreasing by 94.71%, 86.88%, and 72.88% at T36, respectively. Although Ara degradation was delayed until T24, the utilization by gut microbiota reached 96.72% at T36 in WKBC-P2. Additionally, gut microbiota started to use Glc and Gal at T12, and utilized 96.53% and 86.57% in WKBC-P2 at T36, respectively. Xyl and GalA in WKBC-P2 were specifically consumed by 33.10% and 60.34% at T36. The degradation of monosaccharides in WKBC-P3 proceeded at a slower rate compared to WKBC-P1 and WKBC-P2. Glc in WKBC-P3 was rapidly consumed, showing a reduction of 61.45% as early as T12. In contrast, Ara was not extensively utilized during the initial phase; instead, it was gradually released from partial polysaccharide degradation, resulting in a temporary accumulation and a slight increase in its concentration at T12. As fermentation progressed, both Ara and Gal were actively metabolized, with utilization rates reaching 86.86% and 61.21% at T24, and remaining at comparable levels by T36.

3.7. Dynamic Changes in SCFA and bSCFA Production During Fermentation

Short-chain fatty acids represent the principal metabolic end-products of gut microbial fermentation of dietary polysaccharides and play a central role in maintaining intestinal homeostasis. These metabolites, particularly acetic, propionic, and butyric acids, serve as energy sources for colonocytes and regulate immune and inflammatory responses. In addition to these major SCFAs, branched-chain fatty acids (bSCFAs) such as isobutyric, isovaleric, and valeric acids are formed through the microbial fermentation of amino acids, reflecting shifts in protein versus carbohydrate fermentation [40]. Therefore, measuring both SCFAs and bSCFAs provides critical insight into the metabolic balance of the gut microbiota and its functional response to dietary substrates.
As shown previously, white kidney bean cotyledon polysaccharides were utilized to varying degrees by the gut microbiota of the elderly, which likely influenced the quantity and profile of the SCFAs produced. To explore this relationship, fermentation of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 was analyzed for total and individual SCFA and bSCFA production, including acetic, propionic, butyric, isobutyric, isovaleric, and valeric acids. As illustrated in Figure 3, significant differences were observed among the polysaccharide types. Total SCFAs concentrations increased steadily over the fermentation period in all groups, with WKBC-P1 and WKBC-P2 producing notably higher levels (12.26 mmol/L and 12.75 mmol/L, respectively) compared with CO, CAP, and WKBC-P3. Conversely, the high GalA content of CAP likely hindered microbial accessibility to its polysaccharide backbone, resulting in lower SCFA production, as previously reported [41].
Figure 3. Concentration of short-chain fatty acids (SCFAs) and branched short-chain fatty acids (BCFAs) by fermentation of CAP, WKBC-P1, WKBC-P2, and WKBC-P3 at T0, T12, T24, and T36. (A) Acetic acid. (B) Butyric acids. (C) Propionic acids. (D) Valeric acids. (E) Isobutyric acid. (F) Isovaleric acid. (G) Total SCFAs. (H) Total BCFAs. (I) Total SCFAs/Total BCFAs. Different capital letters indicate significant differences (p < 0.05) among different groups. Different lowercase letters indicate significant differences (p < 0.05) with fermentation time. CAP, commercial apple polysaccharides.
Increased concentration of total SCFAs was attributed to the accumulation of acetic acid, butyric acid, and propionic acid. Here, acetic acid produced by WKBC-P1, WKBC-P2, and WKBC-P3 was significantly higher than that of CAP and the control by the end of fermentation at T36. In addition, the high amount of acetic acid produced by white kidney bean cotyledon polysaccharides might be associated with their lower molecular weight than CAP. Butyric acid is a key SCFA molecule, with important roles in supplying energy to intestinal epithelial cells and regulating age-related intestinal dysfunction [42]. WKBC-P2 especially increased butyric acid production, and was dramatically higher than CAP, WKBC-P1, and WKBC-P3, reaching a concentration of 1.50 mmol/L. WKBC-P2 also exhibited the most prominent propionic acid production, with a concentration as high as 2.91 mmol/L. Propionic acid not only performs important metabolic functions but also alleviates intestinal inflammation by regulating energy metabolism and immune responses, thereby maintaining intestinal homeostasis [43].
The production of valeric acid, isobutyric acid, and isovaleric acid during fermentation with white kidney bean polysaccharides and CAP was markedly lower than that observed in the CO groups, as shown in Figure 3D–F. Consequently, the total concentration of bSCFAs was also reduced in the polysaccharide-supplemented groups (Figure 3H). This reduction likely reflects a shift in microbial metabolism toward carbohydrate fermentation, as the availability of fermentable polysaccharides decreases the microbial reliance on amino acid catabolism. In contrast, the higher bSCFAs levels detected in CO controls may result from a limited supply of fermentable carbohydrates, which promotes the microbial degradation of branched-chain amino acids. Although no significant differences in bSCFAs concentrations were detected among the polysaccharides supplemented groups, the overall levels of these metabolites in CAP, WKBC-P1, WKBC-P2, and WKBC-P3 remained consistently lower than those observed in the CO groups. This pattern suggests that, during fermentation, gut microbiota of the elderly preferentially metabolize the supplied polysaccharides as carbohydrate substrates rather than shifting toward protein fermentation pathways.

3.8. The Relationship Between Structural Characteristics of White Kidney Bean Cotyledon Polysaccharides and Prebiotic Potential

Based on the above observations, relationships were built between the gut microbiota composition and fermentation characteristics of white kidney bean cotyledon polysaccharides. Data on gut microbiota composition, SCFAs production, and the monosaccharides content of the polysaccharides fermentation group at T12–T36 were involved. Pearson correlation analysis and a heatmap were employed to visualize the relationships, and the results with significance are shown in Figure 4. As illustrated, Bacteroides_H and Parabacteroides_B showed significant positive correlations (p < 0.05) with the majority of SCFAs production, while exhibiting significant negative correlations (p < 0.05) with monosaccharides such as Gal and Glc. Bifidobacterium exhibited similar patterns as Bacteroides_H and Parabacteroides_B, showing significant negative correlations with neutral sugars like Ara, Gal, and Glc (p < 0.05), while showing only positive correlation with acetate acid production (p < 0.05). Conversely, Escherichia showed a highly significant negative correlation with SCFAs production (p < 0.01) and a highly significant positive correlation with neutral sugars like Ara, Gal, and Glc (p < 0.01). Romboutsia_B, Blautia_A, and Gemmiger_A also exhibited significant negative correlations with SCFAs production, including propionic acid and butyric acid (p < 0.05). Blautia_A and Gemmiger_A showed significant negative correlations with Xyl (p < 0.05). The significant correlation results of Romboutsia_B, Blautia_A, and Gemmiger_A were less significant than the relative abundances of Romboutsia_B, Blautia_A, and Gemmiger_A, which were at a very low level among the polysaccharide groups. Moreover, correlation analysis of monosaccharides such as GlcA and Rha was also of limited significance due to their extremely low abundance in white kidney bean cotyledon polysaccharides.
Figure 4. Correlation analysis of fermentation characteristics of white kidney bean cotyledon polysaccharides and gut microbiota in genus level. * p < 0.05, ** p < 0.01.

4. Discussion

The gut serves as a key target organ for improving health in the elderly, and dysbiosis of the gut microbiota has been linked to age-associated chronic diseases [6]. Multiple studies indicated that polysaccharides extracted from natural resources can function as prebiotics to improve gut microbiota composition and exert anti-aging effects, with their efficacy dependent on polysaccharide structure [44]. This study employed an ex vivo model to investigate the prebiotic activity of white kidney bean cotyledon polysaccharides obtained through different extraction methods and the relationship with polysaccharide structure, providing a basis for the subsequent development of nutritional supplements for the elderly.
Different extraction methods would cause variance in the composition and structure of polysaccharides [45]. Unlike soybean soluble polysaccharides, which primarily consisted of Gal as the main monosaccharide, the polysaccharides extracted from white kidney bean cotyledon in this study were mainly composed of Ara and Glc [46]. Specifically, WKBC-P1 and WKBC-P3 were primarily composed of Ara, while WKBC-P2 was mainly enriched in Glc. Acidic extraction might significantly enrich Ara by disrupting non-covalent bonds in the cell wall. This is consistent with previous findings showing that Ara commonly occurred in the furanose form within the side chains of polysaccharides, where its glycosidic linkages were relatively labile and could be readily hydrolyzed and released under acidic extraction conditions [47]. Additionally, chelating agent extraction might effectively release more side chain structures by breaking metal ion bridges within polysaccharide molecules, which contributed to the accumulation of Ara [48]. However, alkaline extraction had a weak effect on Ara release and tended to release a large amount of Glc, with the molar ratio of Glc notably higher than WKBC-P1 and WKBC-P3. Alkaline extraction might disrupt hydrogen bonds and metal ion bridges that form cross-linking structures, thereby releasing glucose from cellulose or starch [49,50]. Whereas, due to the limited cleavage of the main polysaccharide backbone and partial de-esterification under alkaline extraction, WKBC-P2 had a high molecular weight [51]. In contrast, the low molecular weight of WKBC-P1 can be attributed to the glycosidic bond cleavage caused by acid hydrolysis [52]. Chelating agent extraction primarily promoted polysaccharide release by disrupting metal ion bridges and loosening the cross-linked structure of the cell wall matrix, while minimizing degradation of the molecular chains. This resulted in the highest molecular weight and a uniform molecular weight distribution [48]. As reported before, polysaccharides with different molecular weights exhibited distinct biological activities. Compared to high-molecular-weight polysaccharides, low-molecular-weight polysaccharides were more readily degraded and utilized by gut microbiota [53]. These may indicate that extraction methods lead to distinct fermentation properties attributed to structural characteristics of polysaccharides.
Distinct prebiotic activities were observed in different white kidney bean cotyledon polysaccharide structures. WKBC-P1 and WKBC-P2, in particular, had a strong potential to restore and maintain a balanced gut microbiota in the elderly. This is evidenced by a significantly improved relative abundance of Bifidobacterium, Bacteroides_H, and Parabacteroides_B, as well as a reduced growth and proliferation of Escherichia and Clostridium_T by WKBC-P1 and WKBC-P2. Bifidobacterium is a well-established producer of SCFAs and plays a central role in maintaining intestinal health [6]. The observed stimulatory effects of white kidney bean polysaccharides on Bifidobacterium proliferation were likely related to their relatively high content of fermentable neutral sugars, particularly Ara and Glc, which served as preferred carbon sources for this beneficial genus [54]. Bacteroides, as key commensal bacteria in the human gut, is equipped with a broad spectrum of polysaccharide-degrading enzymes and glycosidases that enable them to efficiently break down complex carbohydrates. In this way, Bacteroides might contribute to the maintenance of intestinal homeostasis by preventing inflammatory gastrointestinal conditions and alleviating metabolic disturbances associated with aging [55]. Additionally, Parabacteroides could also provide protection against aging-related bacterial infections [56]. Such advantages of WKBC-P1 and WKBC-P2 in regulating microbial communities might be benefited from their structural characteristics. WKBC-P1 and WKBC-P2 possessed more compact structures, as illustrated by SAXS analysis. Previous research by Tingirikari et al. reported that a more compact molecular conformation can promote bacterial adhesion to polysaccharide surfaces, thereby enhancing enzymatic hydrolysis and improving fermentation efficiency [57]. Furthermore, SEM analysis of WKBC-P1 and WKBC-P2 revealed irregular porous particles and irregular stacking of the layered structure, which were also more conducive to bacterial colonization [58]. These observed differences in microbial communities, scattering patterns, and cross-linking networks further suggest that the extraction methods exert a substantial influence on polysaccharide aggregation behavior, structural stability, and ultimately their biological activity [59].
As a result of microbial regulation, WKBC-P1 and WKBC-P2 also significantly improved the production of SCFAs. WKBC-P1 produced the highest amount of acetic acid at T36, which might be associated with the high abundance of Bifidobacterium, corresponding to the results in correlation analysis. Acetic acid accumulation in aged individuals has been shown to significantly extend lifespan and improve age-related disorders, including intestinal inflammation and redox status [60]. The observation may suggest the potential of WKBC-P1 as an anti-inflammatory and anti-oxidative material for the elderly. WKBC-P2 especially accumulated butyric acid with fermentation, which correlated with its enrichment of Bacteroides_H capable of metabolizing polysaccharides to produce butyric acids [61]. Furthermore, WKBC-P2 also enriched beneficial bacteria such as Bifidobacterium and Parabacteroides_B, collectively contributing to its high butyric acid yield. WKBC-P3 exhibited a weaker regulatory effect on beneficial bacteria after fermentation and promoted the growth of pathogens such as Clostridium_T, resulting in significantly lower SCFAs concentrations than WKBC-P1 and WKBC-P2. This may be associated with molecular weight differences. Low-molecular-weight polysaccharides outperformed high-molecular-weight polysaccharides in promoting the growth of beneficial bacteria (such as Bifidobacteria and Bacteroides_H) and inhibiting potential pathogens, while also producing more SCFAs during fermentation [61]. Elevated concentrations of bSCFAs, particularly isobutyric and isovaleric acids, are commonly associated with protein fermentation and have been linked to unfavorable intestinal conditions, including inflammation and epithelial stress [62,63]. Although all polysaccharides significantly reduced bSCFA concentrations with fermentation, bSCFAs produced by WKBC-P2 fermentation were higher compared to the other two groups. This may be attributed to the high protein content in WKBC-P2. When the fermentable carbohydrates were consumed by the end of fermentation, gut microbiota shifted to utilize protein present in crude polysaccharides [64].
Dynamic changes in monosaccharides during fermentation revealed that the gut microbiota favored utilizing abundant neutral sugars (e.g., Ara and Glc) present in white kidney bean cotyledon polysaccharides. We discovered that white kidney bean cotyledon polysaccharides WKBC-P1, WKBC-P2, and WKBC-P3 improved the relative abundance of Bifidobacterium through degradation of Ara, Gal, and Glc. There was substantial Glc consumption in WKBC-P2, consistent with the metabolic profile of Bacteroides, which secretes β-glucosidase and preferentially cleaves glycosidic bonds containing glucose residues [65]. WKBC-P1 exhibited extensive utilization of neutral sugars during the early fermentation stage. We speculate this might be associated with its low molecular weight, compact molecular conformation, and porous structure. Such structural specificity results in distinct monosaccharide degradation and microbial community changes among all three white kidney bean cotyledon polysaccharides, providing a foundation for future studies on the structure-function relationship of polysaccharides within aging-related microbial communities. However, we acknowledge certain limitations in this study. For example, the ex vivo fermentation model cannot fully replicate the human intestinal environment containing a mucus layer and mimicking host–microbe interactions. To elucidate donor-specific variations, individual fermentation of fecal samples could be achieved with more donor recruitment. Additionally, the purity of the polysaccharides could be further improved to diminish the possible role of proteins and to define the fine structures of the polysaccharides.

5. Conclusions

In this study, we systematically investigated the regulatory effects of white kidney bean cotyledon polysaccharides, which were obtained via different extraction methods from the gut microbiota of the elderly through ex vivo fermentation. Results revealed that the structure–function characteristics of these polysaccharides have a significant effect on gut microbial metabolic activities and production of SCFAs. FT-IR proved that WKBC-P1, WKBC-P2, and WKBC-P3 all contained typical groups of polysaccharides. WKBC-P1 and WKBC-P3 were abundant in Ara, while WKBC-P2 was rich in Glc. In addition, the relatively low Dmax value, molecular weight, and irregular rough surface of WKBC-P1 and WKBC-P2 may facilitate microbiota fermentation. In particular, WKBC-P1 was rapidly utilized and increased the relative abundance of Bifidobacterium as well as the production of SCFAs. WKBC-P2 was gradually degraded, particularly enriched Bacteroides_H and Parabacteroides_B, and promoted the production of butyric acid. In contrast, WKBC-P3 exhibited a much slower fermentation rate. Our findings suggest positive effects of WKBC-P1 and WKBC-P2 on gut microbial community balance and metabolic activity. This knowledge provides a theoretical basis for the development of polysaccharide-based dietary supplements for the elderly.

Author Contributions

Conceptualization, C.K. and B.W.; methodology, Y.H. and X.L.; software, Y.H., J.S., and X.L.; validation, C.K., Y.H., and X.L.; formal analysis, C.K., Y.H., and J.S.; investigation, C.K., Y.H., J.S., and X.L.; resources, B.W.; data curation, C.K. and Y.H.; writing—original draft preparation, C.K. and Y.H.; writing—review and editing, C.K., Y.H., and B.W.; visualization, C.K. and Y.H.; supervision, B.W. and P.d.V.; project administration, C.K. and P.d.V.; funding acquisition, C.K. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the National Natural Science Foundation of China (32201954).

Institutional Review Board Statement

The studies involving human participants were reviewed and approved by the Ethical Committee for Scientific Research, School of Food and Health, Beijing Technology and Business University (NO. 253, 9 December 2025).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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