Remodeling of Bamboo (Phyllostachys edulis) Shoot Polysaccharides by Monascus purpureus Fermentation Enhances Antioxidant Protection in Caco-2 Cells

Abstract

Bamboo shoot polysaccharides possess promising bioactivities, but their high molecular weight and complex branched structures limit their functional efficacy. In this study, bamboo (Phyllostachys edulis) shoots were fermented with Monascus purpureus ATCC16426 to obtain a novel neutral polysaccharide (FBSP-1). Monosaccharide composition analysis indicated that FBSP-1 comprised glucose (Glc), xylose (Xyl), arabinose (Ara), galactose (Gal), and mannose (Man). Glycosidic linkage and nuclear magnetic resonance (NMR) spectroscopy data revealed that the backbone of FBSP-1 mainly consisted of →2)-α-Manp-(1→, →4)-α-Glcp-(1→, →3)-β-Xylp-(1→, →3)-α-Araf-(1→ and →2)-α-Araf-(1→. Compared with unfermented polysaccharide BSP-1, fermentation markedly altered the monosaccharide profile, increasing Glc, Xyl and Man, decreasing Ara and Gal, and reducing the molecular weight. In H₂O₂-treated Caco-2 cells, FBSP-1 significantly alleviated oxidative damage by reducing intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, while enhancing the activities of superoxide dismutase (SOD) and glutathione reductase (GR), demonstrating pronounced antioxidant protective effects. Overall, this study demonstrates that Monascus fermentation is an effective strategy for the structural remodeling of bamboo shoot polysaccharides, enabling the enhancement of their antioxidant functionality and supporting their potential application as functional ingredients in plant-based antioxidant foods.

1. Introduction

Bamboo shoots (Phyllostachys edulis) are widely consumed plant-based foods rich in polysaccharides, dietary fiber, and essential micronutrients [1,2,3]. Among these components, bamboo shoot polysaccharides are regarded as the principal contributors to their antioxidant [4], hypoglycemic [5], immunomodulatory [6], and other bioactivities [7]. However, native bamboo shoot polysaccharides typically possess high molecular weights and complex branched structures, resulting in limited solubility, diffusivity, and bioaccessibility. These properties, in turn, impair their dispersion in food systems, interactions with digestive environments, and overall biological efficacy, ultimately restricting their functional performance in food and nutritional applications [1]. Therefore, developing mild and food-compatible strategies to tailor the molecular structure of bamboo shoot polysaccharides is crucial for enhancing their bioactivity and promoting their high-value utilization.

Polysaccharide bioactivity is strongly influenced by molecular weight, monosaccharide composition, and glycosidic linkage patterns [8,9]. Although chemical modification methods, such as carboxymethylation or sulfonation, can alter structural features, they often involve harsh conditions or organic reagents, restricting their suitability for food-related applications [10]. In contrast, microbial fermentation offers a green, and controllable strategy for polysaccharide remodeling, enabling enzyme-mediated depolymerization and structural reorganization under mild conditions to enhance biological functionality [11,12,13]. Monascus purpureus, a food-grade fungus with both nutritional and medicinal relevance, secretes various polysaccharide-degrading enzymes during fermentation [14,15]. Previous studies have demonstrated that Monascus fermentation markedly enhances the bioactivities of plant-derived polysaccharides. For example, Liu et al. [16] discovered that Monascus-fermented Poria cocos polysaccharides exhibit significant immunomodulatory activity. Wang et al. [17] reported that a Monascus-derived polysaccharide (MP-1) significantly alleviated inflammatory responses by suppressing the expression of pro-inflammatory cytokines. Based on these findings, applying Monascus purpureus fermentation to bamboo shoots may represent an effective strategy to modulate the structure of bamboo shoot polysaccharides and enhance their bioactivity.

Caco-2 cells, which closely recapitulate the human intestinal epithelium, are widely employed to assess the intestinal antioxidant and cytoprotective effects of dietary bioactives [18,19,20]. As excessive ROS drive intestinal oxidative injury, evaluating Monascus fermentation–remodeled bamboo shoot polysaccharides for their ability to modulate ROS and reinforce antioxidant defenses in Caco-2 cells is of significant nutritional relevance.

Since Monascus-fermented bamboo shoot polysaccharides remain unexplored, this study hypothesizes that Monascus purpureus fermentation may induce structural reorganization of these polysaccharides, enhancing their antioxidant and cytoprotective activities. Bamboo shoots were fermented with Monascus purpureus ATCC 16426 to isolate a novel neutral polysaccharide (FBSP-1), which was compared with the unfermented BSP-1 in terms of structure and biological activity. Monosaccharide composition analysis, molecular weight determination, methylation profiling, and NMR spectroscopy were used to elucidate fermentation-induced structural modifications. Furthermore, the H₂O₂-induced Caco-2 oxidative stress model was employed to assess antioxidant and cytoprotective effects, confirming that Monascus fermentation enhances the antioxidant activity of bamboo shoot polysaccharides. This work presents an effective fermentation-based approach to modifying bamboo shoot polysaccharides and highlights their potential as functional ingredients in plant-based antioxidant foods.

2. Materials and Methods

2.1. Materials and Reagents

The fresh bamboo shoots (Phyllostachys edulis) were purchased from Hunan Jinzhusun Agricultural Development Co., Ltd. (Changsha, China). Monascus purpureus ATCC16426 was stored at −80 °C in our laboratory. NaBD₄, CH₃I, Trifluoroacetic Acid (TFA), 1-Phenyl-3-methyl-5-pyrazolone (PMP), monosaccharide standards, DEAE-52 cellulose, Sephadex G-100, and dialysis membranes (MD44, 3500 Da) were purchased from Aladdin (Shanghai, China). Caco-2 cells were obtained from Xiangya Hospital of Central South University, China.

2.2. Bamboo Shoot Fermentation by Monascus purpureus

The fermentation process of bamboo shoots was performed according to the method reported by Liu et al. [16]. Fresh bamboo (Phyllostachys edulis) shoots were freeze-dried, ground, and sieved through a 100-mesh sieve to obtain bamboo shoot powder. Monascus purpureus ATCC 16426 was initially cultured on potato dextrose agar (PDA) medium at 30 °C for 5 days, and a spore suspension was prepared for seed culture. Bamboo shoot powder was mixed with 1% yeast extract, dispersed in distilled water (1:6, g/mL), and autoclaved at 121 °C for 20 min. The mixture was then fermented under optimized conditions: 7 days at 30 °C, initial pH 6.0, and inoculum size 6%, which were selected based on preliminary optimization experiments aimed at maximizing bamboo shoot polysaccharide yield. The detailed optimization data are presented in Figure S1.

2.3. Extraction of Bamboo Shoot Polysaccharides

Following a previously reported method with slight modifications [7], fermented bamboo shoot samples were extracted with deionized water (1:20, g/mL) at 70 °C for 3 h. After centrifugation (4500 rpm, 10 min), the residues underwent repeated extraction under the same conditions for five successive cycles. The pooled supernatants were vacuum-concentrated and precipitated with four volumes of absolute ethanol at 5 °C overnight. The precipitate was recovered by centrifugation, redissolved in deionized water, and deproteinized by the Sevag method three times. Protein removal was confirmed by measuring the absorbance at 280 nm. The solution was subsequently decolorized with resin, and subjected to dialysis (MWCO 3500 Da) against deionized water for 48 h. The retentate was freeze-dried to obtain crude fermented bamboo shoot polysaccharides (FBSP). Non-fermented bamboo shoot polysaccharides (BSP) were obtained by the same procedure. The yield of FBSP was calculated as the mass of obtained FBSP relative to the mass of bamboo shoots, expressed as a percentage, while the yield of BSP was calculated as the mass of obtained BSP relative to the mass of bamboo shoots, also expressed as a percentage.

2.4. Isolation and Purification of FBSP-1 and BSP-1

According to the method reported by Li et al. [16], FBSP (100 mg) was dissolved in distilled water and separated on a DEAE-52 column with NaCl gradients (0, 0.05, 0.1, and 0.3 M) at 1.0 mL/min. The 0 M NaCl fraction was collected and purified on a Sephadex G-100 column with ultrapure water. The main fractions were concentrated and freeze-dried to afford FBSP-1. Following the same procedure, the non-fermented bamboo shoot polysaccharide (BSP-1) was purified. The yield of FBSP-1 was calculated as the percentage of obtained FBSP-1 relative to FBSP, while the yield of BSP-1 was calculated as the percentage of obtained BSP-1 relative to BSP.

2.5. Monosaccharide Composition Analysis

The monosaccharide composition was determined using a PMP-derivatized high- performance liquid chromatography (HPLC) method [8]. Each polysaccharide sample (5.0 mg) was prepared at 10.0 mg/mL, and the solution (400 μL) was hydrolyzed with 4.0 M TFA (800 μL) at 110 °C for 2 h. After nitrogen drying to remove residual acid, the hydrolysate was redissolved in 800 μL of ethanol. A 400 μL aliquot was derivatized by treatment with 0.3 M NaOH (400 μL) and 0.5 M PMP in methanol (400 μL) at 70 °C for 50 min. The mixture was adjusted to neutrality with 0.3 M HCl (400 μL), diluted to 1.0 mL, and extracted with chloroform (1.0 mL × 3) to remove excess PMP. The aqueous phase was passed through a 0.22 μm membrane before HPLC analysis. Monosaccharide standards (Rha, Ara, Xyl, Man, Glc, and Gal) were derivatized similarly.

2.6. Determination of Molecular Weight

The molecular weights of the BSP-1 and FBSP-1 were determined by high-performance gel permeation chromatography (HPGPC) [7]. Polysaccharide solutions (5 mg/mL) were filtered using 0.22 µm filters before analysis. Separation was achieved using 0.05 M NaCl at 0.65 mL/min, with the column maintained at 40 °C and a 30 μL injection volume. Dextran standards (1–670 kDa) were analyzed under the same conditions to establish a calibration curve (Figure S2). Sample molecular weights were inferred from retention times relative to standards.

2.7. Spectral Analysis

The conformational structure of bamboo shoot polysaccharides was analyzed using Congo red as previously reported [16]. FBSP-1 (0.5 mg/mL) was mixed with the same volume of 50 µM Congo red in each tube, followed by NaOH solutions addition to final concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 M. The mixtures were incubated at 25 °C for 30 min, and the maximum absorption wavelength (λmax, 400–600 nm) was recorded.

BSP-1 and FBSP-1 were characterized by ultraviolet (UV) (UV-2600, Shimadzu, Kyoto, Japan) and Fourier transform infrared (FT-IR) spectroscopy (IRAffinity-1, Shimadzu, Kyoto, Japan). UV spectra were obtained from 200 to 600 nm using a UV-1900PC spectrometer (Shimadzu, Kyoto, Japan). For FT-IR detection, 2 mg of each sample was blended with KBr (200 mg), finely ground, and compressed into pellets for scanning between 4000–400 cm⁻¹.

2.8. Scanning Electron Microscopy (SEM) Analysis

The samples (BSP-1 and FBSP-1) were sputter-coated with gold prior to observation. Their surface morphology was examined using a Regulus 8020 SEM (Hitachi, Tokyo, Japan) at 10 kV and 3000× magnification.

2.9. Methylation Analysis

Methylation analysis of FBSP-1 was performed using a modified Hakomori method [21]. FBSP-1 (10 mg) and NaOH (30 mg) were dissolved in anhydrous DMSO (2 mL) and stirred for 30 min. CH₃I (1 mL) was added dropwise under nitrogen, and the reaction proceeded in the dark for 2 h. The methylated mixture was extracted with CH₂Cl₂, dried, hydrolyzed with 0.5 M TFA at 70 °C for 4 h, reduced with NaBD₄, acetylated, and the alditol acetates were extracted with CH₂Cl₂ for GC-MS analysis.

2.10. NMR Analysis

Following the method reported by Dai et al. [4], FBSP-1 (30 mg) was dissolved in D₂O (0.5 mL). NMR spectra were acquired on a 400 MHz spectrometer (Bruker AM-400, Bruker, Billerica, MA, USA). 1D spectra (¹H and ¹³C NMR) and 2D spectra (HSQC, COSY, HMBC, and NOESY) were recorded. All spectra were processed and analyzed using MestReNova software (version 14.1.0, Mestrelab Research, Santiago de Compostela, Spain).

2.11. Cell Culture

Human colorectal cancer cell Caco-2 from Xiangya Medical College were authenticated by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination using a commercial detection kit (MycoAlert, Lonza, Walkersville, ME, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) at 37 °C in a 95% air and 5% CO₂ environment. Cell morphology was observed with an inverted microscope (DMI3000B, Leica Corporation, Wetzlar, Germany).

2.12. MTS Assay

Cells in the logarithmic growth phase were trypsinized, counted with an automated cell counter (TC20™, Bio-Rad, Hercules, CA, USA), and seeded into 96-well plates at a density of 2.5 × 10⁴ cells/well (100 μL). When cells grew to an appropriate degree, BSP-1 or FBSP-1 at different concentrations (0, 12.5, 25, 50, 100, 200, and 400 μg/mL) were added into the plate with or without H₂O₂ (100 μM). After 24 h, MTS solution was added to the wells, followed by incubation at 37 °C for 30 min. The optical density was measured at 490 nm using a Multiskan SPECTRUM reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.13. ROS Staining

ROS content in H₂O₂-induced Caco-2 cells was assessed after polysaccharide treatment using the procedure reported by Qin et al. [22].

2.14. Biochemical Assessments

Cells were adjusted to 2.5 × 10⁴ cells/well in a 96-well plate. When cells reached an appropriate confluence, different concentrations of FBSP-1 and H₂O₂ (100 μM) were added into the plate. After 24 h, the cells were digested and collected. The activities of SOD, GR and the content of MDA were measured using the different kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

2.15. Hoechst 33258 Staining

Cells were adjusted to 3 × 10⁵ cells/well in a 6-well plate. When the cells reached an appropriate confluence, different concentrations of FBSP-1 and H₂O₂ (100 μM) were added. Cells were then fixed with paraformaldehyde and stained with Hoechst 33258 (Beyotime Institute of Biotechnology, Beijing, China) for 15 min. Fluorescence images were acquired using a Nikon 80i fluorescence microscope (Nikon Corporation, Tokyo, Japan).

2.16. Flow Cytometry

Apoptosis was assessed in Caco-2 cells treated with FBSP-1 and H₂O₂ using the Tali™ Apoptosis Assay Kit–Annexin V Alexa Fluor^® 488 (Invitrogen™, Life Technologies, Milan, Italy) a BD Accuri C6 flow cytometer (BD, Biosciences, San Jose, CA, USA). Caco-2 cells were treated with polysaccharides and 100 μM H₂O₂ for 24 h, resuspended in 100 µL 1× Annexin binding buffer, and incubated with 5 µL Annexin V-FITC and 1 µL PI for 5 min in the dark before flow cytometry analysis.

2.17. Statistical Analysis

All experiments were performed in at least three independent replicates, and results are presented as mean ± SD. Normality and homogeneity of variance were assessed prior to statistical analysis. Data were analyzed by one-way ANOVA with Tukey’s post hoc test using SPSS 23 (SPSS Inc., Chicago, IL, USA). Confidence interval is 95%. Statistical significance was defined as p < 0.05 and highly significant as p < 0.01. Graphs were generated using GraphPad Prism 6.

2.18. In Vitro Free Radical Scavenging Assays

The in vitro antioxidant activities of BSP-1 and FBSP-1 were assessed using •DPPH, ABTS⁺, and hydroxyl radical (•OH) scavenging assays following established protocols with slight modifications [23]. Polysaccharide solutions at various concentrations were incubated with the respective radical systems, and the absorbance was measured at the corresponding characteristic wavelengths. Radical scavenging activity was calculated as the percentage decrease in absorbance relative to the control.

3. Results and Discussion

3.1. Isolation and Purification Analysis

Crude polysaccharides (FBSP) were obtained by Monascus fermentation with a yield of 16.85%. FBSP was subsequently purified using a DEAE-52 cellulose column, and the eluates were monitored using the phenol–sulfuric acid method [17]. The elution profile was generated based on the characteristic absorbance peaks at 490 nm (Figure 1A). The ultrapure water-eluted fraction showed the most intense peak and was therefore collected as the major component. This fraction was further purified by Sephadex G-100 gel filtration, and the major fraction identified from the elution profile was collected to obtain FBSP-1 with a yield of 24.58% (Figure 1B). Using the same purification procedure, the unfermented crude polysaccharide (BSP) was obtained with a yield of 5.21%, while the yield of the purified fraction BSP-1 was 21.52%. Notably, this purification strategy, together with prior decolorization and dialysis (MWCO 3500 Da), effectively removes low-molecular-weight compounds, including pigments and most fermentation-derived secondary metabolites, thereby ensuring that FBSP-1 mainly consists of high-molecular-weight polysaccharides. The sugar contents of FBSP-1 and BSP-1 were 94.72 ± 0.42% and 93.25 ± 0.53%, respectively (Table S1), indicating a high degree of polysaccharide purity. Such high purity minimizes interference from proteins, phenolics, and other small reducing molecules that may otherwise cause nonspecific radical-scavenging effects, thereby enabling a more reliable attribution of the observed antioxidant activity to the polysaccharide fractions themselves [24].

Figure 1.

Purification and characterization of FBSP-1 and BSP-1. (A) Purification on DEAE-52. (B) Purification on Sephadex G-100. (C) Monosaccharide composition analysis of HPLC. (D) Congo red analysis. Each measurement was repeated at least three times.

3.2. Monosaccharide Composition and Content Analysis

The monosaccharide composition of FBSP-1 underwent pronounced alterations following fermentation compared with BSP-1 (Figure 1C and Table 1). Before fermentation, BSP-1 consisted of Glc, Ara, Gal, Xyl and Man in a molar ratio of 50.29 ± 0.78, 21.88 ± 0.58, 20.19 ± 0.51, 6.63 ± 0.19, and 1.01 ± 0.03, respectively, with Glc as the predominant constituent. After fermentation, FBSP-1 was composed of Glc, Xyl, Ara, Gal, and Man in molar ratios of 69.56 ± 0.95, 15.10 ± 0.42, 6.18 ± 0.19, 4.96 ± 0.09 and 4.20 ± 0.12, respectively. Notably, fermentation did not alter the types of monosaccharides present but led to a pronounced increase in Glc, Xyl, and Man contents [25], accompanied by a marked reduction in Ara and Gal levels. These results indicate that Monascus purpureus fermentation mediates the biotransformation of bamboo shoot polysaccharides, resulting in pronounced changes in monosaccharide composition and relative abundance. During fermentation, selective microbial utilization of specific saccharide moieties, together with polysaccharide chain reorganization, may account for the reduced proportions of Ara and Gal and the relative enrichment of Glc, Xyl, and Man. Similar fermentation-induced shifts have been reported for other plant-derived polysaccharides [16]. Alterations in monosaccharide composition may further influence polysaccharide bioactivity, as structural features are closely associated with antioxidant properties [9]. Consistent with previous reports, Monascus fermentation has been shown to substantially remodel the structure of plant-derived polysaccharides and enhance their bioactivity [17,26].

Table 1.

Composition of BSP-1 and FBSP-1.

Samples	Mw(kDa)	Monosaccharide Composition (mol%)
		Ara	Xyl	Man	Glc	Gal
BSP-1	22.30	21.88 ± 0.58	6.63 ± 0.19	1.01 ± 0.03	50.29 ± 0.78	20.19 ± 0.51
FBSP-1	8.84	6.18 ± 0.19	15.10 ± 0.42	4.20 ± 0.12	69.56 ± 0.95	4.96 ± 0.09

Note: Data are expressed as mean ± SD (n = 3).

3.3. Congo-Red Analysis

The changes in the maximum absorption wavelength (λ_max) of FBSP-1 and BSP-1 upon complexation with Congo red in NaOH solutions of varying concentrations are shown in Figure 1D. The λ_max exhibited a red shift with increasing NaOH concentration, reaching a maximum at 0.20 M NaOH. This red shift reflects the presence of a triple-helix conformation in the polysaccharide molecules [27]. Subsequently, a further increase in NaOH concentration caused λ_max to decrease. These results indicate that fermentation preserves the primary structure of bamboo shoot polysaccharides, which retain a stable triple-helix conformation. The triple-helix conformation helps maintain the structural stability and ordered chain arrangement of polysaccharide molecules, thereby increasing the spatial accessibility of active functional groups such as hydroxyl and carboxyl groups. This facilitates their interactions with ROS and consequently enhances biological functions. Such a structure–function relationship has been well documented in previous studies [28].

3.4. Molecular Weight Analysis

Polysaccharides with high molecular weight and poor water solubility generally exhibit low absorption efficiency in vivo. This limits their application in food and drug products. In contrast, polysaccharides with molecular weight below 10 kDa often display enhanced bioactivity. As shown in Figure 2A,B and Table 1, the molecular weight of the polysaccharides was determined using HPGPC. The results revealed that the unfermented polysaccharide BSP-1 had a molecular weight of 22.30 kDa, whereas that of the fermented product FBSP-1 was reduced to 8.84 kDa, demonstrating that fermentation markedly reduced the molecular weight of the native polysaccharides. The polydispersity indices (M_w/M_n) of BSP-1 and FBSP-1 were 1.16 and 1.09, respectively, indicating a relatively uniform molecular weight distribution. Notably, Monascus produces extracellular polysaccharides with molecular weights typically exceeding 100 kDa under liquid-fermentation conditions [29]. The present results indicate that FBSP-1 and Monascus extracellular polysaccharides were effectively separated by DEAE-52 ion-exchange chromatography and Sephadex G-100 gel filtration, confirming that FBSP-1 originates from bamboo shoot polysaccharides rather than fungal exopolysaccharides. Low molecular weight polysaccharides are often associated with enhanced biological activities, including antioxidant, anti-inflammatory, and lipid-lowering effects. This observation is consistent with a previous study on Monascus-fermented quinoa, which demonstrated that low-molecular-weight polysaccharides produced during fermentation can significantly alleviate hyperlipidemia [12]. These findings suggest that fermentation-induced structural modifications of polysaccharides can enhance their physiological functions. Similarly, during the fermentation of Ganoderma lucidum, a reduction in polysaccharide molecular weight was accompanied by improved hydroxyl and superoxide anion radical scavenging capacities [30]. This further demonstrates that reducing the molecular weight of the polysaccharide can effectively enhance their biological activity.

Figure 2.

Physicochemical characterization of FBSP-1 and BSP-1. (A,B) The molecular weight distribution by HPGPC; (C) UV spectrum; (D) FT-IR spectrum; (E,F) SEM images of BSP-1 and FBSP-1 with magnifications of 3000×; (G) GC-MS analysis of FBSP-1 and the glycosyl residues associated with peaks 1–12 are presented in Table 2.

3.5. UV and FT-IR Spectral Analysis

As shown in Figure 2C, both BSP-1 and FBSP-1 displayed a prominent peak at 200 nm, which was characteristic of polysaccharides. No signals were observed at 260, 280, or 510 nm, confirming the absence of nucleic acids, proteins, and flavonoids [31]. The FT-IR spectra (Figure 2D) revealed a broad absorption band at 3421 cm⁻¹, attributable to O-H stretching vibrations, along with peaks at 2927 cm⁻¹ and 1650 cm⁻¹ corresponding to C-H and C=O stretching and bending vibrations, respectively [32]. The absorption peaks at 1387 cm⁻¹ were related to C-H bending vibrations. The strong bands at 1269, 1150, and 1033 cm⁻¹ within the 1200–1000 cm⁻¹ range were assigned to C-O-C and C-O-H stretching vibrations, reflecting the presence of pyranose rings in polysaccharides [33]. Notably, the band at 1650 cm⁻¹ was indicative of C=O groups in glucans, suggesting the presence of β-glucan structures [34]. Additionally, weak absorptions at 917, 847, and 616 cm⁻¹ suggest the coexistence of α- and β-anomeric configurations in the pyranose units of FBSP-1 [35].

3.6. SEM Analysis

The surface morphologies of BSP-1 and FBSP-1 were examined using SEM. BSP-1 exhibited a honeycomb-like sheet structure at 3000× magnification (Figure 2E). Fermentation markedly altered the morphology of FBSP-1, producing a loosened, filamentous structure with bead-like features (Figure 2F). Compared with the unfermented BSP-1, this morphological remodeling may increase the specific surface area, improve dispersibility and solubility, and enhance the accessibility of active functional groups such as hydroxyl and carboxyl groups, thereby contributing to enhanced antioxidant effects [36].

3.7. Methylation and GC-MS Analysis

The structure of the purified polysaccharide fraction FBSP-1 from fermented bamboo shoots was elucidated by methylation analysis coupled with GC-MS. The primary structural features of the unfermented bamboo shoot polysaccharide BSP-1 (also referred to as BSDF-1) have been comprehensively reported in previous studies [37]. The total ion chromatogram of FBSP-1 was shown in Figure 2G. Based on ion signal interpretation in conjunction with relevant literature and the CCRC database, the types and molar ratios of methylated sugar derivatives were determined. These data allow the structure of the glycosidic bond in FBSP-1 to be inferred. Twelve distinct glycosidic linkages were identified, among which five were predominant (Table 2 and Figure S3). Glucose residues were mainly present as →4)-α-Glcp-(1→, α-Glcp-(1→, and →4,6)-α-Glcp-(1→, with →4)-α-Glcp-(1→ being the most abundant (38.86%). Xylose residues were mainly present as →4)-β-Xylp-(1→ and →3,4)-β-Xylp-(1→, representing 14.87% of the total linkages, while arabinose residues accounted for 6.47%. These results indicate that Glc, Xyl, and Ara are the predominant monosaccharides in FBSP-1, consistent with previous monosaccharide composition analyses. Overall, methylation analysis revealed that the FBSP-1 is primarily composed of Glc, Xyl and Ara residues interconnected through 1→3, and 1→4 glycosidic linkages to form the backbone structure. Li et al. [37] reported that the unfermented bamboo shoot polysaccharide BSDF-1 consisted of Glc, Gal, and Ara, with 12 distinct glycosidic linkages. Among these, the →4)-β-Glcp-(1→ linkage predominated, accounting for 48.70%, while Gal residues mainly occurred as →4)-β-Galp-(1→, with a proportion of 16.36%. Based on this previously reported structural information, comparison with FBSP-1 indicates that fermentation not only alters the monosaccharide composition of bamboo shoot polysaccharides but also markedly reshapes their glycosidic linkage patterns. It makes FBSP-1 with structural features distinct from those of BSP-1.

Table 2.

Methylation analysis of FBSP-1.

Peak No.	Rt (min)	PMAAs	Glycosidic Linkages	Molar Ratio (mol%)	Mass Fragments (m/z)
1	11.73	1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol	1-Araf	4.31	43, 59, 71, 87, 102, 118, 129, 145, 160
2	14.54	1,3,4-tri-O-acetyl-2,5-di-O-methyl arabinitol	1,3-Araf	1.04	43, 59, 71, 86.9, 98.9, 117.9, 128.9, 146.9, 159.9, 172.9, 201.9, 233
3	15.70	1,4,5-tri-O-acetyl-2,3-di-O-methyl xylitol	1,4-Xylp	7.50	43, 59, 71, 87, 102, 118, 128.9, 144.9, 162, 173, 189
4	16.86	1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol	1-Glcp	19.42	43, 59, 71, 87, 102, 118, 129, 145, 161, 174, 205
5	17.51	1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol	1-Galp	3.07	43, 60, 71, 86.9, 101.9, 117.9, 128.9, 144.9, 160.9, 173, 204.9
6	18.23	1,3,4,5-tetra-O-acetyl-O-methyl xylitol	1,3,4-Xylp	7.37	43, 59, 74, 84.9, 98.9, 118, 126.9, 141.9, 159.9, 172.9, 186.9, 201, 261
7	18.37	1,2,4,5-tetra-O-acetyl-3-O-methyl arabintiol	1,2,5-Araf	1.12	44, 60, 70.9, 86.9, 98.9, 117.9, 128.9, 144.8, 172.9, 189.9
8	19.62	1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol	1,2-Manp	2.34	43, 60, 71, 87.9, 100.9, 117.9, 128.9, 144.9, 160.9, 173.9, 189.9
9	20.20	1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol	1,4-Glcp	38.86	43.1, 59.1, 75, 87, 99, 118, 131, 143, 162, 173, 203, 233.1
10	20.59	1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol	1,6-Glcp	3.63	43, 60, 71, 86.9, 101.9, 118, 128.9, 142.9, 162, 173, 188.9, 217, 233
11	23.41	1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl glucitol	1,4,6-Glcp	10.29	43.1, 60, 72, 84.9, 102, 110.9, 118, 127, 141.9, 158.9, 187, 201, 231, 261
12	24.19	1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl mannitol	1,2,6-Manp	1.05	43, 60, 73.9, 86.9, 100.9, 117.9, 128.9, 138.9, 159.9, 173.9, 188.9, 202, 234, 245

3.8. NMR Analysis

The structure of FBSP-1 was extensively analyzed using NMR spectroscopy. The ¹H NMR signals were primarily distributed between 3.0 and 6.0 ppm (Figure 3A). Signals from sugar ring protons appeared mainly in the δ 3.0–4.2 ppm range, while anomeric proton peaks at δ 5.28, 5.26, 5.22, 5.20, 5.17, 5.10, 5.07, 5.00, 4.84, 4.52, 4.51, and 4.47 were located in the 4.4–6.0 ppm region. The ¹³C NMR spectrum (Figure 3B) showed resonances at δ 60–120 ppm; major anomeric carbon signals at δ 109.00, 108.19, 107.96, 103.37, 102.94, 101.75, 101.53, 101.44, 101.16, 99.85, 99.51, and 98.56 were clustered in the δ 95–120 ppm range, whereas ring carbons were located at δ 60.00–90.00 ppm.

Figure 3.

NMR spectra of FBSP-1. (A) ¹H NMR (B) ¹³C NMR (C) HSQC (D) COSY (E) HMBC (F) NOESY.

Anomeric carbons and protons were identified from ¹H and ¹³C NMR spectra. In the HSQC spectrum, the cross-peak at δ_H/δ_C 5.22/101.44 ppm indicated the anomeric position of residue A (Figure 3C). The COSY spectrum showed H2-H6 shifts for residue A at δ 3.51, 3.72, 3.52, 3.86, and 3.75 ppm (Figure 3D). Based on HSQC correlations and H2-H6 shifts, carbon shifts C2-C6 were assigned as 71.60, 73.00, 78.84, 70.88, and 61.16 ppm. The downfield shift in C-4 at 78.84 ppm confirmed a (1→4) glycosidic linkage in residue A. For residue B, the anomeric proton at δ H 5.20 ppm and COSY cross-peaks between H-1/H-2, H-2/H-3, H-3/H-4, H-4/H-5, and H-5/H-6 revealed chemical shifts in H2-H6 at 3.54, 3.70, 3.92, 3.69, and 3.40 ppm. Carbon shifts C1-C6 were assigned as δ 101.16, 71.91, 72.79, 76.57, 72.99, and 70.37 ppm. The downfield shifts in C-4 and C-6 suggested (1→4,6) linkages in residue B. The chemical shifts and linkage patterns for residues D, E, F, G, I, J, K, L, M, and N were determined similarly (Table 3).

Table 3.

NMR signal attribution of FBSP-1 glycosidic bond.

Peak	Residues	Chemical Shift (ppm)
		H1/C1	H2/C2	H3/C3	H4/C4	H5/C5	H6/C6
A	1,4-Glcp	5.22/101.44	3.51/71.60	3.72/73.00	3.52/78.84	3.86/70.88	3.75/61.16
B	1,4,6-Glcp	5.2/101.16	3.54/71.91	3.70/72.79	3.92/76.57	3.69/72.99	3.40/70.37
D	1,3,4-Xylp	4.47/102.94	3.21/73.59	3.35/73.10	3.53/71.51	4.08/66.84	-
E	1,2,5-Araf	4.90/107.96	4.05/81.01	3.85/76.70	3.98/83.87	3.77/66.65	-
F	1,6-Glcp	4.84/99.51	3.45/73.10	3.62/74.30	3.90/70.70	3.81/71.50	3.64/70.51
G	1,3-Araf	5.28/100.72	4.07/80.95	3.99/84.62	4.13/84.33	3.66/60.87	-
I	1,4-Xylp	4.51/101.75	3.22/74.15	3.44/74.15	3.57/79.20	3.36/54.20	-
J	1-Araf	5.07/108.19	4.15/81.28	3.95/77.35	4.14/83.82	3.69/64.02	-
K	1-Galp	4.52/103.37	3.23/71.90	3.71/73.43	3.90/69.6	3.68/77.33	3.72/61.16
L	1-Glcp	5.17/101.53	3.51/72.38	4.04/70.18	38.7/74.15	3.47/72.39	3.71/61.18
M	1,2-Manp	5.26/99.85	3.81/79.60	3.98/72.66	3.61/69.23	3.75/75.56	3.66/62.45
N	1,2,6-Manp	5.10/98.56	4.15/81.45	3.86/71.03	3.68/70.25	3.67/75.36	3.78/66.29

The HMBC and NOESY spectra were used to further elucidate the substitution sites, backbone architecture, and linkage sequence of the sugar residues in the polysaccharide (Figure 3E,F). For the main chain, a series of characteristic cross-peaks were observed at δ 5.10/78.84 and 5.22/3.52 ppm [N(H1)/A(C4), A(H1)/A(H4)], δ 5.22/73.10 ppm [A(H1)/D(C3)], δ 4.47/3.52 ppm [D(H1)/A(H4)], δ 5.22/76.57 ppm [A(H1)/B(C4)], δ 5.20/84.62 ppm [B(H1)/G(C3)], and δ 5.28/81.01 ppm [G(H1)/E(C2)]. Based on these peaks, the backbone of FBSP-1 was deduced as →2)-α-Manp-(1→[→(4)-α-Glcp-(1→)]₅→3)-β-Xylp-(1→4)-α-Glcp-(1→4)-α-Glcp-(1→3)-α-Araf-(1→2)-α-Araf-(1→. In the side chains, a cross-peak at δ 5.26/66.29 ppm [M(H1)/N(C6)] revealed a →1)-α-Manp-(2→ branch attached to the main chain through O-6 of residue N. Additional cross-peaks at δ 5.07/79.20 ppm [J(H1)/I(C4)], δ 4.51/3.64 ppm [I(H1)/F(H6)], and δ 4.84/71.51 ppm [F(H1)/D(C4)] corresponded to a branched fragment of →1)-α-Araf-(1→4)-β-Xylp-(1→6)-α-Glcp-(1→4→, connected via O-4 of residue D. Cross-peaks at δ 4.52/78.84 ppm [K(H1)/A(C4)] and δ 5.22/70.37 ppm [A(H1)/B(C6)] indicated a →1)-β-Galp-(1→4)-α-Glcp-(1→6→ side chain linked through O-6 of residue B. Finally, δ 5.17/78.84 ppm [L(H1)/A(C4)] and δ 5.22/3.77 ppm [A(H1)/E(H5)] suggested a →1)-α-Glcp-(1→4)-α-Glcp-(1→5→ side chain connected via O-5 of residue E.

In conjunction with the above analyses and relevant literature [38], the primary structure of FBSP-1 was proposed (Figure 4). The main chain consists of →2)-α-Manp-(1→[→(4)-α-Glcp-(1→]₅→3)-β-Xylp-(1→4)-α-Glcp-(1→4)-α-Glcp-(1→3)-α-Araf-(1→2)-α-Araf-(1→ residues. FBSP-1 contained three types of side chains: (i) the branch →1)-α-Araf-(1→4)-β-Xylp-(1→6)-α-Glcp-(1→ is linked to the main chain via the O-4 position of the →3,4)-β-Xylp-(1→ residue. (ii) the branch →1)-β-Galp-(1→4)-α-Glcp-(1→ is attached to the main chain through the O-6 position of the →4,6)-α-Glcp-(1→ residue. (iii) the branch α-Glcp-(1→4)-α-Glcp-(1→ is connected to the main chain via the O-5 position of the →2,5)-α-Araf-(1→ residue. Compared with the unfermented polysaccharide BSDF-1 as reported in previous literature [37], FBSP-1 exhibits significant structural differences. The backbone of BSDF-1 primarily consists of →4)-β-Galp-(1→4)-β-Glcp-(1→, with terminal fragments of α-Araf-(1→5)-α-Araf-(1→) attached to the main chain. In contrast, FBSP-1 features a more complex backbone structure composed of Manp, Glcp, Xylp, and Araf as the core components, along with diverse branching structures. These structural differences are likely attributed to the enzymatic systems secreted by Monascus purpureus during fermentation. It may have restructured and modified the original polysaccharide backbone, resulting in altered monosaccharide composition and linkage patterns. This transformation leads to the distinct structural features of FBSP-1 compared to water-extracted polysaccharides.

Figure 4.

Proposed (A) main structure of FBSP-1 and (B) its repeating unit.

3.9. Effects of Polysaccharides on the Viability of Caco-2 Cells

To evaluate the potential cytotoxicity of BSP-1 and FBSP-1, Caco-2 cells were treated with different concentrations of polysaccharide (12.5, 25, 50, 100, 200, and 400 μg/mL). After 24 h of incubation, no morphology alterations were observed (Figure 5A,C). Consistently, MTS assays demonstrated that neither BSP-1 nor FBSP-1 significantly affected cell viability within the tested concentration range of 12.5–400 μg/mL (Figure 5B,D). These results collectively indicate that BSP-1 and FBSP-1 are non-cytotoxic to Caco-2 cells under the tested concentration range.

Figure 5.

The effect of BSP-1 and FBSP-1 on cell viability of Caco-2 cells. (A) Microscopic image and (B) MTS results of Caco-2 cells treated with different concentrations of BSP-1 (0 (CK), 12.5, 25, 50, 100, 200, 400 µg/mL); The (C) microscopic image and (D) MTS results of Caco-2 cells treated with different concentrations of FBSP-1 (0 (CK), 12.5, 25, 50, 100, 200, 400 µg/mL). a: untreated control; b–g: 12.5 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL and 400 µg/mL BSP-1/FBSP-1. No significant statistical differences were observed in panels (B,D) (p > 0.05).

3.10. Effects of Polysaccharides on H₂O₂-Induced Caco-2 Cells

Based on the effects of H₂O₂ concentration and exposure time on cell viability (Table S2), in conjunction with relevant literature [39], a gut oxidative stress model was established in Caco-2 cells using 100 µM H₂O₂. After 24 h of treatment, noticeable morphological damage and a reduction in cell number were observed. MTS results showed that H₂O₂ reduced cell viability by approximately 18% (p < 0.01). The in vitro antioxidant capacities of BSP-1 and FBSP-1 were initially assessed. BSP-1 at concentrations of 12.5 and 25 µg/mL showed no protective effect on H₂O₂-injured Caco-2 cells (Figure 6A,B), whereas treatment with 50–400 µg/mL BSP-1 significantly restored cell viability. Compared with the H₂O₂ treatment group, 200 µg/mL BSP-1 produced an extremely significant improvement (p < 0.01) in cell viability. For FBSP-1, the antioxidant effect appeared stronger in vitro. At 25 µg/mL, FBSP-1 significantly alleviated (p < 0.05) the reduction in viability caused by H₂O₂ (Figure 6C,D); At 50 µg/mL, it produced an extremely significant improvement (p < 0.01). At different concentrations of FBSP-1 (0, 12.5, 25, 50, 100, 200, and 400 µg/mL), the viabilities of H₂O₂-induced Caco-2 cells were 81.45 ± 2.83%, 82.10 ± 6.44%, 86.70 ± 2.06%, 91.75 ± 3.09%, 93.68 ± 2.65%, 93.42 ± 2.52%, and 94.39 ± 5.19%.

Figure 6.

The comparison of the antioxidant activity between BSP-1 and FBSP-1. (A) Microscopic image and (B) MTS results of Caco-2 cells treated with BSP-1 and H₂O₂. (C) Microscopic image and (D) MTS results of Caco-2 cells treated with FBSP-1 and H₂O₂. a: untreated control; b: 100 µM H₂O_2, c–h: 100 µM H₂O₂ in combination with 12.5 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL and 400 µg/mL BSP-1/FBSP-1. (E,F) ROS staining in Caco-2 cells treated with (E) BSP-1 (F) or FBSP-1. a: untreated control, b: 100 µM H₂O₂, c–e: 100 µM H₂O₂ in combination with 12.5, 25, and 50 µg/mL BSP-1/FBSP-1, respectively. (G,H) The relative quantification of ROS staining. Vs. control group, ##: p < 0.01; vs. H₂O₂ treatment group, *: p < 0.05, **: p < 0.01. ns: not significant. Data are presented as mean values with 95% confidence intervals (CI).

To further compare the antioxidant activity of BSP-1 and FBSP-1 in vitro, we evaluated their effects on H₂O₂-induced ROS accumulation in Caco-2 cells. ROS staining enables visualization of intracellular oxidative stress by detecting changes in fluorescence intensity that reflect intracellular ROS levels, thereby allowing qualitative and quantitative evaluation of ROS accumulation. ROS staining results revealed that 12.5 and 25 µg/mL BSP-1 had no significant effect on H₂O₂-induced ROS elevation (Figure 6E,G and Table S3). In contrast, 50 µg/mL BSP-1 partially reduced ROS content by 13.29% (p < 0.05) relative to the H₂O₂ treatment group. Notably, FBSP-1 exhibited a stronger antioxidant effect: treatment with 12.5, 25, and 50 µg/mL FBSP-1 significantly decreased ROS fluorescence intensity in H₂O₂-induced cells (Figure 6F). Relative to the H₂O₂-treated group, the ROS levels of FBSP-1 treatment group (12.5, 25, and 50 µg/mL) were reduced by 24.40% (p < 0.05), 29.76% (p < 0.01), and 32.74% (p < 0.01), respectively (Figure 6H and Table S3). These results demonstrate that FBSP-1 effectively alleviates H₂O₂-induced oxidative stress at relatively low concentrations and exhibits markedly stronger antioxidant activity than BSP-1. This enhanced effect may be attributed to overall changes in the monosaccharide composition and glycosidic linkage structure of the polysaccharide during fermentation. In particular, alterations in monosaccharide composition, especially the increased proportions of Glc and Man, may be associated with the improvement in antioxidant activity, which is consistent with previous reports [40].

3.11. FBSP-1 Alleviated Oxidative Stress Damage

Based on the ROS staining results, which demonstrated that FBSP-1 exhibited stronger antioxidant activity than BSP-1 at lower concentrations, FBSP-1 was selected for further cellular analyses (Figure 7 and Figure S4). Excessive ROS can trigger oxidative damage, leading to alterations in key oxidative markers. GR was chosen as a representative marker due to its essential role in regenerating reduced glutathione (GSH) within the glutathione redox cycle, which reflects the cellular capacity to maintain redox homeostasis under oxidative stress conditions [41]. In H₂O₂-treated Caco-2 cells, SOD and GR activities were markedly inhibited, while MDA content was significantly elevated (Figure 7A–C and Table S3). These results indicate that FBSP-1 effectively attenuates cellular oxidative stress. The protective effects of FBSP-1 were also evident at the apoptosis level. Hoechst staining revealed that 100 µM H₂O₂ caused Caco-2 cells to shrink and exhibit condensed nuclei (Figure 7D). This damage was progressively alleviated with increasing concentrations of FBSP-1. Flow cytometry further demonstrated that H₂O₂ treatment reduced the average percentage of viable cells to 79.23% (Figure 7E and Figure S4). Subsequent treatment with 12.5, 25, and 50 µg/mL FBSP-1 improved average cell viability to 84.70%, 87.33%, and 90.27%, respectively. Concurrently, both early and late apoptotic cell populations were reduced. These results demonstrate that FBSP-1 effectively mitigates oxidative stress-induced cellular damage and suppresses apoptosis, with protective effects observable even at relatively low concentrations, underscoring its potential as a cytoprotective agent. These results indicate that FBSP-1 effectively alleviates oxidative stress–induced cellular damage and suppresses apoptosis, consistent with previous reports [4,42,43] on the cytoprotective effects of bamboo shoot polysaccharides, and that such protection is evident even at relatively low concentrations, highlighting its application potential. It should be noted that the polysaccharide concentrations used in the Caco-2 experiments in this study were primarily intended for in vitro functional evaluation rather than to directly reflect systemic exposure following dietary intake. Since dietary polysaccharides are generally resistant to digestion and poorly absorbed, their effects are more likely to occur within the intestinal lumen or at the epithelial interface, making the use of relatively higher local concentrations (50–400 μg/mL) in the Caco-2 model reasonable and consistent with previous studies on plant-derived polysaccharides [44,45].

Figure 7.

FBSP-1 alleviated H₂O₂-induced oxidative stress damage in Caco-2 cells. (A) SOD activity, (B) MDA activity and (C) GR activity in H₂O₂-induced Caco-2 cells. (D) Hoechst staining. a: untreated control; b: 100 µM H₂O₂; c–e: 100 µM H₂O₂ in combination with 12.5, 25, and 50 µg/mL FBSP-1, respectively; (E) flow cytometry of H₂O₂-induced Caco-2 cells. a₁: untreated control; b₁: 100 µM H₂O₂; c₁–e₁: 100 µM H₂O₂ in combination with 12.5, 25, and 50 µg/mL FBSP-1, respectively; f: Cell viability. Vs. control group, ##: p < 0.01; vs. H₂O₂ treatment group, *: p < 0.05, **: p < 0.01. ns: not significant. Data are presented as mean values with 95% confidence intervals (CI).

3.12. Free Radical Scavenging Rate In Vitro

BSPs have recently been demonstrated to protect against oxidative stress in various models, mainly by scavenging ROS [4]. In this work, the antioxidant potential of BSP-1 and FBSP-1 was comprehensively assessed using •DPPH, ABTS⁺, and •OH radical scavenging assays (Table S4). Both polysaccharides exhibited a dose-dependent response, with FBSP-1 showing significantly higher scavenging activities than BSP-1. At concentrations of 0.5, 1.0, and 3.0 mg/mL, FBSP-1 achieved radical scavenging efficiencies ranging from 39.42% to 84.95% for •DPPH, 31.69% to 74.06% for ABTS⁺, and 37.13% to 66.86% for •OH, respectively. Within the tested concentration range (0.5–3.0 mg/mL), FBSP-1 showed stronger antioxidative capacity than BSP-1. It should be noted that the above radical-scavenging assays were conducted in simplified, acellular systems, primarily reflecting the intrinsic chemical capacity of the polysaccharides to neutralize free radicals. While these assays do not fully reflect antioxidant effects at the cellular level, they provide a preliminary indication of the antioxidant potential of bamboo shoot polysaccharides.

3.13. The Correlation Between Antioxidant Activity and Polysaccharide Structure

Polysaccharides have complex structures that underpin their diverse biological activities, but these complexities make it challenging to understand their structure-activity relationships. The relationship between FBSP-1 glycosidic linkage features and its antioxidant activity was elucidated using Pearson correlation analysis, with the results visualized as a heatmap (Figure 8).

Figure 8.

Heatmap Analysis. (A) Heatmap depicting the relationship between partial structures of BSP-1 and FBSP-1 and their antioxidant activity. Data are shown as mean ± SD (n = 3). Red and blue indicate positive and negative correlations, respectively. Correlation coefficients were calculated using Pearson analysis. (B) Heatmap of FBSP-1 partial structures, with red and blue circles highlighting statistically positive and negative correlations with antioxidant activity (p < 0.05).

Monosaccharide composition and glycosidic linkages exerted distinct influences on the antioxidant process. Man content correlated positively with •DPPH radical scavenging (r = 0.986), indicating that Man-rich polysaccharides generally exhibit enhanced antioxidant activity. This enhancement may be attributed to favorable hydroxyl configurations and increased accessibility of active sites [40,46], which is consistent with the markedly improved antioxidant activity observed for FBSP-1 after fermentation. Glc exhibited a concentration-dependent scavenging effect on •DPPH radicals (r = 0.538). Xyl was positively correlated with ABTS⁺ scavenging (r = 0.985), Gal was linked to enhanced antioxidation enzyme activities SOD (r = 0.980) and GR (r = 0.928), and reduced lipid peroxidation MDA (r = −0.757).

Further analysis revealed that the Glc residues of Glc(p)-(1→ and →4)-Glc(p)-(1→ were strongly positively correlated with •OH (r = 0.984, 0.990) and SOD (r = 0.983, 0.991). These findings suggest that →4)-Glc(p)-(1→ glycosidic linkages may enhance polysaccharide antioxidant activity by improving solubility and conformational stability, thereby increasing the spatial accessibility of hydroxyl and other reactive functional groups and facilitating their interactions with ROS. This structure–function relationship has also been supported by previous study [47,48]. In addition, →6)-Glc(p)-(1→ and →4,6)-Glc(p)-(1→ linkages showed positive correlations with •DPPH scavenging activity (r = 0.984 and 0.896, respectively), suggesting that branched Glc residues may contribute to enhanced electron-donating capacity [7,49].

Collectively, the antioxidant activity of bamboo shoot polysaccharides is governed by multiple structural factors, including molecular weight, monosaccharide composition, and glycosidic linkage architecture. Pearson correlation analysis revealed significant associations between specific monosaccharide features and glycosidic linkage types with both radical-scavenging capacity and cellular antioxidant indicators. Notably, FBSP-1, obtained after Monascus fermentation, exhibited enhanced antioxidant activity compared with the unfermented BSP-1. These findings suggest that fermentation enhances antioxidant potential through global remodeling of polysaccharide structures, including changes in monosaccharide composition, glycosidic linkages, and overall molecular architecture. Taken together with the results from in vitro radical-scavenging assays and the Caco-2 cell model, these results indicate that fermentation-induced structural modifications play a critical role in the improved antioxidant activity and cytoprotective effects of FBSP-1.

From a bioaccessibility perspective, fermentation-induced structural modifications may influence the in vivo behavior of polysaccharides. Variations in molecular weight, monosaccharide composition, and glycosidic linkage architecture are known to influence physicochemical properties such as solubility, conformational flexibility, and resistance to gastrointestinal digestion, which are important factors governing polysaccharide bioaccessibility [50]. In this context, the reduced molecular weight and altered linkage features of FBSP-1 could potentially affect its interactions with intestinal epithelial cells or its accessibility within gut-associated biological environments. Although these effects were not directly assessed in vivo, the enhanced cellular antioxidant responses observed suggest that fermentation-driven structural remodeling may improve both the bioaccessibility and functional efficacy of bamboo shoot polysaccharides.

Despite the comprehensive structural and functional analyses performed in this study, several limitations should be acknowledged. The antioxidant activity was evaluated using in vitro assays and the Caco-2 cell model, which may not fully reflect the complexity of in vivo systems. Additionally, fermentation-derived small molecules that could contribute to antioxidant effects were not analyzed, as the focus was on polysaccharide structural modifications.

4. Conclusions

In conclusion, this study demonstrates that fermentation with Monascus purpureus ATCC16426 effectively modifies the molecular structure of bamboo (Phyllostachys edulis) shoot polysaccharides, enhancing their antioxidant properties. Fermentation altered the monosaccharide composition and reduced the molecular weight. Detailed structural analysis through methylation and NMR spectroscopy revealed key features of FBSP-1, including the main chain configuration and glycosidic linkages, which provided crucial insights into the structure-activity relationship. Functional assays showed that FBSP-1 significantly reduced ROS and MDA levels and enhanced SOD and GR activities in H₂O₂-induced Caco-2 cells, demonstrating stronger antioxidant protection than BSP-1. These results provide a basis for further investigation into fermented bamboo shoot polysaccharides as potential functional ingredients, particularly in plant-based food systems or health-oriented formulations targeting oxidative stress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods15040704/s1, Figure S1: Fermentation condition optimization; Figure S2: Standard curve of dextran; Figure S3: Mass spectra (A-L) of peak PMAAs for FBSP-1; Figure S4: Additional representative flow cytometry plots; Table S1: Chemical composition of BSP-1 and FBSP-1; Table S2: Effects of H₂O₂ concentration and exposure time on cell viability; Table S3: Antioxidative activity of FBSP-1 and WBSP-1 on Caco-2 cells in vitro; Table S4: Free radical scavenging rate in vitro of FBSP-1.

Author Contributions

F.L. (Fang Long): Conceptualization, Writing—original draft, Methodology, Investigation. Z.H.: Writing—original draft, Methodology, Investigation. H.L.: Methodology, Data curation. Z.C.: Methodology, Investigation. S.L.: Investigation, Validation. Y.Z.: Validation. A.L.: Data curation, Writing—review and editing. F.L. (Feijun Luo): Data curation, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

The authors gratefully acknowledge the funds from the Hunan Provincial Natural Science Foundation (2022JJ50256) and the Key Project of State Key R & D Program, China (2022YFF1100200).