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. 2023 Dec 10;21:101058. doi: 10.1016/j.fochx.2023.101058

Structural and digestive characters of a heteropolysaccharide fraction from tea (Camellia sinensis L.) flower

Dan Chen a, Ao Wang a, Jialiang Lv a, Chao Tang a, Chang-hai Jin a, Jun Liu a, Xiaoxiong Zeng b, Li Wang c,⁎
PMCID: PMC10765012  PMID: 38178927

Highlights

  • •

    TFPS-1-3p was a typical heteropolysaccharide composed of Rha, Ara, Gal and GalA.

  • •

    [→4)-α-d-GalpA-(1 → ]n and → 4)-α-d-Galp-(1 → 2,4)-α-l-Rhap-(1 → as the main linkages.

  • •

    Branched linkages included 1,6-β-Galp, 1,3,6-β-Galp, 1,5-α-Araf and 1,3,5-α-Araf.

  • •

    TFPS-1-3p could not be degraded under simulated human gastrointestinal conditions.

  • •

    TFPS-1–3 degradation was attributed to gut microbes and generated SCFAs.

Keywords: Tea flower, Polysaccharide, Structure, Digestibility, Bacteroides

Abstract

Tea (Camellia sinensis L.) flower polysaccharides (TFPS) have various health-promoting functions. In the present work, the structure of a purified TFPS fraction, namely TFPS-1-3p, and its in vitro digestive properties were investigated. The results demonstrated that TFPS-1-3p was a typical heteropolysaccharide consisting of rhamnose (Rha), arabinose (Ara), galactose (Gal) and galacturonic acid (GalA) with a molecular weight of 47.77 kDa. The backbone of TFPS-1-3p contained → 4)-α-d-GalpA(-6-OMe)-(1 → 4)-α-GalpA-(1 → and → 4)-α-d-GalpA(-6-OMe)-(1 → 2,4)-α-l-Rhap-(1 → linkages. The branch linkages in TFPS-1-3p contained → 6)-β-d-Galp-(1→, →3,6)-β-d-Galp-(1→, →5)-α-l-Araf-(1 → and → 3,5)-α-l-Araf-(1 →. Subsequently, TFPS-1-3p could not be degraded under simulated human gastrointestinal conditions but could be of use to human fecal microbes, thereby lowering the pH and increasing the production of short-chain fatty acids (SCFAs) of the gut microenvironment and altering the composition of the gut microbiota. The relative abundance of Fusobacterium_mortiferum Megasphaera_elsdenii_DSM_20460, Bacteroides thetaiotaomicron, Bacteroides plebeius and Collinsella aerofaciens increased significantly, potentially contributing to the degradation of TFPS-1-3p.

1. Introduction

Dietary polysaccharides are distributed in a wide range of plants, animals, and microorganisms. It is widely recognized that dietary polysaccharides with low toxicity have multiple benefits for health, such as antioxidant, anti-obesity, anti-diabetic, antitumor, hepatoprotective, immunomodulatory and probiotics-like activities, and thus have received extensive attention (Chen et al., 2021, Chen et al., 2022, Lee et al., 2022). Physico-chemical and function characteristics of dietary polysaccharides vary with their sources, extraction methods and structural characteristics (Chen, et al., 2022). Polysaccharides can be structurally classified into homogeneous and heterogeneous polysaccharides in view of their monosaccharide compositions (Yao, Wang, Yin, Nie, & Xie, 2021). Homopolysaccharides are composed of a uniform type of sugar monomer, such as starch, cellulose, or glycogen. Heteropolysaccharides are comprised of two or more different monosaccharide units linked by linear or branched glycosidic linkages. However, the restricted use of polysaccharides as dietary supplements has been limited to date because of their low purity, high heterogeneity, and ambiguous structure–function relationships (Tang, et al., 2019). Understanding the chemical structure of dietary polysaccharides remains challenging, which severely limits their application (Chen et al., 2022, Yao et al., 2021).

Most dietary polysaccharides have complex glycosidic linkages and are difficult for the body's digestive enzymes to break down. The human lower gastrointestinal tract is colonized by a complex community of trillions (1014) of individual microorganisms. This complex ecosystem is largely driven by a broad structural diversity of indigestible dietary polysaccharides (Briggs, Grondin, & Brumer, 2021). For example, carbohydrate-active enzymes (CAZymes) that target the carbohydrate-rich human diet and indigestible dietary polysaccharides present in the gut are encoded by many members of the gut flora. Gut microbes grow on complex and diverse polysaccharides, and gut microbial taxa may compete or cooperate in glycosyl catabolism, thus forming a complex ecological network across different trophic niches (Tang, et al., 2019). This quantitative and functional diversity of polysaccharide utilization systems determines a taxon's polysaccharide utilization profile, be it a “specialist” or “generalist” (Briggs, et al., 2021). Gut microbiota ferment non-digestible polysaccharides to produce short-chain fatty acids (SCFAs) and other metabolites that are captured by the colonic endothelium and released into the circulation, where they can have multiple beneficial effects on hosts (Frampton, Murphy, Frost, & Chambers, 2020). Maintaining a balanced gut ecology has been implicated in the regulation of human diseases (de Vos et al., 2022, Fan and Pedersen, 2021, Hasain et al., 2020), and is one of the important operational roles of numerous dietary polysaccharides, accompanied by their own metabolic processes in the body.

In the past two decades, tea (Camellia sinensis L.) flowers have been found to be a valuable new resource that has attracted much attention in Japan and China (Chen, et al., 2020). In this context, tea flower polysaccharides (TFPS) are typical substances with various health-promoting functions, including antioxidant, anti-obesity, hepatoprotective, anti-allergic, and immunomodulatory activities (Chen, et al., 2019). Our previous work revealed that a purified polysaccharide fraction, named TFPS-1-3p, could enhance the immunoreactions in cyclophosphamide-induced immunosuppression mice and could serve as a potential immunomodulator to improve host’s health (Chen, et al., 2019). Given that TFPS-1-3p consisted of carbohydrates (33.96 ± 0.04 %, glucose equivalent), uronic acids (78.79 ± 1.51 %, glucuronic acid equivalent), and proteins (0.75 ± 0.14 %), and was made up of the following galacturonic acid (GalA), arabinose (Ara), galactose (Gal), and rhamnose (Rha) with a molar ratio of 74.05: 14.56: 7.92: 3.47 (Chen, et al., 2019). However, the structure of TFPS-1-3p remains elusive. Therefore, the structure of TFPS-1-3p was characterized using high-performance gel permeation chromatography (HPGPC), methylation analysis, and nuclear magnetic resonance (NMR) spectroscopy. Additionally, the digestive properties of TFPS-1-3p were investigated in vitro under simulated human gastrointestinal and colonic conditions. The aim of this work is to provide a basic structural characterization and digestive properties for further exploitation of TFPS-1-3p.

2. Materials and methods

2.1. Materials and chemicals

Dried tea flowers (variety, Longjing 43, harvested in November 2019 in Hangzhou, Zhejiang, China) were made available by the Department of Tea Science, Zhejiang University (Hangzhou, China). DEAE-Fast Flow and Sephadex G-100 were acquired by GE Healthcare Life Sciences. 3, 5-Dinitrosalicylic acid (DNS) was acquired by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glucose was acquired by Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). α-Amylase (from human saliva), gastric lipase, and pancreatin were acquired by Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Pepsin, trypsin, and bile salt were bought by Solarbio Science & Technology Co., Ltd. (Beijing, China). All other reagents used were of at least analytical grade.

2.2. Preparation of TFPS-1-3p

The preparation of TFPS-1-3p was in accordance with our report (Chen, et al., 2019). Dried flowers were pulverized and pre-treated twice with 80 % aqueous ethanol at 1:20 (w/v). The pre-treated dried powder of tea flowers was then extracted twice with a distilled water (1:25, w/v) at 96 °C for 4 h each, and then centrifuged (3000 × g, 15 min) to collect the supernatants, and after concentration and precipitation with anhydrous ethanol, the crude TFPS extract was obtained. The crude TFPS was dissolved to filter through a 0.1 μm membrane (Merck Millipore, Germany), and the filtrate was precipitated with four times the volume of anhydrous ethanol to obtain the TFPS-1 extract after centrifugation (3000 × g, 15 min), dialysis, and lyophilization. Then, TFPS-1 (200 mg) was dissolved in deionized water (5.0 mL), applied to a DEAE-Fast Flow column (2.6 × 30 cm) and eluted stepwise with sodium chloride solution (0, 0.1, 0.3 and 0.5 mol/L) at a flow rate of 1.0 mL/min to obtain three fractions: TFPS-1–1, TFPS-1–2, and TFPS-1–3, respectively. TFPS-1–3 was further applied to a Sephadex G-100 column (1.6 × 100 cm) and eluted with 0.1 mol/L of NaCl solution at a flow rate of 15 mL/h, yielding TFPS-1-3p (Fig. S1).

2.3. The weight-average molecular weight (Mw) of TFPS-1-3p

The Mw of TFPS-1-3p was determined by HPGPC using an Agilent 1200 series instrument (Agilent Technologies, Santa Clara, CA, USA) fitted with a refractive index detector and a TSK-G4000PWXL column (7.8 mm × 300 mm, Tosoh Crop., Tokyo, Japan) (Yuan, et al., 2015). The mobile phase was 0.1 mol/L NaCl at a flow rate of 0.5 mL/min and the temperature of column oven was 35 °C. The Mw of TFPS-1-3p was estimated using standard dextran with known Mw of 6.2, 10, 21.7, 48.8, 113, 200, 348 and 736 kDa.

2.4. Methylation and GC–MS analysis

The glycosidic linkage of TFPS-1-3p was analyzed by methylation and the partially methylated alditol acetate (PMAA) derivatives were analyzed by GC–MS according to the reported method (Sims, Canachan, Bell, & Hinkley, 2018). TFPS-1-3p (5 mg) was dissoluted in deionized water (1 mL), combined with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (1 mL, 100 mg/mL) and allowed to react for 2 h. After adding 1 mL of 2 mol/L imidazole, the solution was mixed with 1 mL of 30 mg/mL NaBH4 or NaBD4 and further reacted for 3 h and then terminated with acetic acid (100 μL). The resultant solution was dialyzed and lyophilized to provide carboxyl-reduced TFPS-1-3p for methylation. Carboxyl-reduced TFPS-1-3p was disbanded in 500 μL of dimethyl sulfoxide (DMSO), and mixed with 1 mg of NaOH and then allowed to react for 30 min. The solution was merged with 50 μL of methyl iodide and stored in the dark for 1 h, and then merged with 1 mL of water and 2 mL of dichloromethane. The phase of dichloromethane was collected, rinsed three times with water, and dried with nitrogen. The dried sample was merged with 100 μL of 2 mol/L trifluoroacetic acid solution and hydrolyzed at 121 °C for 90 min and then dried. This dried sample was mixed with 50 μL of 2 mol/L ammonium hydroxide and 50 μL of 1 mol/L NaBD4 and allowed to react at room temperature for 2.5 h. Acetic acid (20 μL) was used to stop the reaction, and the solution was rinsed four times in methanol and dried under nitrogen. The dried sample was then mixed with acetic anhydride (250 μL) and reacted at 100 °C for 2.5 h. 1 mL of water and 500 μL of dichloromethane were added sequentially, and the phase of dichloromethane was harvested after three times washes with water and analyzed on a GC–MS (7890A-5977B, Agilent Technologies Inc., CA, USA) platform fitted with a BPX70 column (30 m × 0.25 mm × 0.25 µm, SGE, Australia). The separation ratio was 10:1. Carrier gas was supplied at a flow rate of 1.0 mL/min using high purity helium. The initial column oven temperature was 140 °C for 2.0 min, and the temperature was ramped to 230 °C with a 3 °C/min program and maintained for 3 min. Analyte was detected in the mass scan range (m/z) in full scan mode (SCAN): 30–600. According to the standardized data in the CCRC Spectral Database for PMAA derivatives, the glycosyl linkages of TFPS-1-3p were identified.

2.5. NMR analysis

TFPS-1-3p was suspended in 0.5 mL of deuterium oxide (D2O, 99.9 % D) and freeze-dried three times to replace hydrogen. The 100 mg of dried sample was disoluted in 1 mL of D2O, and 1D (1H NMR and 13C NMR) and 2D (homonuclear 1H/1H correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC) and nuclear Overhauser effect spectroscopy (NOESY)) NMR spectra were acquired using a Bruker 600 MHz NMR instrument (Bruker Corp., Fallanden, Switzerland) at 298 K with 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TMSP) as the internal standard. The NMR spectra were calibrated by HDO hydrogen δH at δ 4.70 ppm, methyl carbon of TMSP δC at δ −1.80 ppm, respectively.

2.6. In vitro digestive properties of TFPS-1-3p

The in vitro digestion properties of TFPS-1-3p were investigated under the simulated human gastrointestinal and colonic conditions.

2.6.1. Simulated oral and gastrointestinal digestion

The in vitro oral and gastrointestinal digestion was performed as protocol described with a few modifications (Brodkorb, et al., 2019). TFPS-1-3p was formulated 1:1 (wt/wt) with simulated salivary fluid (with or without salivary amylase) in the oral phase and incubated with stirring at pH 7 to simulate chewing of the meal. Simulated salivary fluid consisted of KCl (15.1 mmol/L), KH2PO4 (3.7 mmol/L), NaHCO3 (13.6 mmol/L), NaCl, MgCl2(H2O)6 (0.15 mmol/L), (NH4)2CO3 (0.06 mmol/L), HCl (1.1 mmol/L), CaCl2(H2O)2 (1.5 mmol/L), and salivary amylase (if present, 150 U/mL). The oral boluses were then formulated 1:1 (v/v) with simulated gastric fluid with or without gastric enzymes (pepsin and gastric lipase) and incubated for 6 at pH 3.0 h with stirring. Simulated gastric fluid consisted of KCl (6.9 mmol/L), KH2PO4 (0.9 mmol/L), NaHCO3 (25 mmol/L), NaCl (47.2 mmol/L), MgCl2(H2O)6 (0.12 mmol/L), (NH4)2CO3 (0.5 mmol/L), HCl (15.6 mmol/L), CaCl2(H2O)2 (0.15 mmol/L), pepsin (if present, 4000 U/mL) and gastric lipase (if present, 120 U/mL). Gastric chyle was then formulated 1:1 (v/v) with simulated intestinal fluid with or without pancreatin and bile salts, and incubated for another 6 h at pH 7. Simulated intestinal fluid consisted of KCl (6.8 mmol/L), KH2PO4 (0.8 mmol/L), NaHCO3 (85 mmol/L), NaCl (38.4 mmol/L), MgCl2(H2O)6 (0.33 mmol/L), HCl (8.4 mmol/L), CaCl2(H2O)2 (0.6 mmol/L), pancreatin (if present, 200 U/mL) and bile salt solution (if present, 20 mmol/L). Each experiment was the subject of five independent replicates.

2.6.2. Simulated colonic fermentation

The in vitro colonic fermentation of TFPS-1-3p was investigated as the described protocol (Zhou, et al., 2018). The fresh feces were obtained from four healthy individuals (one female and three males) who were in good physical condition and off antibiotics for at least 3 months. Each fresh fecal samples were formulated equally and homogenized in autoclaved phosphate-buffered saline (0.1 mmol/L, pH 7.2) to obtain 10 % (w/v) fecal slurry. The supernatants were immediately transferred to anaerobic flasks as microbial donors after centrifugation (500 × g, 5 min). TFPS-1-3p was diluted 10:1 (w/v) with and the autoclaved nutrient medium and this mixture was diluted 9:1 (v/v) of microbial donors to start the fermentation phase in an anaerobic incubator (Yuejin Medical Optical Instruments Factory, Shanghai, China) and incubated at 37 °C for 24 h. The nutrient medium contained 2.0 g/L of yeast extract, 2.0 g/L of peptone, 0.1 g/L of NaCl, 0.04 g/L of K2HPO4, 0.01 g /L of CaCl2·6H2O, 0.01 g/L of MgSO4·7H2O, 2.0 g/L of NaHCO3, 0.02 g/L of hemin, 0.5 g/L of cysteine HCl, 0.5 g/L of bile salts, 1.0 mg/L of resazurin, 0.2 % (v/v) of Tween 80 and 0.001 % (v/v) of vitamin K1. Following the parallel conditions, equal amounts of fructooligosaccharide (FOS) and blank addition (BLK) were used as controls. The in vitro colonic fermentation phase at 0 h was maintained as the Origin group. Each experiment had five independent replications.

2.6.3. Changes of residual total carbohydrates and reducing sugar content during the simulated digestion and fermentation process

Total carbohydrate content was measured by the phenol–sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Reducing sugar content was measured by DNS method (Miller, 1959). Glucose was used as the standard.

2.7. Effect of TFPS-1-3p on the gut microenvironment

The pH value during the colonic fermentation phase was detected using a pH meter (Mettler-Toledo Instruments Co., Ltd. Shanghai, China). The colonic fermentation chyme at 0 h and 24 h was centrifuged at 5000 × g for 5 min, the supernatants were obtained and formulated 1:1 (v/v) with 25 mmol/L 2-ethylbutyric acid (an internal standard). SCFAs was measured according to the reported method (Tian, et al., 2016). The mixture was formulated 1:1 (v/v) with 0.15 mmol/L of oxalic acid solution. 1 μL of the supernatant was obtained after centrifugation (5000 × g, 5 min) and injected onto an Agilent 6890 N GC system fitted with a HP-INNOWAX column (30 m × 0.25 mm × 0.25 μm, Agilent) and a flame ionization detector for the measurement of acetate, propionate, n/i-butyrate and n/i-valerate. Lactate was measured using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s protocol. Meanwhile, colonic fermentation chyme precipitates at 0 h and 24 h were collected for microbial DNA extraction, followed by amplification of the V3-V4 regions of 16S rDNA on the Illumina MiSeq sequencing platform. Fecal microbiota composition was assessed based on QIIME microbiota analysis (Langille, et al., 2013).

2.8. Statistical analysis

All data are displayed as the mean ± standard deviation (SD) or box-whisker plots. Statistical significance between groups was analyzed by one-way ANOVA procedure followed by Tukey test using SPSS 22 software (IBM). A value of p < 0.05 was considered a significant difference. Linear discriminant analysis effect size (LEfSe) of microbial diversity was assessed on an online platform (https://huttenhower.sph.harvard.edu/galaxy/).

3. Results and discussion

3.1. Determination of the Mw of TFPS-1-3p

As shown in Fig. 1A, a single and symmetric peak was observed in the HPGPC chromatogram. Based on the calibration with dextran standard, the Mw of TFPS-1-3p was estimated to be 47.77 kDa. In another study, Han, et al. (2011) reported that the Mw of two polysaccharide fractions from tea flowers ranged from 10.71 to 167.5 kDa. The differences in Mw of TFPS could be associated with varieties, purification processes and test methods (Chen, et al., 2020).

Fig. 1.

Fig. 1

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Structural information identification of TFPS-1-3p. (A) HPGPC chromatogram, (B) 1H NMR spectra, (C) 13C NMR spectra, (D) HSQC NMR spectra, (E) COSY NMR spectra, (F) HMBC NMR spectra, (G) NOESY NMR spectra, and (H) hypothetical structure.

3.2. Methylation analysis

Methylation analysis and GC–MS were used to investigate the glycosyl linkages of TFPS-1-3p. The glycosyl linkages of Rha, GalA, Gal, and Ara are displayed and summarized in Table 1 in accordance with the retention times and GC–MS mass fragments (Fig. S2 and S3, Supplementary Material). The peaks of PMAA derivatives of TFPS-1-3p were recognized as nine linkage modes including Araf(1→, Galp(1→, GalpA(1→, →5)Araf(1→, →2,4)Rhap(1→,→4)GalpA(1→, →6)Galp(1→, →3,5)Araf(1→, and → 3,6)Galp(1 → with the molar ratio of 3.17: 5.26: 7.10: 2.29: 3.27: 71.23: 2.32: 2.48: 2.88 (The calculated data are shown in Table S1, Supplementary Material). The proportions of Rhap, GalpA, Galp and Araf in the methylation analysis were 1: 23.94: 3.20: 2.43. These results demonstrated that TFPS-1-3p was a typical heteropolysaccharide. The constituent sugar data indicated a higher proportion of Ara than the methylation data, which was likely due to issues with the methodological issues and resulting loss of some arabinosyl residues during the methylation procedure.

Table 1.

Methylation analysis and mode of linkage of TFPS-1-3p.

Methylated alditol acetate Type of linkage Retention time Molar ratio Mass fragments (m/z)
1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol Araf(1→ 5.678 3.17 59, 71, 87, 102, 118, 129, 145, 161
1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol Galp(1→ 9.514 5.26 59, 71, 87, 118, 129, 145, 161, 205
1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol GalpA(1→ 9.514 7.10 59, 71, 88, 118, 131, 147, 163, 207
1,4,5-tri-O-acetyl-2,3-di-O-methyl arabinitol →5)Araf(1→ 10.157 2.29 59, 87, 102, 118, 129, 162, 189
1,2,4,5-tetra-O-acetyl-6-deoxy-3-O-methyl rhamnitol →2,4)Rhap(1→ 11.951 3.27 74, 88, 102, 117, 130, 143, 160, 190, 203
1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol →4)GalpA(1→ 13.297 71.23 73, 87, 102, 118, 131, 175, 205, 235
1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol →6)Galp(1→ 14.976 2.32 59, 87, 102, 118, 129, 162, 189, 233
1,3,4,5-tetra-O-acetyl-2-O-methyl arabinitol →3,5)Araf(1→ 18.207 2.48 85, 118, 127, 159, 201, 261
1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl galactitol →3,6)Galp(1→ 18.389 2.89 59, 74, 87, 101, 118, 129, 160, 189, 202, 234, 305
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3.3. NMR spectra

To further explore the structure of TFPS-1-3p, 1D (1H and 13C) and 2D (COSY, HSQC, HMBC and NOESY) NMR spectra (Fig. 1B–F) were performed. As shown in Table 2, nine residues (labeled as A-I) were carefully distinguished. The 1H NMR spectral signals in Fig. 1B were mainly between 3.0 and 5.5 ppm. Certain sugar residues or groups can be identified by the characteristic signals of 1H NMR. For example, the methyl proton signal of 6-position deoxysugar is displayed in the high-field region of δ 0.8–1.4 ppm; the methyl proton signal of acetyl (CH3COO– or -OAc) appears in the low-field region of δ 1.8–2.2 ppm; the methyl proton signal of methyl ester (–COOCH3 or -COOMe) is located at δ 3.0–3.8 ppm (Yao, et al., 2021). The δH in the region of 4.4–4.95 ppm and 4.95–5.5 ppm was evidence for the presence of both α- and β-configured glycosidic linkages in TFPS-1-3p. In addition, the signals of the anomeric proton occurred primarily between 4.0 and 5.5 ppm, including δ4.92, 4.78, 5.17, 4.36, 4.4, 4.5, 5.09, 5.01, and 4.96 ppm equivalent to H-1 of A, B, C, D, E, I, F, and G. The 13C NMR spectrum of the TFPS-1-3p was displayed in Fig. 1C, multiple anomeric carbons in the region of δ 90–111 ppm including δ100.64, 101.31, 104.36, 104.38, 105.89, 108.53, 108.68 and 110.61 corresponding to C-1 of C/A, B, D, E, F, H, I and G. It has been suggested that the reduced-terminal end groups belong to α-GalpA and β-GalpA based on the C-1 signal peaks at δ93.62 and 97.83 ppm, respectively (Petersen, Meier, Duus, & Clausen, 2008).

Table 2.

1H and 13C chemical shifts from identified 2D NMR spectra of TFPS-1-3p.

Glycosyl residues H1
 H2
 H3
 H4
 H5
 H6a
 H6b OMe
C1 C2 C3 C4 C5 C6
A → 4)-α-GalpA-(1→ 4.92 3.61 3.87 4.27 4.72
100.64 69.11 69.79 79.46 72.12 175.86
B → 4)-α-d-GalpA-6-OMe-(1→ 4.78 3.58 3.87 4.44 5.02 3.66
101.31 69.26 69.29 79.8 71.65 172.15 54.17
C → 2,4)-α-l-Rhap-(1→ 5.17 3.98 3.79 3.43 3.87 1.12
100.64 77.24 71.81 80.13 69.81 17.82
D → 6)-β-d-Galp-(1→ 4.36 3.4 3.63 3.97 3.79 3.61 3.90
104.36 72.19 73.29 69.55 74.82 70.22
E → 3,6)-β-d-Galp-(1→ 4.4 3.48 3.61 4.01 3.79 3.61 3.90
104.38 71.2 81.51 69.55 74.82 70.22
F β-d-Galp-(1→ 4.5 3.52 3.63 3.97 3.63 3.61 3.66
105.89 71.09 73.65 69.55 76.63 63.26
G α-l-Araf-(1→ 5.09 4.06 3.81 4.15 3.57/3.69
110.61 83.04 77.61 80.41 62.32
H → 3,5)-α-l-Araf-(1→ 5.01 3.99 3.89 4.17 3.66/3.75
108.53 82.41 85.28 83.47 67.59
I → 5)-α-l-Araf-(1→ 4.96 3.99 3.88 4.17 3.66/3.75
108.68 82.37 77.77 83.47 67.59
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Detailed structural information could be obtained from the 2D NMR spectrum, a powerful technique for characterizing the structure of polysaccharides. Nine signals of the anomeric (carbon) atoms at 4.92 (100.64), 4.78 (101.31), 5.17 (100.64), 4.36 (104.36), 4.4 (104.38), 4.5 (105.89), 5.09 (110.61), 5.01 (108.53), and 4.96 (108.68) ppm from 1H- and 13C NMR spectra were found to be further resolved by 2D NMR spectroscopy. On the basis of the interpretation of the 1D, 2D NMR spectra and the data in the earlier works (Chen et al., 2021, Yao et al., 2021, Yuan et al., 2020), nine types of glycosyl residues linkages in TFPS-1-3p have been assigned including → 4)-α-GalpA-(1→, →4)-α-d-GalpA-6-OMe-(1→, →2,4)-α-l-Rhap-(1→, →6)-β-d-Galp-(1→, →3,6)-β-d-Galp-(1→, terminal β-d-Galp-(1→, terminal α-l-Araf-(1→, →3,5)-α-l-Araf-(1 → and → 5)-α-l-Araf-(1 → and their signals were presented in Table 2.

The example of residue A, designated → 4)-α-GalpA-(1→, was considered. In the HSQC spectrum (Fig. 1D), the signals of the anomeric protons and carbon atoms were observed at 4.92/100.64 ppm, indicating that residue A is in the α-configuration (Chen et al., 2021, Yuan et al., 2020). In the 1H–1H COSY spectrum (Fig. 1E), the cross peaks A H1/H2 (4.92/3.61), A H2/H3 (3.61/3.87) and A H3/H4 (3.87/4.27) were observed. Subsequently, the carbon signals including δ100.64, 69.11, 69.79, 79.46, and 72.12 ppm, which were assigned to C-1, C-2, C-3, C-4 and C-5 of residue A, could be further mapped in the HSQC spectrum (Fig. 1D). The cross peak δ 4.72/175.86 ppm of H-5 and C-6 was found in the HMBC spectrum (Fig. 1F). δC 175.86 ppm is the characteristic peak of the C-6 position in the non-esterified GalA residue. In this case, the chemical shifts of C-1 and C-4 moved to the lower panel, indicating that the residue was occupied at the C-1 and C-4 positions of the sugar nucleus according to certain published literature reports (Chen et al., 2021, Chen et al., 2022, Patova et al., 2019, Petersen et al., 2008, Yao et al., 2021, Yuan et al., 2020). Taken together, the sugar residue A has been recognized as → 4)-α-d-GalpA-(1 →.

The δC 177 ppm was the characteristic peak of the C-6 position in the non-esterified GalA residue, and the characteristic peak of the C-6 position in the esterified GalA residue was located near δC 172.15 ppm (Petersen, et al., 2008). In the HSQC spectrum (Fig. 1D), the signal peak at δ 3.66/54.16 ppm was found. The signal at δH 3.66 ppm was the methyl proton of the methyl ester (-COOMe), and the characteristic peak at δC 54.16 ppm represented the methyl carbon attached to the C-6 position in the methyl-esterified GalA residue (Petersen, et al., 2008). These characteristic signals indicated the presence of -COOMe in GalA residue, thus the sugar residue B has been recognized as → 4)-α-d-GalpA-6-OMe-(1 →.

In the HMBC spectrum (Fig. 1F), a variety of residue connections can be inferred. The OMe/B C-6, A H-1/C/C−2, C H-1/A/C-4, A H-5/A/C-6, G H-1/H C-3, H H-1/I C-5, I H-1/E C-3, E H-1/D C-6, F H-1/E C-3, and F H-1/H C-5 were recognized as the linkages of −6-OMe-(1 → 4)-α-d-GalpA-6-OMe-(1→, →4)-α-GalpA-(1 → 2,4)-α-l-Rhap-(1→ (A-C), →2,4)-α-l-Rhap-(1 → 4)-α-GalpA-(1→ (C-A), →4)-α-GalpA-(1 → 4)-α-GalpA-(1→ (A-A), α-l-Araf-(1 → 3,5)-α-l-Araf-(1→ (G-H), →3,5)-α-l-Araf-(1 → 5)-α-l-Araf-(1→ (H-I), →5)-α-l-Araf-(1 → 3,6)-β-d-Galp-(1→ (I-E), →3,6)-β-d-Galp-(1 → 6)-β-d-Galp-(1→ (E-D), β-d-Galp-(1 → 3,6)-β-d-Galp-(1→ (F-E), β-d-Galp-(1 → 3,5)-α-l-Araf-(1→ (F-H), respectively. And in the NOESY spectrum (Fig. 1G), certain inter-residual cross-peaks were also identifed: the A H-1/A H-4, C H-1/A H-4, and D H-1/C H-4, suggesting that the linkages of → 4)-α-GalpA-(1 → 4)-α-GalpA-(1→ (A-A), →2,4)-α-l-Rhap-(1 → 4)-α-GalpA-(1→ (C-A), →6)-β-d-Galp-(1 → 2,4)-α-l-Rhap-(1→ (D-C) existed.

Consequently, the hypothetical structure of TFPS-1-3p was proposed as shown in Fig. 1H. TFPS-1-3p is a polysaccharide with the main linkage of [→4)-α-d-GalpA(-6-OMe)-(1 → 4)-α-GalpA-(1 → ] and [→4)-α-d-GalpA(-6-OMe)-(1 → 2,4)-α-l-Rhap-(1 → ] and the branched chains including the linkage of → 6)-β-d-Galp-(1 → and → 3,6)-β-d-Galp-(1 → linkages) and (→5)-α-l-Araf-(1 → and → 3,5)-α-l-Araf-(1 →. There is a small amount of methyl esterification and O-2 or O-3 acetylated substitutions in the backbone of TFPS-1-3p. Similarly, given that most TFPS are mainly composed of Rha, Ara, GalA and Gal with small amounts of Glc, GalA, Xyl, depending on the extraction and separation method (Chen, Chen, Sun, Zeng, & Ye, 2020). The chemical structure of two purified TFPS fractions (namely TFPS-1 and TFPS-2) has been reported previously (Han, et al., 2011). Hereinto, TFPS-1 (167.5 kDa) contained α-l-Rhap, α-d-Galp, α-d-GalpA, α-d-Xylp, α-d-Glcp and β-d-Glcp residues. TFPS-2 (10.1 kDa) was built from α-l-Rhap, α-l-Arap, αD-Xylp, α-d-Glcp, and α-d-GlcpA residues. It has been reported that structural characteristics could give rise to differences in the degradation process and bioactivity of polysaccharides. In particular, complex polysaccharides are theoretically difficult to be degraded thus could reach to the distal gut as a “prebiotic”. Thus, the degradation properties and the effect of TFPS-1-3p on the gut microenvironment deserve to be addressed.

3.4. In vitro digestive properties of TFPS-1-3p

As shown in Table 3, there was no significant difference (p > 0.05) on the content of residual total carbohydrates and reducing sugars during the simulated saliva and gastrointestinal fluid digestion, indicating that TFPS-1-3p cannot be degraded by human saliva and gastrointestinal fluid. α-Amylase is the predominant active enzyme in saliva, capable of breaking the α-(1 → 4) linkages between glucose residues in starch and glycogen (Roberts & Whelan, 1960). Digestive enzymes in the human gastrointestinal digestive tract, including pepsin, lipase, and pancreatic enzymes, have a limited ability to degrade polysaccharides with complex linkage bonds (Brodkorb, et al., 2019). It was similar to the majority of plant-derived polysaccharides, such as from Fuzhuan brick tea (Chen, et al., 2017) and Chinese wolfberry (Zhou, et al., 2018), which structurally did not contain glycan α-(1 → 4) linkage and thus cannot be degraded by human saliva and gastrointestinal fluid. Subsequently, the TFPS-1-3p that has escaped depolymerization by enzymes of the upper human digestive system could safely reach the colon. The levels of residual total carbohydrates and reducing sugars were significantly lower during the fermentation phase (p < 0.05). These results suggested that TFPS-1-3p could be exploited by gut microbes. It has been widely recognized that gut microbes have the ability to encode a large number of CAZymes specifically designed to break down undigested polysaccharides as nutrients for their own growth has been widely recognized (Ndeh & Gilbert, 2018). A plausible hypothesis is that TFPS-1-3p was a dietary nutrient of the gut microbiota and was involved in regulating the gut microenvironment during the colonic fermentation.

Table 3.

The content of residual total carbohydrate and reducing sugars during the in vitro gastrointestinal digestion and colonic fermentation process.

Process Duration (h) Total residual carbohydrate (%) The residual reducing sugars (%)
Oral phase 0 100.00 ± 0.35a* 100.00 ± 3.01a
0.5 100.37 ± 0.53a 98.74 ± 3.86a
1 99.42 ± 0.46a 96.80 ± 3.98a
Gastric phase 0 100.00 ± 0.21a 100.00 ± 2.14a
2 99.75 ± 0.03a 98.15 ± 6.12a
6 99.87 ± 0.54a 98.60 ± 7.73a
Intestinal phase 0 100.00 ± 0.29a 100.00 ± 3.31a
2 100.13 ± 0.32a 100.00 ± 4.73a
6 99.92 ± 0.23a 99.89 ± 2.04a
Colonic phase 0 100.00 ± 0.39a 100.00 ± 1.70a
6 85.43 ± 0.34b 86.76 ± 3.13b
12 70.67 ± 0.26c 78.59 ± 3.40c
24 42.44 ± 0.11d 52.35 ± 2.51d
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*Different letters indicate significant differences between data within the same column (p < 0.05).

3.5. Effects of TFPS-1-3p on gut microenvironment

Emerging studies have found that the benefits of consuming a high-fiber diet are that gut microbes can metabolize indigestible polysaccharides to create SCFAs., thereby lowering the intracellular pH, and thus improving the gut microenvironment and body health (Lee, et al., 2022). SCFAs including lactate, acetate, propionate, n/i-butyrate and n/i-valerate, are the major metabolites of polysaccharides produced by gut microbiota. Currently, the levels of lactate, acetate, propionate, n/i-butyrate and n/i-valerate were measured during the colonic fermentation phase to evaluate the effects of TFPS-1-3p on the gut microenvironment and to provide further evidence for the degradation of TFPS-1-3p by gut microbiota. As the results were shown in Table 4, the levels of SCFAs enhanced with increasing fermentation time, among which acetate, propionate, and n-butyrate were the predominant metabolites. The concentrations of acetate and n-butyrate were significantly higher in the TFPS-1-3p group than in the BLK and FOS groups at different fermentation times (p < 0.05). Propionate and n-valerate appeared in all groups after 12 h of fermentation, and i-valerate and i-butyrate appeared after 24 h of fermentation, but propionate, i-butyrate, i-valerate and n-valerate were not detected in the Origin group and each treatment group after 6 h of fermentation. The content of total acids in the TFPS-1-3p group was higher than in the FOS and BLK groups at all stages of the fermentation process. In particular, after 24 h of fermentation, the total acids content of the TFPS-1-3p group (63.10 ± 0.46 mmol/L) was almost twice as high as that of the FOS group (33.30 ± 1.30 mmol/L) and three times as high as that of the BLK group (21.12 ± 0.83 mmol/L). SCFAs have been extensively implicated in the prevention of gut diseases and maintenance of gut health by modulating the immune system, lowering luminal pH to inhibit pathogenic intestinal bacteria, and boosting the mineral bioavailability (such as calcium and magnesium) (van der Hee & Wells, 2021). Additionally, Table 4 showed that the pH values of the FOS and BLK groups decreased significantly from 0 to 6 h, and then did not decrease further by increasing the fermentation time. Meanwhile, pH value gradually decreased within 24 h, which was in agreement with the downward trend of residual sugar content in the TFPS-1-3p group. Consequently, TFPS-1-3p can be released slowly to regulate the gut microenvironment without sudden changes in a short period of time.

Table 4.

The concentrations of SCFAs and the pH value during the colonic fermentation.

Duration (h) Treatment SCFAs (mmol/L)
 pH value
Lactate Acetate Propionate i-Butyrate n-Butyrate i-Valerate n-Valerate Total acids
0 Origin 1.00 ± 0.01f* 7.41 ± 0.02a -# – 1.77 ± 0.03a – – 10.18 ± 0.05a 7.00 ± 0.01f
6 FOS 2.53 ± 0.01i 11.82 ± 0.13e – – 2.29 ± 0.07b – – 16.65 ± 0.98 cd 4.62 ± 0.02a
BLK 0.75 ± 0.06e 8.24 ± 0.36b – – 2.60 ± 0.07c – – 11.59 ± 0.85b 6.61 ± 0.02e
TFPS-1-3p 0.82 ± 0.07e 13.60 ± 0.89f – – 4.85 ± 0.09e – – 19.26 ± 0.74de 6.04 ± 0.01d
12 FOS 1.99 ± 0.02 h 12.24 ± 0.82de 1.34 ± 0.03a – 2.43 ± 0.10bc – 1.27 ± 0.07b 18.28 ± 0.62d 4.65 ± 0.02a
BLK 0.26 ± 0.02c 9.97 ± 0.29c 1.00 ± 0.08a – 2.80 ± 0.11 cd 0.42 ± 0.02b 0.85 ± 0.02a 15.28 ± 0.93c 6.62 ± 0.02e
TFPS-1-3p 0.33 ± 0.00d 20.50 ± 1.00 g 1.19 ± 0.12a – 8.50 ± 0.11f – 1.47 ± 0.09c 31.98 ± 0.92 g 5.60 ± 0.01c
24 FOS 1.81 ± 0.02 g 13.94 ± 0.69f 12.17 ± 0.57d 0.03 ± 0.00a 2.36 ± 0.19b 0.32 ± 0.01a 2.67 ± 0.04d 33.29 ± 1.30 g 4.68 ± 0.02a
BLK 0.03 ± 0.00a 10.22 ± 0.94 cd 4.46 ± 0.31b 0.74 ± 0.04c 2.89 ± 0.09d 1.54 ± 0.04d 1.23 ± 0.00b 21.12 ± 0.83f 7.67 ± 0.08 g
TFPS-1-3p 0.12 ± 0.02b 37.70 ± 0.28 h 6.69 ± 0.38c 0.54 ± 0.04b 14.21 ± 0.20 g 0.82 ± 0.01c 3.02 ± 0.04e 63.10 ± 0.46 h 5.40 ± 0.01b
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#Not detected.

*Different letters indicate significant differences between data within the same column (p < 0.05).

The gut microbiota is a large colony of microorganisms’ resident in the gut ecosystem, whose role in human health is to maintain a dynamic balance with the host and to play local and distant roles in key physiological processes. Polysaccharides are the most common nutritional components of the gut microbiome and are intimately involved in host wellness (Brodkorb, et al., 2019). During colonic fermentation, polysaccharides drive the growth of specified beneficial gut bacteria, modify the gut microbial profile, and alter local and remote host physiology to reduce disease incidence (Ho Do, Seo, & Park, 2021). In this paper, the effect of TFPS-1-3p on gut microbes was monitored by 16S rDNA high-throughput sequencing analysis. A totally 1,240,460 valid reads were acquired from 20 samples (62,023 ± 2,628 reads per sample). α-diversity refers to the average species diversity within a sample in the environment. The Sobs and Shannon indices are commonly used to reflect species α-diversity, and their values are proportional to community richness and diversity, respectively. As shown in Fig. 2A, the Sobs and Shannon indices of both the FOS and TFPS-1-3p groups were significantly lower than those of the BLK and Origin groups (p < 0.05), indicating that microbial fermentations of FOS and TFPS-1-3p were unable to increase community richness and diversity. This was in accordance with that described by (Zhou, et al., 2018), which was due to the selective induction of proliferation of specific microorganisms by FOS and TFPS-1-3p.

Fig. 2.

Fig. 2

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The composition and diversity of gut microbiota. (A) α-diversity (Sobs and Shannon) indices, (B) LEfSe analysis, (C) Relative abundance of differentiated taxa at the species level. Different letters marked on the box (A) or column (C) within the same color indicate significant differences at p < 0.05 by Tukey test.

To obtain a clearer picture of the specific species variation, the LEfSe analysis, an indicator of β-diversity, was used to reveal taxonomic differences between treatments. As shown in Fig. 2B, the dominant taxa in the Origin group were Lentisphaerae and Candidatus_Saccharibacteria, Actinomycetaceae, Micrococcaceae, Bacillales_Incertae_Sedis_XI, Prevotellaceae, Enterococcaceae, Leuconostocaceae, Streptococcaceae, Clostridiaceae_1, Clostridiales_Incertae_Sedis_XI, Clostridiales_Incertae_Sedis_XIII, Eubacteriaceae, Peptostreptococcaceae, Ruminococcaceae, Victivallaceae, Methylobacteriaceae, Oxalobacteraceae, Pasteurellaceae and Moraxellaceae. The dominant species in the BLK group were Bacteroidetes, Proteobacteria, Fusobacteria, Synergistetes, Bacteroidaceae, Porphyromonadaceae, Acidaminococcaceae, Fusobacteriaceae, Desulfovibrionaceae, Succinivibrionaceae, Enterobacteriaceae and Synergistaceae. The dominant species in the FOS group were Bifidobacteriaceae, Rikenellaceae, Erysipelotrichaceae, Veillonellaceae and Sutterellaceae. While the dominant species in the TFPS-1-3p group were Coriobacteriaceae and Lachnospiraceae. Furthermore, the variation of microbes at the species level were shown in Fig. 2C. A total of 14 species of gut microorganisms differed significantly among the different groups. Faecalibacterium prausnitzii is one of the most predominant anaerobic strains in the human gut and has been shown to be a powerful probiotic for fight against inflammatory bacteria (Parsaei, Sarafraz, Moaddab, & Ebrahimzadeh Leylabadlo, 2021). A previous study revealed that F. prausnitzii could be enhanced by fermenting high-methoxyl pectins rather than low-methoxyl pectins (Larsen, et al., 2019). F. prausnitzii is capable of the acquisition and degradation of various β-mannooligosaccharides (Lindstad Lars, et al., 2021). However, in vitro culture and passage through the rough conditions of the gastrointestinal tract posed many difficulties due to its extremely sensitive characteristic (Phùng, et al., 2022). In this study, F. prausnitzii was significantly decreased (p < 0.05) after 24 h of in vitro fermentation in comparison to the Origin group. Additionally, TFPS-1-3p fermentation significantly reduced (p < 0.05) the relative abundance of F. prausnitzii when compared to the other groups. The limitation of F. prausnitzii after TFPS-1-3p fermentation might be associate with its structural characteristics, which is not abundant in β-mannooligosaccharides. Conversely, the relative abundance of Fusobacterium mortiferum, Megasphaera elsdenii DSM 20460, Collinsella aerofaciens, Bacteroides thetaiotaomicron and Bacteroides plebeius increased significantly (p < 0.05) after 24 h of TFPS-1-3p fermentation compared to the Origin and FOS groups, which may be related to the degradation of TFPS-1-3p. Bacteroides spp. is the mainstay in the human gut microbiota for metabolizing complex polysaccharides in the diet, dedicating up to 20 % of its genome to complex polysaccharide metabolism in the form of genes encoding primarily CAZymes such as polysaccharide lyases, glycoside hydrolases, polysaccharide transport and binding proteins, and carbohydrate sulfate lyases (Hindson, 2019). A versatile core genetic locus was found toorchestrate the metabolism of multiple glycans by B. thetaiotaomicron (Ndeh, et al., 2020). Similarly, B. plebeius have gained polysaccharide utilization loci enabling the hybrid galactan metabolism (Robb, et al., 2022). F. mortiferum has long been shown to encode 6-phospho-alpha-glucosidase (EC 3.2.1.122) that belongs to Family 4 of the glycosylhydrolase (Bouma, Reizer, Reizer, Robrish, & Thompson, 1997). Subsequently, the degradation products or fragments of TFPS-1-3p were converted into SCFAs. M. elsdenii DSM 20460 can convert metabolites of other microorganisms (for example, Trichoderma reesei could secrete cellulase to degrade cellulose into soluble sugars) into various SCFAs (Shahab, et al., 2020). The fact that C. aerofaciens was one of the butyrate-producing bacteria was proved (Qin, et al., 2019). Therefore, F. mortiferum, M. elsdenii DSM 20460, B. thetaiotaomicron, B. plebeius and C. aerofaciens probably contributed to the degradation of the undigested heteropolysaccharide TFPS-1-3p and thus the gut microenvironment was modulated. However, the degradation pathways and polysaccharide utilization loci of TFPS-1-3p need to be further explored.

4. Conclusion

TFPS-1-3p that was isolated from TFPS was a typical heteropolysaccharide with a molecular weight of 47.77 kDa. The linkages of TFPS-1-3p mainly contained → 4)-α-d-GalpA(-6-OMe)-(1 → 4)-α-GalpA-(1 → and → 4)-α-d-GalpA(-6-OMe)-(1 → 2,4)-α-l-Rhap-(1 → with a little methyl esterification and (O-2 or O-3) acetylated substitutions. The branched chains consisted of linkages of → 6)-β-d-Galp-(1 → and → 3,6)-β-d-Galp-(1 → linkages) and (→5)-α-l-Araf-(1 → and → 3,5)-α-l-Araf-(1 →. TFPS-1-3p was resistant to degradation under simulated human gastrointestinal conditions but was degradable after in vitro colon fermentation. Thus, the gut microenvironment was changed with decreased pH and enhanced production of SCFAs, and the composition of the gut microbiota was altered. F. mortiferum, M. elsdenii DSM 20460, B. thetaiotaomicron, B. plebeius and C. aerofaciens potentially contributed to the degradation of TFPS-1-3p.

CRediT authorship contribution statement

Dan Chen: Project administration, Writing – original draft, Writing – review & editing, Funding acquisition. Ao Wang: Data curation, Investigation. Jialiang Lv: Validation, Visualization. Chao Tang: Supervision. Chang-hai Jin: Methodology. Jun Liu: Data curation. Xiaoxiong Zeng: Conceptualization, Resources. Li Wang: Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by the National Natural Science Youth Foundation of China (32102027), Anhui Natural Science Youth Foundation (2108085QC144), Natural Science Research Project of Colleges and Universities in Anhui Province (KJ2021A0143), Natural Science Youth Foundation of Anhui Agricultural University (2020zd17), Introduction and Stabilization Project of Talent Project of Anhui Agricultural University (rc352006), Shennong New Talent Project of Anhui Agricultural University (rc352102), and Scientific research start-up fee of Yangzhou University (137012022).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2023.101058.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (948.5KB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.docx (948.5KB, docx)

Data Availability Statement

Data will be made available on request.

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