Separation and Structural Characterization of a Novel Exopolysaccharide from Rhizopus nigricans

The present study aims to analyze the structural characterization and antioxidant activity of a novel exopolysaccharide from Rhizopus nigricans (EPS2-1). For this purpose, EPS2-1 was purified through DEAE-52, Sephadex G-100, and Sephadex G-75 chromatography. The structural characterization of EPS2-1 was analyzed using high-performance gel permeation chromatography (HPGPC), Fourier transform infrared spectroscopy (FT-IR), methylation analysis, nuclear magnetic resonance (NMR) spectra, transmission electron microscope (TEM), and atomic force microscope (AFM). The results revealed that EPS2-1 is composed of mannose (Man), galactose (Gal), glucose (Glc), arabinose (Ara), and Fucose (Fuc), and possesses a molecular weight of 32.803 kDa. The backbone of EPS2-1 comprised →2)-α-D-Manp-(1→ and →3)-β-D-Galp-(1→, linked with the O-6 position of (→2,6)-α-D-Manp-(1→) of the main chain is branch α-D-Manp-(1→6)-α-D-Manp-(1→, linked with the O-6 positions of (→3)-β-D-Galp-(1→) of the main chain are branches →4)-β-D-Glcp-(1→ and →3)-β-D-Galp-(1→, respectively. Finally, we demonstrated that EPS2-1 also shows free radical scavenging activity and iron ion reducing ability. At the same time, EPS2-1 could inhibit the proliferation of MFC cells and increase the cell viability of RAW264.7 cells. Our results suggested that EPS2-1 is a novel polysaccharide, and EPS2-1 has antioxidant activity. In addition, EPS2-1 may possess potential immunomodulatory and antitumor activities. This study promoted the application of EPS2-1 as the functional ingredients in the pharmaceutical and food industries.


Introduction
A polysaccharide is a type of natural polymer of more than 10 monosaccharides connected through glycosidic linkages; it is a class of biopolymers with diverse structures [1]. Natural polysaccharides have been used in the food, cosmetics, and the pharmaceutical industry for many years due to their excellent biological activity. Several natural polysaccharides have been reported to have anti-diabetic, antiviral, anti-aging, and immunomodulatory activities [2], which exist widely in plants, animals, and microorganisms [3].
Previously, scientists have successfully isolated numerous polysaccharides with excellent biological activity from fungi [4]. Some fungal polysaccharides demonstrate antitumor activities. For example, a polysaccharide from mycelia of Phellinus linteus could inhibit the proliferation of S-180 sarcoma cells, increase the expression level of Bax and Cytochorme c, and induce apoptosis in cells [5]. Meanwhile, a polysaccharide from Sanghuangporns vaninii could inhibit cell viability and cell colony formation of NCI-H460 cells, which induced apoptosis of lung cancer cells [6]. At the same time, many fungal polysaccharides possess immunostimulatory activity. The polysaccharide from Ganoderma promoted T lymphocyte proliferation and increased the content of IL-2 and IFN-γ of cells, and this effect was realized

Molecular Weight and Monosaccharide Composition
The HPGPC profile of EPS2-1 showed only one narrow symmetrical peak, indicating that EPS2-1 is a homogeneous polysaccharide. The weight-average molecular weight (Mw) of EPS2-1 was estimated to be 32.803 kDa, and the polydispersity ratio was 1.345 ( Figure 3A). The monosaccharide composition of EPS2-1 was analyzed using high-performance ion exchange chromatography (HPIC). EPS2-1 was composed of Man, Gal, Glc, Ara, and Fuc with a ratio of 0.519:0.204:0.065:0.031 and 0.029, respectively, which indicated that Man and Gal are the major monosaccharide components (Table 1).

Molecular Weight and Monosaccharide Composition
The HPGPC profile of EPS2-1 showed only one narrow symmetrical peak, indicating that EPS2-1 is a homogeneous polysaccharide. The weight-average molecular weight (Mw) of EPS2-1 was estimated to be 32.803 kDa, and the polydispersity ratio was 1.345 ( Figure 3A). The monosaccharide composition of EPS2-1 was analyzed using high-performance ion exchange chromatography (HPIC). EPS2-1 was composed of Man, Gal, Glc, Ara, and Fuc with a ratio of 0.519:0.204:0.065:0.031 and 0.029, respectively, which indicated that Man and Gal are the major monosaccharide components (Table 1).

Molecular Weight and Monosaccharide Composition
The HPGPC profile of EPS2-1 showed only one narrow symmetrical peak, indicating that EPS2-1 is a homogeneous polysaccharide. The weight-average molecular weight (Mw) of EPS2-1 was estimated to be 32.803 kDa, and the polydispersity ratio was 1.345 ( Figure 3A). The monosaccharide composition of EPS2-1 was analyzed using high-performance ion exchange chromatography (HPIC). EPS2-1 was composed of Man, Gal, Glc, Ara, and Fuc with a ratio of 0.519:0.204:0.065:0.031 and 0.029, respectively, which indicated that Man and Gal are the major monosaccharide components (Table 1).

Molecular Weight and Monosaccharide Composition
The HPGPC profile of EPS2-1 showed only one narrow symmetrical peak, indicating that EPS2-1 is a homogeneous polysaccharide. The weight-average molecular weight (Mw) of EPS2-1 was estimated to be 32.803 kDa, and the polydispersity ratio was 1.345 ( Figure 3A). The monosaccharide composition of EPS2-1 was analyzed using high-performance ion exchange chromatography (HPIC). EPS2-1 was composed of Man, Gal, Glc, Ara, and Fuc with a ratio of 0.519:0.204:0.065:0.031 and 0.029, respectively, which indicated that Man and Gal are the major monosaccharide components (Table 1).   Man and Gal are major monosaccharide components in many fungal polysaccharides, such as the polysaccharide from Cordyceps militaris and Sanghuangporus vaninii [6]. According to previous studies, EPS1-1 was composed of Glc, Man, Gal, and Fru with a Mw of 9.7 kDa. The molecular weight and monosaccharide composition of EPS2-1 were different from EPS1-1, which indicated that EPS2-1 is a novel polysaccharide. The differences might be due to the different concentrations of NaCl in anion-exchange chromatography.

FT-IR and UV Spectrum
A broad and strong absorption at 3383.54 cm −1 was attributed to the O-H stretching vibration, and the absorption at 2935.44 cm −1 was due to the C-H stretching vibration. The band at 1051.54 cm −1 resulted from the absorption of pyranoside, and the peak at 810.08 cm −1 suggested the existence of the Mannose ( Figure 3C) [18]. Moreover, the UV spectra suggested that there was no absorption at 260 and 280 nm, indicating the absence of protein and nucleic acid in EPS2-1 ( Figure 3B).

Methylation Analysis
Methylation analysis was used to determine the type of linkage of monosaccharide residues. After the methylated product was hydrolyzed, reduced, and acetylated, the composition was analyzed by GC-MS [19]. The methylation analysis results are summarized in Table 2 (Table 2).

Molecular Morphology
Molecular morphology is used to demonstrate the structure of macromolecules intuitively. A main linear chain with side chains was enriched in EPS2-1, which was in agreement with the results of GC-MS and NMR analysis. Thus, EPS2-1 was a branched polysaccharide [26]. The linear chains of EPS2-1 were entangled together with each other, which made EPS2-1 look like a bow tie ( Figure 6A) [27]. It has been reported that the entangled chains were beneficial for improving the biological activities of glycans [28].
Atomic force microscopy (AFM) is an effective method to observe conformation and provide three-dimensional structure information of macromolecules [29]. AFM revealed that EPS2-1 exhibits chain conformation of regular line clusters ( Figure 6B). The height of single-chain polysaccharides was usually between 0.1 and 1.0 nm, and the height of EPS2-1 ranged from −3.0 nm to 3.0 nm. AFM indicated that EPS2-1 has branches and may interweave with each other due to van der Waals and other forces between molecular chains [30].   HMBC and NOESY spectroscopy were used to analyze various sugar residues' connection sites and sequence [3]. In the HMBC spectrum ( Figure 5C), the anomeric proton of residue B had a cross-peak signal with its C-2, which corresponded to →2)-α-D-Manp-(1→2)-α-D-Manp-(1→. As shown in the NOESY spectrum ( Figure 5D Figure 5E).

Molecular Morphology
Molecular morphology is used to demonstrate the structure of macromolecules intuitively. A main linear chain with side chains was enriched in EPS2-1, which was in agreement with the results of GC-MS and NMR analysis. Thus, EPS2-1 was a branched polysaccharide [26]. The linear chains of EPS2-1 were entangled together with each other, which made EPS2-1 look like a bow tie ( Figure 6A) [27]. It has been reported that the entangled chains were beneficial for improving the biological activities of glycans [28].
Atomic force microscopy (AFM) is an effective method to observe conformation and provide three-dimensional structure information of macromolecules [29]. AFM revealed that EPS2-1 exhibits chain conformation of regular line clusters ( Figure 6B). The height of single-chain polysaccharides was usually between 0.1 and 1.0 nm, and the height of EPS2-1 ranged from −3.0 nm to 3.0 nm. AFM indicated that EPS2-1 has branches and may interweave with each other due to van der Waals and other forces between molecular chains [30].  Atomic force microscopy (AFM) is an effective method to observe conformation and provide three-dimensional structure information of macromolecules [29]. AFM revealed that EPS2-1 exhibits chain conformation of regular line clusters ( Figure 6B). The height of single-chain polysaccharides was usually between 0.1 and 1.0 nm, and the height of EPS2-1 ranged from −3.0 nm to 3.0 nm. AFM indicated that EPS2-1 has branches and may interweave with each other due to van der Waals and other forces between molecular chains [30].

In Vitro Antioxidant Activities of EPS2-1
Free radicals can trigger the progression of senescence-related diseases, such as Alzheimer's disease, diabetes, and cancer. Free radicals can accept electrons or hydrogen atoms from antioxidants to get a stable status [31]. Therefore, antioxidants are widely used in the pharmaceutical and food industries. Hence, DDPH, ABTS, hydroxyl radicals, and FRAP assays were used to evaluate the antioxidant activity of EPS2-1 in this study [32].
The scavenging effects of EPS2-1 on free radicals are illustrated in Figure 7A. The DPPH-scavenging rate of EPS2-1 in this study was as high as 20.55% at the concentration of 5 mg/mL. EPS2-1 scavenged ABTS in a dose-dependent manner, at the concentration of 5 mg/mL, the scavenging rate reached 12.09%. The scavenging rate of the hydroxyl radical by EPS2-1 at 5 mg/mL was 13.76%. Antioxidants can reduce Fe 3+ to Fe 2+ , hence, FRAP assay was used to detect the reducing power of antioxidants. The reducing power of EPS2-1 was shown in Figure 7B and indicated by the FRAP value. With the increase of EPS2-1 concentration, the FRAP value was increased in a concentration-dependent manner. The FRAP value of EPS2-1 ranged from 0.051 mmol to 0.610 mmol, the FRAP value at 4 mg/mL and 5 mg/mL were significantly higher than the control group.

Effect of EPS2-1 in the Proliferation of Tumor and RAW264.7 Cells
To explore the potential immunomodulatory and antitumor activities of EPS2-1, tumor and RAW264.7 cells were treated with EPS2-1 for 24 h. Then, the CCK-8 assay was used to detect the effect of EPS2-1 on cell viability. As shown in Figure 8A, EPS2-1 significantly decreased the cell viability of MFC cells, where the viability of MFC cells was 89.68%, 74.99%, 36.09%, and 40.60% at concentrations ranging from 0.1 to 0.8 mg/mL. At the same time, EPS2-1 and EPS1-1 could not affect the cell viability of human lung cancer cells SPCA1 and H1299 (p > 0.05) ( Figure 8C,D). Furthermore, EPS2-1 and EPS1-1 (0.1 to 0.8 mg/mL) could increase the cell viability of RAW264.7 cells ( Figure 8B). Compared with the control group, EPS2-1 significantly increased the cell viability of RAW264.7 cells by up to 135.04% at 0.2 mg/mL. FRAP assays were used to evaluate the antioxidant activity of EPS2-1 in this study [32].
The scavenging effects of EPS2-1 on free radicals are illustrated in Figure 7A. The DPPH-scavenging rate of EPS2-1 in this study was as high as 20.55% at the concentration of 5 mg/mL. EPS2-1 scavenged ABTS in a dose-dependent manner, at the concentration of 5 mg/mL, the scavenging rate reached 12.09%. The scavenging rate of the hydroxyl radical by EPS2-1 at 5 mg/mL was 13.76%. Antioxidants can reduce Fe 3+ to Fe 2+ , hence, FRAP assay was used to detect the reducing power of antioxidants. The reducing power of EPS2-1 was shown in Figure 7B and indicated by the FRAP value. With the increase of EPS2-1 concentration, the FRAP value was increased in a concentration-dependent manner. The FRAP value of EPS2-1 ranged from 0.051 mmol to 0.610 mmol, the FRAP value at 4 mg/mL and 5 mg/mL were significantly higher than the control group.

Effect of EPS2-1 in the Proliferation of Tumor and RAW264.7 Cells
To explore the potential immunomodulatory and antitumor activities of EPS2-1, tumor and RAW264.7 cells were treated with EPS2-1 for 24 h. Then, the CCK-8 assay was used to detect the effect of EPS2-1 on cell viability. As shown in Figure 8A, EPS2-1 significantly decreased the cell viability of MFC cells, where the viability of MFC cells was 89.68%, 74.99%, 36.09%, and 40.60% at concentrations ranging from 0.1 to 0.8 mg/mL. At the same time, EPS2-1 and EPS1-1 could not affect the cell viability of human lung cancer cells SPCA1 and H1299 (p > 0.05) ( Figure 8C,D). Furthermore, EPS2-1 and EPS1-1 (0.1 to 0.8 mg/mL) could increase the cell viability of RAW264.7 cells ( Figure 8B). Compared with the control group, EPS2-1 significantly increased the cell viability of RAW264.7 cells by up to 135.04% at 0.2 mg/mL.

Isolation and Purification of Exopolysaccharides from Rhizopus Nigricans
The crude exopolysaccharides were extracted from R. nigricans according to previously reported methods [33]. Briefly, R. nigricans was cultured in Potato Dextrose Broth (PDB) for 7 d at 28 °C, then the fermentation liquor of R. nigricans was precipitated with four volumes of ethanol for 48 h. The crude polysaccharide was obtained after deproteinization, decoloration, dialysis, and lyophilization. The lyophilized polysaccharide powder was dissolved in 0.02 M Tris-HCl (pH 7.4), which was purified by column chromatography of DEAE-52 Sepharose, followed by Tris-HCl buffer and a stepwise elution using NaCl (0 and 0.3 M) at a flow rate of 1 mL/min [34]. Two buffer-eluted fractions were col-

Isolation and Purification of Exopolysaccharides from Rhizopus Nigricans
The crude exopolysaccharides were extracted from R. nigricans according to previously reported methods [33]. Briefly, R. nigricans was cultured in Potato Dextrose Broth (PDB) for 7 d at 28 • C, then the fermentation liquor of R. nigricans was precipitated with four volumes of ethanol for 48 h. The crude polysaccharide was obtained after deproteinization, decoloration, dialysis, and lyophilization. The lyophilized polysaccharide powder was dissolved in 0.02 M Tris-HCl (pH 7.4), which was purified by column chromatography of DEAE-52 Sepharose, followed by Tris-HCl buffer and a stepwise elution using NaCl (0 and 0.3 M) at a flow rate of 1 mL/min [34]. Two buffer-eluted fractions were collected, and then purified through Sephadex G-100 and Sephadex G-75 columns with ultra-pure water. The major peaks were collected and lyophilized, named EPS1-1 and EPS2-1, respectively [35]. Finally, total carbohydrate content was measured by the phenol-sulfuric acid method [36,37].

Monosaccharide Composition Analysis
High-performance ion exchange chromatography (HPIC) was used to analyze the monosaccharide composition of EPS2-1, the experimental methods referred to the previously reported methods [39]. Briefly, EPS2-1 was hydrolyzed using 10 mL TFA (3 M). After dissolving in methanol to remove residual TFA, the dried hydrolysates were dissolved in distilled water and injected into HPIC. The HPIC system was equipped with Dionex Carbopac PA20 column (ICS5000, 150 mm × 3.0 mm × 10 µm, Thermo Fisher Scientific, Co., Ltd., MA, USA), with an injection volume of 5 µL.

FT-IR and UV Spectroscopy
The FT-IR spectra of EPS2-1 (KBr pellets) was obtained using a Nicolet 10 spectrometer (Thermo Fisher Scientific, Co., Ltd., MA, USA) in the frequency range of 4000-500 cm −1 . A 5 mg sample of EPS2-1 was dissolved in 5 mL of distilled water, and then the sample solution was filtered through a 0.22 µm filtration membrane. Then, the UV spectroscopy of EPS2-1 was analyzed using an Ultrospec 7000 spectrometer (GE, MA, USA) [40].

Methylation and GC-MS Analysis
The methylation and GC-MS analysis were performed according to the previously reported method [21]. Briefly, EPS2-1 was dissolved in 1 mL of DMSO, and 20 mg sodium hydroxide was added under nitrogen protection. Then 3.6 mL of methyl iodide was added dropwise, and the reaction was quenched with 2 mL distilled water. The methylated product was hydrolyzed with 1 mL of trifluoroacetate (2 M, 121 • C, 90 min). Next, the hydrolysis sample was reduced with 1 M NaBH 4 . Acetic acid was used to stop the reaction, then 0.25 mL of acetic anhydride was added. Finally, the acetylation sample was dissolved in 3 mL of chloroform, and the dichloromethane phase was harvested. The obtained product was detected using the GC-MS-QP 2010 system (Shimadzu, Kyoto, Japan), which was equipped with a RXI-5 SIL MS column.

NMR Analysis
A sample of 50 mg of EPS2-1 was dissolved in 0.5 mL of D 2 O for repeated freeze-drying, and the treated samples were dissolved in 0.5 mL of D 2 O [41]. The 1D NMR ( 1 H NMR and 13 C NMR) and 2D NMR of EPS2-1 were analyzed using the Bruker Avance 600 MHz NMR instrument. The 2D NMR included HSQC spectroscopy, HMBC spectroscopy, COSY spectroscopy, and NOESY spectroscopy.

Morphological Analysis
For transmission electron microscopy (TEM), 1 mL of EPS2-1 (1 µg/mL) was filtered through the 0.22 µm filtration membrane. Next, 10 µL of the solution was transferred to a 400-mesh copper grid (carbon-coated) and dried naturally [42]. Then, the sample was observed in JEM 2100F (JOEL, Tokyo, Japan). For the atomic force microscope (AFM) analysis, 10 µL of EPS2-1 (1 µg/mL) was added to the surface of mica flakes, then the sample was analyzed through an AFM (Bruker, Karlsruhe, Germany).

Antioxidant Activity In Vitro
The DPPH radical assay, ABTS radical assay, hydroxyl radical assay, and the ferric reducing antioxidant power (FRAP) assay were measured according to the previously described methods [43,44]. For the DPPH radical scavenging assay, 0.25 mL of the sample and 1.25 mL of DPPH-ethanol solution (0.3 mM) were incubated in the dark at 28 • C for 30 min. Then, the absorbance was measured at 515 nm [45]. For FRAP assay, the absorbance of the reaction solution at 593 nm represented the FRAP of samples [46].

Cell Viability Assay
Cells were seeded in 96-well plates (6 × 10 3 cells/well) for 24 h. After being treated with polysaccharides for 24 h, the CCK-8 reagent was added. The absorbance was detected at 450 nm using Microplate Reader (BioTek, Winooski, VT, USA).

Statistical Analysis
The difference between groups was evaluated by t-test and one-way ANOVA through GraphPad Prism 8.0. p values < 0.05 were considered statistically significant. All results were expressed as mean ± SD.