Structural characterization, antioxidant activity, and fermentation characteristics of Flammulina velutipes residue polysaccharide degraded by ultrasonic assisted H₂O₂-Vc technique

Graphical abstract

Abstract

Adhere to the concept of low-carbon environmental protection and turning waste into treasure, polysaccharides from Flammulina velutipes residue polysaccharide (FVRP) has been developed and possesses diverse bioactivities, comprising antioxidant, hypoglycemic, and relieving heavy metal damage, which still has the disadvantages of high molecular weight and low bioavailability. The current work is the first to prepare a degraded polysaccharide (FVRPV) from FVRP by ultrasonic assisted H₂O₂-Vc technique in order to reduce its molecular weight, thereby improving its activity and bioavailability. Our results found that the molecular weight and average particle size were declined, but the monosaccharide composition and characteristic functional group types of FVRPV had no impact. The structural changes of polysaccharides analyzed by XRD, Congo Red test, I₂-KI, SEM, and methylation analysis indicated that the surface morphology and glycosidic bond composition of FVRPV possessed longer side chains and a greater number of branches with an amorphous crystal structure devoid of a triple helix configuration, and had experienced notable alterations after ultrasonic assisted H₂O₂-Vc treatment. Meanwhile, the in vitro antioxidant capacity of FVRPV had significantly increased compared to FVRP, implying ultrasonic assisted H₂O₂-Vc technique maybe a effective method to enhance the bioactivity of polysaccharides. In addition, the content of polysaccharide, reducing sugar, and uronic acid in FVRPV was significantly decreased, but antioxidant capacity of fermentation broth was stronger by in vitro human fecal fermentation. The 16S rDNA sequencing data displayed that FVRPV can enrich probiotics and reduce the abundance of pathogenic bacteria through different metabolic pathways mediated by gut microbiota, thereby exerting its potential probiotic effects. The interesting work provides a novel degraded polysaccharide by ultrasonic assisted H₂O₂-Vc technique, laying a foundation for developing FVRPV as a new antioxidant and prebiotic.

1. Introduction

Polysaccharides are a class of natural macromolecular active substances comsist of multiple monosaccharides and their derivatives, mainly possessing bioactivities, including antioxidant, anti-tumor, anti-inflammatory, and prebiotic effects [1], [2]. However, owing to the high molecular weight, polysaccharides are difficult to break through the cell barrier and penetrate the organism to exhibit bioactivities, resulting in low bioavailability and the limitation of biological characteristics [3]. Therefore, green and efficient degradation methods have always been a hotspot in the research of polysaccharides. Polysaccharide derivatives with different structural types can be obtained through chemical, physical, and biological methods [4]. Structure adjustment of polysaccharides, as well as the type, quantity, and position of substituent, not only can reduce the molecular weight, but alter their structural properties, thereby enhancing their biological activities [5]. The biological degradation generally refers to enzymatic degradation. Due to the high cost and limited source of specific enzymes, non-specific enzymes are usually used for enzymatic degradation [6]. Ultrasonic assistant method is the commonly used physical degradation method [7]. Chemical degradation approaches mainly comprise acid degradation and oxidative degradation [8].

Although the above approaches are widely used, a single method is still some limitations [3]. Therefore, researchers focus on combination methods to improve the degradation efficiency of polysaccharides, such as ultrasonic combined with non-metallic Fenton reaction [9], [10], ultrasonic assistant H₂O₂/enzyme degradation method, microwave assistant H₂O₂/hydrothermal degradation method, microwave assistant enzyme/acid degradation method, free radical combined ultrasonic degradation, etc. [11], [12]. The combination of ultrasound and H₂O₂ degradation method has shown a more significant function than individual degradation methods. This composite method mainly destroys the glycosidic bonds between monosaccharides, with less impact on the primary structure of polysaccharides. Meanwhile, the more hydroxyl groups generated during the degradation process enhance the bioactivity of polysaccharides [13]. The particle size and molecular weight of pectin polysaccharides degraded by ultrasonic assistant H₂O₂ method were significantly reduced as the ultrasonic time went, but the degree of degradation of pectin polysaccharides further increased [14]. Ultrasonic assisted H₂O₂-Vc technique is also suitable for the degradation of Tremella fuciformis polysaccharides, which possessed a lower molecular weight and stronger in vitro antioxidant capacity based on Caenorhabditis elegans model [15].

Flammulina velutipes is a worldwide cultivated edible and medicinal mushroom, possessing multiple bioactivities, thereby has been gained widespread attention in the biochemistry and pharmacology field [16]. With the promotion of industrial cultivation of Flammulina velutipes, the yield of Flammulina velutipes residue (FVR) continues to increase. However, traditional incineration and burial methods not only pollute the environment, but also lead to significant waste of nutrients in the FVR [17]. Flammulina velutipes residue polysaccharide (FVRP) is the primary active ingredient of FVR, exhibits antioxidant, anti-diabetes nephropathy, anti-virus, anti-heavy metal toxicity and other biological activities [18]. However, FVRP also faces the problem of low bioavailability due to its high molecular weight [19]. Therefore, this study utilized ultrasonic assisted H₂O₂-Vc technique to obtain FVRPV degraded from FVRP for the first time, and analyzed the physicochemical characteristics, antioxidant capacity and in vitro fermentation characteristics FVRPV.

2. Materials and methods

2.1. Materials and reagents

FVRP was extracted before and saved in the laboratory [17]. Hydroxybenzene, ABTS, DPPH, Vc and standards of SCFAs (chromatography-pure grades) were supported by Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China). FOS was supported by Beijing Solarbio Life Sciences Co., Ltd (Beijing, China). Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Gal-UA and Glc-UA were supported by Sigma-Aldrich (Shanghai) Trading Co., Ltd (Shanghai, China). Other chemicals are of analytically pure grades. Human fecal were contributed by 6 healthy individuals (3 males and 3 females, BMI 18.5–24, 23–27 years old) meeting specific inclusion criteria, who have not any history of intestinal diseases or taken antibiotics within the past 90 days.

2.2. Preparation of FVRPV

The degradation of FVRP was performed pursuant to the reference [13]. Dissolved FVRP solution (5 mg/mL) was adjusted the pH to 3.0, and then mixed with 0.035 % H₂O₂ solution and 0.03 % Vc. The mixture was treated with ultrasound at 80 ℃ and 600 W for 30 min by Ultrasonic cleaner SCIENTZ SB-800DT, (Ningbo, China), and then was adjusted the pH to neutral. The reaction solution was then dialysis and freeze-dried to acquire the degraded polysaccharide (FVRPV).

2.3. Determination of physicochemical properties and monosaccharide composition of FVRPV

The content determination method of polysaccharide, reducing sugar, protein, uronic acid and total phenolic substances in FVRPV was referred to the study of Nie et al. [20], determined by Multifunctional Enzyme Labeling Instrument (TECAN-Spark, Switzerland). The monosaccharide composition of FVRPV was tested referred to the report of Li et al. [21]. Firstly, 2 mL of FVRPV solution of 5 mg/mL was underwent hydrolysis treatment with trifluoroacetic acid. Next, under alkaline conditions, the degradation solution was derivatized with PMP and separated by High-performance anion-exchange chromatography (HPAEC) equiped with a pulsed amperometric detector (PAD, Dionex ICS 5000 system) on a Dionex CarboPac PA20 anion-exchange column (150 × 3.0 mm, 10 μm). The injection volume is 5 μL. Solvent system: mobile phase A (H₂O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), and the flow rate was kept at 0.5 mL/min and the column temperature was sustained at 30 ℃. Gradient program: 95: 5: 0, V/V at 0 min, 85: 5: 10, V/V at 26 min, 85: 5: 10 V/V at 42 min, 60: 0: 40, V/V at 42.1 min, 60: 40: 0, V/V at 52 min, 95: 5: 0, V/V at 52.1 min, 95: 5: 0, V/V at 60 min.

2.4. Particle size distribution and Zeta potential of FVRPV

Firstly, 1 mg/mL FVRPV solution was prepared and dispersed with ultrasonic treatment. Next, a laser particle size analyzer (NANO ZS90, Malvern, UK) was used to examine the particle size, PDI and Zeta potential of FVRPV.

2.5. Determination of relative molecular weight (Mw)

The homogeneity and molecular weight of FVRPV were determined by high performance gel permeation chromatography (HPGPC-ELSD) equipped with RID-10A parallax refraction detector and TSK-gel G-3000PWXL column (7.8 × 300 mm). 10 μL of FVRPV solution (2 mg/mL) was filtered with 0.22 μm filter membrane. Chromatographic conditions were as follows: mobile phase was deionized water, flow rate was 0.6 mL/min, column temperature was 35 ℃ [22]. Dextran standard products with relative molecular weight (Mw) of 12 kDa, 633 kDa, 126 kDa, 287 kDa and 556 kDa were prepared into 2 mg/mL solution was detected according to the above chromatographic conditions. The molecular weight of FVRPV was calculated according to the retention time.

2.6. Structural features of FVRPV

2.6.1. FT-IR Spectroscopy assay

FT-IR assay was conducted referred to the method proposed by Yan et al. [23]. The dried FVRPV powder was mingled with spectral grade potassium bromide powder evenly, and then pressed into a tablet for FI-TR assay on a Nexus 470 FT-IR spectrometer (Thermo Nicolet, USA).

2.6.2. XRD analysis

The crystal structures of FVRP and FVRPV were determined using X-ray diffraction method. In brief, the dried sample powder was uniformly placed on a glass plate and scanned at a rate of 2°/min over a 2θ range of 5–90° by X-ray polycrystalline diffractometer (Rigaku SmartLab SE, Japan) [24].

2.6.3. Congo red examination

Congo red examination was conducte in light to the antecedent report [25]. 1 m L of FVRPV solution (2 mg/mL) was mingled with 1 mL of Congo Red solution (200 μg/mL), and then was added different volumes of NaOH with the final volume of 4 mL to make the concentration of NaOH from 0 to 0.5 M and then reacted at room temperature for 5 min. Full wavelength scanning from 400 to 700 nm were recorded by Multifunctional Enzyme Labeling Instrument (TECAN-Spark, Switzerland), and the maximum absorption wavelength (λ_max) was used for curve drawing.

2.6.4. I₂-KI assay

2 mL of FVRPV (1 mg/mL) was mingled and reacted with 1.2 mL potassium iodide reagent (0.2 % KI, and 0.02 % I₂, w/v) at room temperature for 10 min. Spectra were recorded in the range 300–700 nm by Multifunctional enzyme Labeling Instrument (TECAN-Spark, Switzerland) [26]

2.6.5. Characterization of surface morphology of FVRPV

FVRPV powder was sprayed with gold powder, and then detected on Scaning electron microscopy system Regulus 8230 (SEM) (Hitachi, Japan) under voltage of 3.0 kV to analyze the surface morphology of FVRPV with an image magnification of 1000 and 5000 times.

2.7. Analysis of methylated glycosidic bond of FVRP and FVRPV

The method of methylation analysis was modified from the of Li et al. [27]. The reduction of FVRP was conducted on Automatic reduction of carboxyl groups instrument provided by Bo Rui Saccharide Biotech Co., Ltd (Yangzhou, China). Subsequently, FVRP and FVRPV were analyzed for total methylation by converting partially methylated polysaccharides into their partially methylated alditol acetate (PMAA) form via hydrolysis, reduction, and acetylation. The glycosidic bonds of PMAA were further analyzed by GC–MS system (Thermo Fisher Scientific 1300–7000, USA) using an HP-INNOWAX column (30 m × 0.32 mm × 0.25 μm). The programmed heating conditions were as follows: the initial temperature of the column incubator was 140 °C, rising to 230 °C at the rate of 1 °C/min. The inlet temperature was set to 250 °C and the detector temperature was 250 °C. The carrier gas was helium, and the flow rate was 1 mL/min. The type and proportion of glycosidic bonds were identified by comparing the mass spectra of the peaks in the PMAA database.

2.8. In vitro antioxidant activity determination

ABTS radical clearance rate, DPPH radical clearance rate, reducing ability, and hydroxyl radical clearance rate of FVRPV were measured referred to the sequential approach [17], which were detailed described in Supplement file, and positive drug was Vc.

2.9. The fermentation characteristics of FVRPV

2.9.1. In vitro fermentation

Sterile FVRPV (10 mg/mL) and FOS (10 mg/mL) were added, respectively, named FVRPV group and FOS group, which was positive control. Equal amount sterile water added was named CK group. Fresh fecal slurry was prepared in accordance with our previous report [22]. 5 mL of 10 % fecal slurry was appended to each group and fermented in an anaerobic environment at 37 ℃, fermentation samples were gathered at key time points during the fermentation process (0 h, 6 h, 12 h, 24 h, 48 h). The physicochemical characteristics and antioxidant abilities of the fermentation broth were measured according to our preceding methods. FT-IR assay was performed to verify the structural changes of FVRPV during in vitro fermentation, which was extracted after alcohol precipitation, dialysis and freeze-dried.

2.9.2. Detection of pH and SCFAs

The pH of the supernatant of the fermentation liquid at different time points during fermentation were analyzed by a pH meter (INESA, Shanghai, China). 1 mL of fermentation supernatant was thoroughly mingled with 0.2 mL of 25 % metaphosphate solution and centrifuged at 10000 rpm/min for 15 min for the determination of SCFAs content by gas chromatography (Agilent Technologies, USA) supplied with a DB-FFAP column (30 m × 0.32 mm × 0.25 μm) [22]. Nitrogen was employed as the carrier gas, with the flow rate of 19 mL/min, and the injection volume was 1  μL with a split ratio of 50: 1. The initial temperature of 65 ℃, and the hydrogen flame ionization detector was set at a temperature of 250 ℃. Acetic acid, Propionic acid, Isobutyric acid, Isovaleric acid, N-butyric acid and N-valeric acid were used as the standards, and finally the total SCFAs during fermentation were calculated.

2.9.3. 16S rDNA sequencing analysis

The impact of FVRPV on gut microbiota was assessed by 16S rDNA sequencing with the V3-V4 region on 15 samples (5 replicates per group), including FVRPV group, CK group, and FOS group. LEfSe analysis identified statistically significant differences in biomarkers between groups and analyzed differential microbial communities. The differential functional prediction was analyze by PICUST software.

2.10. Data statistical analysis

IBM SPSS (26.0) was utilized for data statistical analysis and variance (ANOVA) analysis to judge the significance of each parameter, with P < 0.05 having statistical significance. The data was represented as mean ± standard deviation, and was plot by GraphPad Prism 8 software.

3. Results and discussion

3.1. Physicochemical properties of FVRPV

FVRPV was obtained from FVRP through ultrasonic assisted H₂O₂-Vc degradation method for the first time, and the recovery rate was 65.21 ± 1.02 %. Table S1 listed the physicochemical properties of FVRPs. Compared with FVRP, FVRPV had significantly increased the polysaccharide content, which was 81.51 ± 0.41 %. Degradation can degrade long-chain polysaccharides to some extent into polysaccharides with short chains, thereby increasing polysaccharide content [28], which was similar to the current results. In addition, after treatment of FVRP with ultrasonic assisted H₂O₂-Vc degradation method, the protein content of FVRPV was significantly decreased, while the content of uronic acid, total phenolics, and reducing sugars did not show significant changes after the ultrasonic assisted H₂O₂-Vc treatment.

The decrease in molecular weight of polysaccharides may enhance the bioactivities, comprising antioxidant, prebiotic, anti-tumor and other capacities [3]. The molecular weight of FVRPV was changed to 297.43 kDa (Fig. 1A, Table S1), while the molecular weight of FVRP was 335.29 kDa [19], showing that the molecular weight of polysaccharides declined after ultrasonic assisted H₂O₂-Vc treatment, which may be due to the breakage of glycosidic bonds in the polysaccharides caused by ultrasound treatment, leading to the release of soluble polysaccharides with lower molecular weight, thereby resulting in a decline in molecular weight [29]. The active free radicals generated by ultrasound and H₂O₂ system may also be the main factor affecting the decrease in the molecular weight of polysaccharides [30]. Our result was consistent with the research findings that the molecular weight of sweet corncob polysaccharides was reduced from the initial 19.71 kDa to 9.49 kDa by a ultrasonic assisted H₂O₂-Vc method [7].

Fig. 1.

Molecular weight (A), Monosaccharide composition (B), FT-IR spectrum (C), XRD curves (D), Congo red assays results (E), I₂-KI assays results (F), and Surface morphology (G) of FVRPV. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The particle size distribution, polymer dispersion index (PDI), and Zeta potential have critical impacts on the stability of polysaccharides [8]. The particle size can affect the metabolism of polysaccharides in organisms, and a decrease in particle size is beneficial for polysaccharides to exhibit their biological capacity [31]. In the current study, the average particle size values of FVRPs were lower than 1000 nm (Table S1), meeting the common particle size standards for polysaccharides [32]. The particle size of FVRP was mainly concentrated between 160–700 nm, with an average particle size of 465.43 nm. The particle size of FVRPV was concentrated between 130–390 nm, with an average particle size of 339.97 nm (Fig. S1). The results illustrated that ultrasonic assisted H₂O₂-Vc degradation reduced the average particle size and changed the particle size distribution range of polysaccharides from Flammulina velutipes residues, suggesting that the biological activity may be enhanced after degradation. Generally speaking, a lower PDI value means a uniform distribution of polysaccharides and the higher stability [3]. The PDI of FVRPV was significantly decreased (Table S1), indicating that the degradation process was effective and FVRPV was relatively uniform and stable. Zeta potential can reflect the state of static charge on the surface of a sample solution. Polysaccharides possessing the smaller the particle size value and the larger absolute value of Zeta potential were easier dispersed in the dissolution system [33]. FVRPV obtained by ultrasonic assisted H₂O₂-Vc treatment had a larger absolute Zeta potential than FVRP, suggesting it was more stable, and easier to disperse.

The monosaccharide composition results showed that FVRPs were heteropolysaccharides with the same monosaccharide types, mainly composed of 9 monosaccharides, including Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Gal UA, and Glc UA [17] (Fig. 1B, Table S1). Ultrasonic assisted H₂O₂-Vc condition did not alter the monosaccharide types of FVRP, but significantly transformed the molar ratio of monosaccharide composition, with a raise in glucose and mannose content and a decrease in galacturonic acid content. The above result implied that degradation preferentially acts on glycosidic bonds near galacturonic acid, while glycosidic bonds near glucose and mannose are more stable [13]. Therefore, the ultrasonic assisted H₂O₂-Vc degradation system caused less damages to the basic structure of polysaccharides during treatment, and the reaction process was relatively stable [34], which was similar to our result. In summary, the basic structure of FVRP remained stable after degradation, which may be crucial for maintaining their biological activity.

3.2. Structural features of FVRPV

FT-IR can reflect the types of functional groups in a substance and the chemical environment, thereby inferring the structure of the compounds. The infrared absorption peaks of FVRPV were similar with that of FVRP (Fig. 1C), suggesting ultrasonic assisted H₂O₂-Vc degradation method did not alter the main functional group types of polysaccharides. The absorption peak appearing near 2927 cm⁻¹ implies the presence of aliphatic C-H bonds in the sample [34]. A wide absorption band near 1420 cm⁻¹ may be caused by the deformation vibration of carbon hydrogen bonds, while two distinct absorption peaks observed in the range of 1100 ∼ 1010 cm⁻¹ may be attributed to the stretching vibration of C-O or C-N bonds [35]. An absorption peak appeared at 808 cm⁻¹, suggested that the sugar residues of FVRP and FVRPV had alpha glycosidic bonds [36]. In summary, similar to FVRPV, FVRPV was a polysaccharide containing alpha glycosidic bonds, implying ultrasonic assisted H₂O₂-Vc technique did not alter the infrared structural characteristics of polysaccharides.

The crystal configuration and crystallinity of FVRPV were determined by the XRD method [37]. The XRD patterns of FVRP and FVRPV exhibited similar results with typical broad diffraction peaks near 21.10° (Fig. 1D), suggesting that both FVRP and FVRPV had an amorphous structure.

Polysaccharides with a triple-helical structure can form a complex with Congo red, leading to an increase in its maximum absorption wavelength [38]. However, compared with the Congo red solution, the mixed solutions of FVRP and FVRPV with Congo red did not show a red shift (Fig. 1E), suggesting the absence of a helical structure. Hence, FVRP and FVRPV did not possess a triple-helical structure.

I₂-KI analysis are commonly used to determine the existance of starch and long chains of polysaccharides. The peak at 350  nm could indicate the presence of longer side chains and more branched chains, while the peak at 565  nm suggests shorter side chains and fewer branched chains [39]. The absorption peaks of FVRP and FVRPV are exclusively detected at 350 nm, with no corresponding peak observed at 565 nm (Fig. 1F). I₂-KI analysis result suggested that both polysaccharides possess longer side chains and more branching. Furthermore, FVRPs did not react with I₂-KI to exhibit the characteristic color reaction of starch, indicating that FVRPs were non-starch polysaccharides.

SEM was applied to examine the microscopic morphological properties of FVRPs (Fig. 1G). FVRP displayed a lamellar structure with a relatively smooth and flat surface, whereas the surface of FVRPV was notably rougher (1000×). At 5000 × magnification, FVRPV revealed an increased number and size of pores compared to FVRP, likely resulting from extensive cavitation activity induced by ultrasound, which aligns with previous report [40].

The methylation analysis result was presented in Table 1 and Fig. S3. The predominant glycosidic bond in FVRP was identified as → 4)-Xylp-(1→(18.2 %), with additional glycosidic bonds comprising Araf-(1→(15.4 %), →4)-Manp-(1→ (11.7 %), Xylp-(1→ (8.3 %), and Galp-(1→ (8.1 %). During the ultrasonic-assisted H₂O₂-Vc degradation process, a significant alteration occurred in the molar percentage of glycosidic bonds within the polysaccharides. The primary glycosidic bond present in FVRPV was → 2,6)-Manp-(1→(36.4 %), →4)-Glcp-(1→(11.5 %) and Araf- (1→ (10.5 %). In addition, the type of glycosidic bond in FVRP also underwent significant changes after ultrasonic degradation. The six glycosidic linkages identified in FVRP were not detected in FVRPV. However, four glycosidic linkages were observed in FVRPV, namely → 2,4)-Xylp-(1→, →3)-Glcp-(1→, →4)-Glcp-(1→, and → 2,6)- Manp-(1→, which were absent in FVRP. These results align with the current literature, which demonstrates that ultrasound can induce the cleavage of glycosidic bonds and decrease molecular weight [41].

Table 1.

GC–MS analysis of methylated FVRP and FVRPV.

PMAA	Glycosidic residues	Molar percentagea		Characteristic ions (m/z)
		FVRP	FVRPV
2,3,5-Me3-Araf	Araf-(1→	15.4	10.5	43,71,87,101,117,129,143,161
2,3,5-Me3-Xylp	Xylp-(1→	8.3	4.2	45,71,87,101,117,129,145,161
2,3,4,6-Me4-Glcp	Glcp-(1→	4.1	6.3	43,71,87,101,117,129,145,161,205
2,3,4,6-Me4-Manp	Manp-(1→	2.5	1.2	43,71,87,101,117,129,145,161,205
2,3-Me2-Araf	→5)-Araf-(1→	5.3	4.9	43,71,87,99,101,117,129,161,189
2,3,4,6-Me4-Galp	Galp-(1→	8.1	4.6	43,71,87,101,117,129,143,161,205
2,3-Me2-Xylp	→4)-Xylp-(1→	18.2	4.5	43,71,87,99,101,117,129,161,189
3,4,6-Me3-Manp	→2)-Manp-(1→	4.3	2.8	43,87,129,161,189
2,3,6-Me3-Galp	→4)-Galp-(1→	3.6	3.5	43,87,99,101,113,117,129,131,161,173,233
2,3,4-Me3-Galp	→6)-Galp-(1→	4.3	2.3	43,87,99,101,117,129,161,173,233
2,4-Me2-Galp	→3,6)-Galp-(1→	1.3	3	43,87,117,129,159,189,233
2-Me1-Fucp	→3,4)-Fucp-(1→	3.3	−	43,87,99,115,117,129,175
2,3,6-Me3-Manp	→4)-Manp-(1→	11.7	−	43,87,99,101,113,117,129,131,161,173,233
2,3,4-Me3-Glcp	→6-Glcp-(1→	2.2	−	43,87,99,101,117,129,161,189,233
2,3,4-Me3-Manp	→6)-Manp-(1→	1.6	−	43,71,87,99,101,117,129,159,161
2,6-Me2-Galp	→3,4)-Galp-(1→	4	−	43,87,99,117,129,143
2,6-Me2-Manp	→3,4)-Manp-(1→	2	−	43,87,99,117,129,143
3-Me1-Xylp	→2,4)-Xylp-(1→	−	2.9	45,87,99,101,117,129,145,189
2,4,6-Me3-Glcp	→3)-Glcp-(1→	−	1.3	43,87,99,101,117,129,161,173,233
2,3,6-Me3-Glcp	→4)-Glcp-(1→	−	11.5	43,87,99,101,113,117,129,131,161,173,233
3,4–Me2-Manp	→2,6)-Manp-(1→	−	36.4	43,87,99,129,189

^aA relative percentage of all derivatives present was cakculated based on the peak area.

In summary, the XRD results showed that both FVRP and its ultrasonic degradation product FVRPV have an amorphous structure. Congo red staining results indicated that neither shows a triple helix structure, while the I₂-KI analysis revealed that both polysaccharides had longer side chains and more branching. Furthermore, the surface of FVRPV appeared to have more pores compared to FVRP under the action of ultrasonic cavitation. The methylation analysis results further indicated that ultrasonic treatment can break down the sugar chains of FVRP, leading to a significant change in the proportion of glycosidic bonds. These changes may be closely related to their physical and chemical properties and biological activity [42].

3.3. Antioxidant ability assay

The in vitro antioxidant abilities of FVRPV was determined by the methods of radical scavenging on ABST, DPPH, hydroxyl, and reducing power assays, which are common indicators to assess the activities of antioxidants [43]. Vc is an unquestioned strong antioxidant [44], and was applied as a positive control in the current study.

FVRPs possessed good antioxidant activity in a dose-dependent way (Fig. 2). The ABTS clearance rate of FVRP reached a maximum of 86.27 %, while FVRPV reached 96.05 %, which was close to 99.76 % of Vc (Fig. 2A), indicating that degradation can enhance the ABTS free radical scavenging activity of FVRP. Similarly, the DPPH scavenging rates of FVRP and FVRPV were 60.36 % and 66.96 % (Fig. 2B), respectively, indicating that ultrasonic assisted H₂O₂-Vc degradation of FVRP can improve the DPPH radical scavenging rate. Reducing power is one of the critical indicators directly reflecting antioxidant capacity [45]. The absorbance of FVRPs showed an upward trend in a concentration manner. At a concentration of 4.0 mg/mL, the reducing powers of FVRP and FVRPV were 0.83 and 1.1 (Fig. 2C), respectively. Therefore, our results indicated that the ultrasonic assisted H₂O₂-Vc condition improved the reducing power of polysaccharides, which was consistent with the research results of Zhang et al. that the antioxidant activity of Tremella fuciformis polysaccharides degraded by H₂O₂-Vc method was significantly improved [46]. The hydroxyl radicals scavenging ability is a crucial indicator for assessing the antioxidant properties of samples [8]. However, FVRPV exhibited significant higher hydroxyl radicals scavenging ability at lower concentration (Fig. 2D). The antioxidant activities of polysaccharides are affected by various factors, such as molecular weight, monosaccharide composition, solubility, and viscosity [47], [48], [49]. Polysaccharides with lower molecular weight have higher content of reducing hydroxyl groups and a larger surface area, making them easy to come into contact with free radicals [50]. Hence, the current study found that the ultrasonic assisted H₂O₂-Vc degradation method had a positive function on reducing the molecular weight and enhancing the antioxidant abilities of FVRP.

Fig. 2.

Antioxidant activity of FVRPV. ABTS radical scavenging activity (A), DPPH radical scavenging activity (B), reducing power (C), hydroxyl radical scavenging activity (D).

3.4. The fermentation characteristics of FVRPV

3.4.1. Changes in physicochemical properties and in vitro antioxidant abilities of fermentation broth

The efficacy of natural active polysaccharides is related to their ability to modulate gut microbiota. The relative abundance of targeted and related bacterial communities involved in polysaccharide metabolism is positively regulated, and the SCFAs produced can regulate the immune response of the body [51]. Therefore, in vitro fermentation experiments were conducted to explore the moderating effects of FVRPV on gut microbiota to reveal the underlying mechanisms of their biological activity. Firstly, the changes in polysaccharide, reducing sugar, and uronic acid content during in vitro fermentation were measured to evaluate the utilization of FVRPV by gut microbiota. As listed in Fig. 3A-C, the polysaccharide content of FVRPV groups significantly decreased after in vitro fermentation. The reducing sugar and uronic acids content were also decreased after long-time fermentation. It was worth noting that the reducing sugar content increased after 12 h of fermentation, and decreased again with fermentation continued. The above data indicated that gut microbiota first utilize FVRPV to produce reducing sugars, which were further exploited by microorganisms and decrease the reducing sugars content [52]. The uronic acid content in FVRPV groups at 48 h was 0.36 ± 0.003 mg/mL, with a decrease of 54.43 %, speculating that FVRPV were exploited by gut microbiota. Similar phenomena had also been observed in the in vitro fermentation assay of polysaccharides from Polygonatum Cyrtonema [53]. In summary, FVRPV can be utilized by gut microbiota to exert potential prebiotic activities. Herein, this study also further evaluated the in vitro antioxidant activity of the fermentation broth. Compared with the CK group, with the extension of fermentation time, the ABTS free radical scavenging rate, DPPH free radical scavenging rate, and reducing ability of FVRPV were significantly improved in a time-dependent way (Fig. 3D-F). With the progress of in vitro fermentation, the clearance rate of ABTS free radicals decreased, while the clearance rate and reducing ability of DPPH free radicals increased significantly. These differences in antioxidant activity may be due to differences in the chemical properties and mechanisms of action of polysaccharides during fermentation [54]. In summary, FVRPV may collaborate with probiotics to regulate the host's redox state, thereby enhancing the antioxidant potential of fermentation broth and possessing positive implications for intestinal and overall health.

Fig. 3.

Total polysaccharide content (A), reducing sugar content (B), uronic acid content (C), ABTS radical scavenging ability (D), DPPH radical scavenging ability (E), reduction ability (F), FT-IR spectrum (G), and SCFAs content (H) of FVRPV after in vitro fermentation. Note: Different lowercase letters indicate significant differences between groups (P < 0.05), while different uppercase letters indicate significant differences within groups (P < 0.05).

3.4.2. Changes in infrared spectral structures

After in vitro fermentation, FT-IR analysis was performed on FVRPV to determine whether in vitro fermentation would affect their structural characteristics. The main functional groups of FVRPV remained unchanged after fermentation for 6 h, 12 h, 24 h, and 48 h (Fig. 3G), which was consistent with the report that the structural characteristics of PPA detected by FT-IR remained unchanged after digestion [55]. These results indicated that FVRPV exhibited stability during the fermentation process, and in vitro fermentation did not alter their infrared spectral structures.

3.4.3. Changes in pH and SCFAs after in vitro fecal fermentation

The change in pH is an important indicator of in vitro fecal fermentation [56]. The pH value of the CK group, the FOS group, and the FVRPV group was all decreased with the progress of fermentation (Fig. S2A). The FOS group exhibited the most significant decrease in pH, followed by the FVRPV groups. The pH value of these tow groups after 48 h of fermentation was 4.06 ± 0.000 and 5.49 ± 0.004, respectively, all lower than the 6.06 ± 0.008 of the CK group. The decrease in pH can inhibit certain pathogenic microorganisms and enhance the absorption of beneficial trace elements in the intestine, including calcium and magnesium [57]. One of the most direct factors affecting the pH value of in vitro fermentation is the change in SCFAs content. SCFAs are the main metabolic products of carbohydrates fermented by gut microbiota, playing a crucial function in protecting host health [58]. The total SCFAs content showed a trend of FOS group > FVRPV group > CK group, which corresponds to the change in pH value of the fermentation broth. The FOS group mainly produced Acetic acid and Propionic acid, while the FVRPV groups mainly promote the accumulation of Acetic acid, Propionic acid, and butyric acid (Fig. 3H). Acetic acid is a crucial energy source for peripheral tissues and the brain, regulating insulin signaling and playing a essential role in fat production and cholesterol synthesis. Propionic acid can suppress cholesterol synthesis, strengthen insulin sensitivity, and play anti-immune suppression functions [59]. The increase in butyric acid may be caused by an increase in the proportion of Firmicutes [60]. In summary, FVRPV had a constructive function on the SCFAs production, possibly by acidifying the intestinal environment with Acetic acid, Propionic acid, and butyric acid, thereby enhancing intestinal immunity.

3.4.4. Influence of FVRPV on gut microbiota composition

Gut microbiota is closely related to human health, principally the immune system and energy metabolism [61]. Polysaccharides impact host activity by altering the composition and metabolic characteristics of gut microbiota [62]. The effect of FVRPV on gut microbiota diversity during in vitro fermentation investigated through high-throughput sequencing analysis of 16S rDNA to evaluate its prebiotic potential. The dilution curve is selected to determine whether the quantity of sequencing data is sufficient to represent the species diversity in the sample and indirectly mirror the abundance of species in the sample [63]. The dilution curves of each group tend to flatten out, suggesting that the sequencing depth was large enough for subsequencing analysis (Fig. S2B).

Alpha diversity was evaluated by Shannon, ACE, Simpson, Chao1, and Sob indices. The Shannon index of FVRPV group was dramatically higher than that of FOS group (Fig. 4A). The Simpson index of FVRPV group was considerably higher than the CK group and FOS group (Fig. 4B). The ACE index and Chao1 index showed that all three groups have good species richness (Fig. S2C-D). In summary, FVRPV had the ability to enrich the species diversity of gut microbiota. Beta diversity analysis can examine the similarity of different samples in species diversity [64]. PC1 and PC2 accounted for 63.83 % and 35.65 % of the overall analysis data, respectively, and there was good differentiation of gut microbiota between the groups (Fig. 4C). PCoA analysis and PLS-DA analysis suggested changes in the structure of gut microbiota among the groups (Fig. 4C-D). To investigate the regulatory function of FVRPV on specific gut microbiota, the relative abundance changes of gut microbiota in each group were analyzed. As shown in Fig. 5A, the total abundance of Proteobacteria in the FVRPV group decreased, while the total abundance of Firmicutes and Bacteroidetes increased at the phylum level in comparison with the CK group.

Fig. 4.

Alpha diversity analysis and Beta diversity analysis. Shannon index (A), simpson index (B), PCoA map (C), PLS-DA map (D).

Fig. 5.

The relative abundance of the gut microbiota at the phylum level (A), the genus level (B), functional predictions for gut microbiota (C).

Proteobacteria is the most diverse bacterial phylum, affecting the host health and resulting in unstable gut microbiota associated with low-grade inflammation, and even chronic colitis [65]. The increase in the ratio of Bacteroidetes to Firmicutes (B/F) is associated with resistance to obesity [66]. In this study, the B/F ratio was increased to 4.0323 in the FVRPV group, and that was 4.2423 in the CK group, implying that FVRPV may have anti-obesity effects. Firmicutes in the intestine can produce SCFAs by fermenting indigestible carbohydrates, thereby promoting the health of the host. FVRPV group possessed a considerable increase in the abundance of Firmicutes in comparison with the CK group, indicating that FVRPV may enhance the SCFAs generation by increasing the abundance of Firmicutes, thereby improving host health, which was consistent with the previous result that FVRPV increased the SCFAs production during in vitro fermentation (Fig. 3H).

At the genus level, the dominant microbiota in the FVRPV group were Bacteroides, Escherichia Shigella, Parabacterides, Phascolarctobacteria, and Fusobacterium (Fig. 5B). Compared with the CK group, FVRPV can dramatically upregulate Lactobacillus. Lactobacillus is the main microbiota and probiotic in a healthy intestinal [67], suggesting FVRPV can enrich probiotics in the intestine. Bacteroides, a genus of bacteria, prevents weight loss and colon problems in Crohn's disease models, reduces mouse obesity, and improves insulin sensitivity, which was dominated in the gut by breaking down polysaccharides and mucins [65], [68]. FVRPV also increased the abundance of Bacteroides, indicating that FVRPV may have the potential to have anti-obesity effects. Alcaligenes has been shown to cause peritonitis [69]. FVRPV can significantly reduce the abundance of Alcaligenes, suggesting that FVRPV may regulate gut microbiota by inhibiting the growth of Alcaligenes, regulating gut microbiota, and thus preventing the risk of peritonitis. In summary, FVRPV can regulate the gut microbiota composition by enriching beneficial bacteria, and inhibiting the growth of harmful bacteria.

Alcaligenes has been shown to cause peritonitis [69]. FVRPV can significantly reduce the abundance of Alcaligenes, suggesting that FVRPV may regulate gut microbiota by inhibiting the growth of Alcaligenes, regulating gut microbiota, and thus preventing the risk of peritonitis. In summary, FVRPV can regulate the gut microbiota composition, enrich beneficial bacteria, and inhibit the growth of harmful bacteria.

LEfSe analysis is a common method for studying the species characteristics that best explain inter group differences between two or more groups, likewise the extent to which these characteristics impact inter group differences. The LDA scores of 48 genera were above 4.0, with 16, 8, and 24 species in the CK group, FOS group, and FVRPV group, respectively (Fig. 6A-B). The differences between the groups were statistically significant. The dominant bacterial genera in the FVRPV group were Bacteroides and Parabacteroides. Bacteroides metabolize polysaccharides and oligosaccharides, providing nutrition and vitamins for hosts and other gut microbiota residents [70]. Parabacterioids are a Gram-negative anaerobic bacterium that has been shown to alleviate inflammatory arthritis [71]. Overall, FVRPV can change the microbiota composition of fecal, particularly enriching beneficial bacteria.

Fig. 6.

Histogram showing LDA-based distribution (A), and Cladogram (B).

KEGG is a comprehensive database that connects genomes, biological pathways, diseases, and drugs, typically selected to annotate the functions of gut microbiota [72]. Compariing with the CK group and FOS group, FVRPV significantly upregulated polysaccharide biosynthesis and metabolism, carbohydrate metabolism, cofactor and vitamin metabolism, terpenoid and polyketide metabolism, as well as the biosynthesis of other secondary metabolites (Fig. 5C), but significantly downregulated 16 pathways, comprising of replication and repair, translation, membrane transport, nucleotide metabolism, amino acid metabolism, transcription, other amino acid metabolism, folding, classification and degradation, cell growth and death, energy metabolism, transport and decomposition metabolism, lipid metabolism, exogenous biodegradation and metabolism, cell movement, and signal transduction. In summary, FVRPV can alter the metabolism pathway of gut microbiota, and changes in these pathways may be a key mechanism for its prebiotic activity.

Oxidative stress can affect the balance of gut microbiota [73]. Gut microbiota has an impact on oxidative stress by means of metabolite synthesis, regulation of antioxidant enzymes, and maintenance of gut homeostasis [74]. Oxidative stress has the potential to influence the gut microbiota by promoting dysbiosis [75], promotes the growth of pathogenic and pro-inflammatory bacteria and reduces the abundance of probiotics [76]. Hence, metabolitemetabolitefermention broth containing FVRPV possessed the higher antioxidant activities. Furthermore, FVRPV changed gut microbiota composition, enriching Bacteroides and Parabacteroides, which may be related with the metabolites produced by microbiota, such as polysaccharides degraded by bacteria and SCFAs. P. goldsteinii, a commensal bacterium whose levels were reduced in mice fed with a high-fat diet [77], was increase by polysaccharides isolated from Hirsutella sinensis [78]. Hirsutella sinensis polysaccharides and Parabacteroides goldsteinii reduce lupus severity in imiquimod-treated mice [79]. PEP-1A from culture broth of Parabacteroides distasonis (P. distasonis) desired immunomodulatory activity [80]. SCSPsj can be fermented by P. distasonis to yield many microbial metabolites, such as organic acids and derivatives, lipids and lipid-like molecules, organoheterocyclic compounds [81]. Hence, FVRPV can promote the stability of gut microbiota through enhancing the antioxidant effects and reduce the adverse effects of oxidative stress on gut microbiota.

However, a novel degraded polysaccharide FVRPV by ultrasonic assisted H₂O₂-Vc technique was only determined by preliminary structural analysis and in vitro fecal fermentation analysis. In the future, FVRPV will be purified by DEAE-52 column and Sephadex G-100 column. The comprehensive structural analysis of purified FVRPV will be conducted by nuclear magnetic resonance. Furthermore, more systematic investigations into the in vivo biological activity of purified FVRPV will also be further discussed with the animal models to clarify its underlying mechanism, especially from the respect of gut microbiota by multiple techniques, such as fecal microbiota transplantation and multi-omics integration analysis (metabolome, microbiome and proteome).

4. Conclusion

A novel degraded polysaccharide FVRPV with ultrasonic assisted H₂O₂-Vc approach was obtained in the current study, and its physicochemical properties, in vitro antioxidant capacity, and in vitro fermentation characteristics was evaluated. The molecular weight and average particle size of FVRPV were significantly declined, but the infrared structural characteristics and monosaccharide composition remained unchanged after ultrasonic assisted H₂O₂-Vc treatment. The results from XRD, Congo red tests and I₂-KI indicated that the crystal structures of FVRP and FVRPV were amorphous, lacking a triple helix configuration, and possessed longer side chains and increased branching. Furthermore, SEM images demonstrated an increase in both the number and size of pores on the surface of FVRPV than FVRP. The methylation results indicated that the glycosidic bond composition of FVRPV had experienced significant alterations compared to FVRP, contributing to a reduction in molecular weight. At the same time, the antioxidant activities of FVRPV were significantly enhanced. In addition, in vitro fecal fermentation data suggested that FVRPV can be exploited by gut microbiota, resulting in a significant reduction in polysaccharide, reducing sugar, and uronic acid content in the fermentation broth, as well as an increase in antioxidant activities. FVRPV can enrich beneficial bacteria, depress the growth of harmful bacteria, affect different metabolic pathways. However, FVRPV exhibited different fermentation characteristics in terms of physicochemical properties, antioxidant activity, and regulation of gut bacterial abundance. Therefore, FVRPV may be developed and utilized as a new antioxidant and prebiotic. However, the utilization of FVRPV in the human gut and its impact on the gut microbiota still require systematic research.

CRediT authorship contribution statement

Yunxiang Que: Writing – original draft, Visualization, Formal analysis, Data curation, Conceptualization. Yao Zhang: Writing – review & editing, Validation. Fengxiang Liang: Validation, Supervision. Liping Wang: Writing – review & editing. Yiting Yang: Software. Jingbo Zhang: Investigation. Wanting Wang: Validation, Supervision. Ying Sun: Writing – review & editing, Funding acquisition. Changjiao Zhong: Writing – review & editing. Haipeng Zhang: Supervision, Funding acquisition. Chengguang He: Writing – review & editing. Lili Guan: Writing – review & editing, Validation, Supervision, Resources. Hongxia Ma: Writing – review & editing, Validation, Supervision, Resources.

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.

Appendix A. Supplementary data

The following are the Supplementary data to this article: