Separation, Purification, Structural Characterization, and Anticancer Activity of a Novel Exopolysaccharide from Mucor sp.

Mucor sp. has a wide range of applications in the food fermentation industry. In this study, a novel exopolysaccharide, labeled MSEPS, was separated from Mucor sp. fermentation broth through ethanol precipitation and was purified by ion-exchange chromatography, as well as gel filtration column chromatography. MSEPS was composed mostly of mannose, galactose, fucose, arabinose, and glucose with a molar ratio of 0.466:0.169:0.139:0.126:0.015 and had a molecular weight of 7.78 × 104 Da. The analysis of methylation and nuclear magnetic resonance results indicated that MSEPS mainly consisted of a backbone of →3,6)-α-d-Manp-(1→3,6)-β-d-Galp-(1→, with substitution at O-3 of →6)-α-d-Manp-(1→ and →6)-β-d-Galp-(1→ by terminal α-l-Araf residues. MTT assays showed that MSEPS was nontoxic in normal cells (HK-2 cells) and inhibited the proliferation of carcinoma cells (SGC-7901 cells). Additionally, morphological analysis and flow cytometry experiments indicated that MSEPS promoted SGC-7901 cell death via apoptosis. Therefore, MSEPS from Mucor sp. can be developed as a potential antitumor agent.


Introduction
Mucor sp., a zygomycete filamentous fungus often found in natural environments such as soils, air, fruits, and vegetables, reproduces rapidly and its hyphae develop densely [1]. Some thermotolerant species (such as M. indicus and M. ramosissimus) are obligate pathogens that affect animal and human health [2,3]. However, proteases, amylases, and lipases, produced by several Mucor species, including M. circinelloides, M. flavus, M. hiemalis, M. mucedo, and M. racemosus, hydrolyze soy components, such as proteins, carbohydrates, and fats, in the process of making classic Asian and African fermented cuisines, such as sufu, ragi, tempeh, furu, and mureha [4][5][6]. Therefore, Mucor sp. plays an essential role in the food fermentation industry. Mucor sp. has also been used in biotechnological processes to produce enzymes, particularly for the biotransformation of diverse substances, such as flavonoids, coumarins, alkaloids, and aromatic compounds, and to identify new active molecules in the pharmaceutical industry or to modify some active components for activity improvement [7,8]. Many researchers have investigated the metabolites of Mucor sp. as a microbial resource. Carvalho et al. reported that whole-cell lipases derived from Mucor circinelloides transesterify saturated short-chain fatty acids, such as lauric acid, offering a low-cost and effective alternative to lipases for industrial, biotechnological, and other applications [9]. The postharvest action of the pathogenic fungus Aspergillus flavus has as lauric acid, offering a low-cost and effective alternative to lipases for industrial, biotechnological, and other applications [9]. The postharvest action of the pathogenic fungus Aspergillus flavus has been inhibited using chitosan, derived from M. circinelloides [10]. Huang et al. reported that the endophytic M. fragilis strain directly produces high yields of two pharmaceutically relevant bioactive chemicals utilized as anticancer and antiviral agents, podophyllotoxin and kaempferol [11].
Fungal polysaccharides, including exopolysaccharides (EPS) and endopolysaccharides, isolated from fruiting bodies, cultured mycelia, and cultured broth, exhibit a variety of biological properties, including antioxidant, antiviral, antitumor, and immunomodulatory activities [12][13][14]. An extracellular polysaccharide of Rhizopus nigricans inhibits the proliferation of colon cancer cells by relieving immunological inflammation in mice [15]. Bacillus subtilis polysaccharides inhibit A549 cell growth and promote cell apoptosis by activating the caspase-3 pathway [16], and the Pantoea alhagi NX-11 EPS exhibits moderate antioxidant capacity [17]. These studies indicate the immense potential of fungal polysaccharides. Additionally, numerous studies have shown that the diversity in the relative molecular weight, monosaccharide composition and content, glycosidic bond type, and sugar-ring configuration in fungal polysaccharides contributes to their structural diversity and functional novelty [18][19][20][21][22]. To date, only a few studies on the isolation, purification, structural characterization, and activity of Mucor fungus polysaccharides have been reported. In this study, an exopolysaccharide was isolated from Mucor sp. (No. CICC 3039) fermentation broth, purified, and characterized. Furthermore, its antitumor activity was studied in vitro. This study can provide a better understanding of the polysaccharides from Mucor sp., facilitating their medical applications to improve human health.

Isolation and Purification of EPS
Crude EPS was obtained from Mucor sp. with a yield of 314 mg/L through ethanol precipitation, decolorization, deproteinization, and dialysis ( Figure 1). It was first fractionated using a DEAE-52 cellulose column, and two predominant fractions were gathered ( Figure 2A). A Sephadex G-100 column was used to purify the fraction eluted with a 0.2 M NaCl solution ( Figure 2B). The main fractions collected were labeled MSEPS, according to the elution profile.

Homogeneity and Molecular Weight
The polysaccharide purity is reflected by the HPGPC spectrum. MSEPS is a homogeneous polysaccharide, as indicated by the single symmetric peak in its HPGPC spectrum ( Figure 3A). The weight-average molecular weight (Mw) of MSEPS was calculated from the standard curve to be approximately 7.78 × 10 4 Da, its number-average molecular weight (Mn) was 5.24 × 10 4 Da, and its Mw/Mn value was 1.48.

FT-IR Spectrum
The infrared spectrum of MSEPS was used to infer the characteristic functional groups in the polysaccharide ( Figure 3B). The broad and strong absorption peak at 3400 cm −1 indicated the presence of the O-H stretching vibration, and the absorption peak at 2940 cm −1 represented the C-H stretching vibration [23]. Additionally, the absorption peak at 1650 cm −1 was due to bound water [24], and the peak at 1420 cm −1 was attributed to the variable angle vibration of C-H [25]. The absorption peak at 1050 cm −1 was caused by the stretching vibration of C-O [26].

Monosaccharide Composition
To identify the monosaccharide components in MSEPS, different monosaccharide standards were run on the IC system, and their retention time were recorded. According to IC analysis, MSEPS was mainly composed of mannose (Man), galactose (Gal), fucose (Fuc), arabinose (Ara), and glucose (Glc) in a molar ratio of 0.466:0.169:0.139:0.126:0.015, with trace amounts of glucuronic acid and galacturonic acid ( Table 1). The monosaccharides Man, Gal, and Ara are frequently found in other fungal polysaccharides [27,28]. Previous studies have shown that the types and proportions of monosaccharides depend on culture conditions, methods of isolation and purification, and the polysaccharide source

Homogeneity and Molecular Weight
The polysaccharide purity is reflected by the HPGPC spectrum. MSEPS is a homogeneous polysaccharide, as indicated by the single symmetric peak in its HPGPC spectrum ( Figure 3A). The weight-average molecular weight (M w ) of MSEPS was calculated from the standard curve to be approximately 7.78 × 10 4 Da, its number-average molecular weight (M n ) was 5.24 × 10 4 Da, and its M w /M n value was 1.48.

Homogeneity and Molecular Weight
The polysaccharide purity is reflected by the HPGPC spectrum. MSEPS is a homogeneous polysaccharide, as indicated by the single symmetric peak in its HPGPC spectrum ( Figure 3A). The weight-average molecular weight (Mw) of MSEPS was calculated from the standard curve to be approximately 7.78 × 10 4 Da, its number-average molecular weight (Mn) was 5.24 × 10 4 Da, and its Mw/Mn value was 1.48.

FT-IR Spectrum
The infrared spectrum of MSEPS was used to infer the characteristic functional groups in the polysaccharide ( Figure 3B). The broad and strong absorption peak at 3400 cm −1 indicated the presence of the O-H stretching vibration, and the absorption peak at 2940 cm −1 represented the C-H stretching vibration [23]. Additionally, the absorption peak at 1650 cm −1 was due to bound water [24], and the peak at 1420 cm −1 was attributed to the variable angle vibration of C-H [25]. The absorption peak at 1050 cm −1 was caused by the stretching vibration of C-O [26].

Monosaccharide Composition
To identify the monosaccharide components in MSEPS, different monosaccharide standards were run on the IC system, and their retention time were recorded. According to IC analysis, MSEPS was mainly composed of mannose (Man), galactose (Gal), fucose (Fuc), arabinose (Ara), and glucose (Glc) in a molar ratio of 0.466:0.169:0.139:0.126:0.015, with trace amounts of glucuronic acid and galacturonic acid ( Table 1). The monosaccharides Man, Gal, and Ara are frequently found in other fungal polysaccharides [27,28]. Previous studies have shown that the types and proportions of monosaccharides depend on culture conditions, methods of isolation and purification, and the polysaccharide source [29,30].

FT-IR Spectrum
The infrared spectrum of MSEPS was used to infer the characteristic functional groups in the polysaccharide ( Figure 3B). The broad and strong absorption peak at 3400 cm −1 indicated the presence of the O-H stretching vibration, and the absorption peak at 2940 cm −1 represented the C-H stretching vibration [23]. Additionally, the absorption peak at 1650 cm −1 was due to bound water [24], and the peak at 1420 cm −1 was attributed to the variable angle vibration of C-H [25]. The absorption peak at 1050 cm −1 was caused by the stretching vibration of C-O [26].

Monosaccharide Composition
To identify the monosaccharide components in MSEPS, different monosaccharide standards were run on the IC system, and their retention time were recorded. According to IC analysis, MSEPS was mainly composed of mannose (Man), galactose (Gal), fucose (Fuc), arabinose (Ara), and glucose (Glc) in a molar ratio of 0.466:0.169:0.139:0.126:0.015, with trace amounts of glucuronic acid and galacturonic acid ( Table 1). The monosaccharides Man, Gal, and Ara are frequently found in other fungal polysaccharides [27,28]. Previous studies have shown that the types and proportions of monosaccharides depend on culture conditions, methods of isolation and purification, and the polysaccharide source [29,30].

Methylation Analysis
The location of linkages between monosaccharide residues in polysaccharides can be determined through methylation analysis. As summarized in Table 2

NMR Spectroscopy
The precise structural features of MSEPS were further elucidated by 1 H, 13 C, 1 H-1 H correlated spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear overhauser effect spectroscopy (NOESY) NMR spectroscopy. In the 1 H-NMR spectrum of MSEPS ( Figure 4A), the peaks at δ 5.21, 5.16, 5.01, 4.99, 4.87, 4.82, 4.57, 4.47, and 4.41 ppm correspond to anomeric protons, which were labeled residues A, B, C, D, E, F, G, H, and I, respectively. The peak at δ 1.12 ppm was assigned to the proton signal of the methyl group in fucose residues. Usually, signals located in the δ 4.8-5.5 ppm region are due to anomeric protons in α-configuration pyranose units. By contrast, the anomeric proton chemical shifts from δ 4.4-4.8 ppm are due to the β-configuration pyranose units [31]. Therefore, the polysaccharide MSEPS contains both αand β-pyranose structures. The other signals at δ 3.2-4.3 ppm were attributed to the sugar-ring protons. In the 13 C spectrum of MSEPS ( Figure 4B), the signals were mainly distributed in the δ 60-110 ppm range. Six anomeric carbon signal peaks were detected at δ 109.20, 104.35, 100.49, 99.32, 99.07, and 95.79 ppm. The chemical shifts of the nonsubstituted C-6 occurred at δ 60-64 ppm, which shifted to δ 65-70 ppm upon substitution at C-6. The signals for C-2, C-3, and C-6 on the sugar ring appeared at δ 70-77 ppm and were shifted to the δ 78-85 ppm range upon substitution [32,33]. The carbon signal at δ 15.37 ppm was attributed to the methyl group in fucose residues.
Additionally, the C-4 position of residue G was replaced, as indicated by movement of the chemical shift of C-4 toward a lower field, to δ 79.66 ppm. Therefore, residue G was confirmed to be →4)-β-D-Galp-(1→ [37]. The chemical shifts from C-1 to C-6 of residue H were acquired from the HSQC spectrum on the basis of the C-H pairs. Residue H was postulated to contain the β-D-Galp fragments because the chemical shift of the heterotopic carbon was δ 104.69 ppm. The downfield shifts of C-3 (δ 81.50 ppm) and C-6 (δ 70.76 ppm) confirmed residue H to be →3,6)-β-D-Galp-(1→ [38]. Similarly, residue I was considered to be β-D-Galp-(1→. In residue B, the hydrogen signal at δ 1.14 ppm and the carbon signal at δ 15.31 ppm corresponded to the methyl group in fucose. Combining this information with results of the methylation analysis, residue B was confirmed to be α-L-Fucp-(1→ [39].
The chemical shifts of carbon and hydrogen for all residues are listed in Table 3. to be α-L-Araf-(1→, due to the chemical shifts of C-1 [35]. The chemical shifts of C-1 and C-3 in residue D were δ 95.78 and δ 80.50 ppm, respectively, which were shifted toward lower fields on substitution. Therefore, it could be inferred that residue D was →3)-α-D- .82 ppm originated from H-1/C-1, H-2/C-2, H-3/C-3, H-4/C-4, H-5/C-5, and H-6a/C-6 in residue F, respectively. The chemical shift of C-6 was shifted toward a lower field (δ 66.82 ppm) compared to that of the unsubstituted C-6 (in the δ 60-64 ppm range). Therefore, residue F was considered to be →6)-α-D-Manp-(1→ [36]. The chemical shift of the heterotopic carbon of residue G was δ 105.80 ppm. According to the literature, the chemical shift of the heterotopic carbon of residue β-D-Galp lies in the δ 103-106 ppm range.
Additionally, the C-4 position of residue G was replaced, as indicated by movement of the chemical shift of C-4 toward a lower field, to δ 79.66 ppm. Therefore, residue G was confirmed to be →4)-β-D-Galp-(1→ [37]. The chemical shifts from C-1 to C-6 of residue H were acquired from the HSQC spectrum on the basis of the C-H pairs. Residue H was postulated to contain the β-D-Galp fragments because the chemical shift of the heterotopic carbon was δ 104.69 ppm. The downfield shifts of C-3 (δ 81.50 ppm) and C-6 (δ 70.76 ppm) confirmed residue H to be →3,6)-β-D-Galp-(1→ [38]. Similarly, residue I was considered to be β-D-Galp-(1→. In residue B, the hydrogen signal at δ 1.14 ppm and the carbon signal at δ 15.31 ppm corresponded to the methyl group in fucose. Combining this information with results of the methylation analysis, residue B was confirmed to be α-L-Fucp-(1→ [39].
The chemical shifts of carbon and hydrogen for all residues are listed in Table 3. to be α-L-Araf-(1→, due to the chemical shifts of C-1 [35]. The chemical shifts of C-1 and C-3 in residue D were δ 95.78 and δ 80.50 ppm, respectively, which were shifted toward lower fields on substitution. Therefore, it could be inferred that residue D was →3)-α-D- .82 ppm originated from H-1/C-1, H-2/C-2, H-3/C-3, H-4/C-4, H-5/C-5, and H-6a/C-6 in residue F, respectively. The chemical shift of C-6 was shifted toward a lower field (δ 66.82 ppm) compared to that of the unsubstituted C-6 (in the δ 60-64 ppm range). Therefore, residue F was considered to be →6)-α-D-Manp-(1→ [36]. The chemical shift of the heterotopic carbon of residue G was δ 105.80 ppm. According to the literature, the chemical shift of the heterotopic carbon of residue β-D-Galp lies in the δ 103-106 ppm range.
Additionally, the C-4 position of residue G was replaced, as indicated by movement of the chemical shift of C-4 toward a lower field, to δ 79.66 ppm. Therefore, residue G was confirmed to be →4)-β-D-Galp-(1→ [37]. The chemical shifts from C-1 to C-6 of residue H were acquired from the HSQC spectrum on the basis of the C-H pairs. Residue H was postulated to contain the β-D-Galp fragments because the chemical shift of the heterotopic carbon was δ 104.69 ppm. The downfield shifts of C-3 (δ 81.50 ppm) and C-6 (δ 70.76 ppm) confirmed residue H to be →3,6)-β-D-Galp-(1→ [38]. Similarly, residue I was considered to be β-D-Galp-(1→. In residue B, the hydrogen signal at δ 1.14 ppm and the carbon signal at δ 15.31 ppm corresponded to the methyl group in fucose. Combining this information with results of the methylation analysis, residue B was confirmed to be α-L-Fucp-(1→ [39].
The chemical shifts of carbon and hydrogen for all residues are listed in Table 3. to be α-L-Araf-(1→, due to the chemical shifts of C-1 [35]. The chemical shifts of C-1 and C-3 in residue D were δ 95.78 and δ 80.50 ppm, respectively, which were shifted toward lower fields on substitution. Therefore, it could be inferred that residue D was →3)-α-D- , and H-6a/C-6 in residue F, respectively. The chemical shift of C-6 was shifted toward a lower field (δ 66.82 ppm) compared to that of the unsubstituted C-6 (in the δ 60-64 ppm range). Therefore, residue F was considered to be →6)-α-D-Manp-(1→ [36]. The chemical shift of the heterotopic carbon of residue G was δ 105.80 ppm. According to the literature, the chemical shift of the heterotopic carbon of residue β-D-Galp lies in the δ 103-106 ppm range.
Additionally, the C-4 position of residue G was replaced, as indicated by movement of the chemical shift of C-4 toward a lower field, to δ 79.66 ppm. Therefore, residue G was confirmed to be →4)-β-D-Galp-(1→ [37]. The chemical shifts from C-1 to C-6 of residue H were acquired from the HSQC spectrum on the basis of the C-H pairs. Residue H was postulated to contain the β-D-Galp fragments because the chemical shift of the heterotopic carbon was δ 104.69 ppm. The downfield shifts of C-3 (δ 81.50 ppm) and C-6 (δ 70.76 ppm) confirmed residue H to be →3,6)-β-D-Galp-(1→ [38]. Similarly, residue I was considered to be β-D-Galp-(1→. In residue B, the hydrogen signal at δ 1.14 ppm and the carbon signal at δ 15.31 ppm corresponded to the methyl group in fucose. Combining this information with results of the methylation analysis, residue B was confirmed to be α-L-Fucp-(1→ [39].
The chemical shifts of carbon and hydrogen for all residues are listed in Table 3. to be α-L-Araf-(1→, due to the chemical shifts of C-1 [35]. The chemical shifts of C-1 and C-3 in residue D were δ 95.78 and δ 80.50 ppm, respectively, which were shifted toward lower fields on substitution. Therefore, it could be inferred that residue D was →3)-α-D- , and H-6a/C-6 in residue F, respectively. The chemical shift of C-6 was shifted toward a lower field (δ 66.82 ppm) compared to that of the unsubstituted C-6 (in the δ 60-64 ppm range). Therefore, residue F was considered to be →6)-α-D-Manp-(1→ [36]. The chemical shift of the heterotopic carbon of residue G was δ 105.80 ppm. According to the literature, the chemical shift of the heterotopic carbon of residue β-D-Galp lies in the δ 103-106 ppm range.
Additionally, the C-4 position of residue G was replaced, as indicated by movement of the chemical shift of C-4 toward a lower field, to δ 79.66 ppm. Therefore, residue G was confirmed to be →4)-β-D-Galp-(1→ [37]. The chemical shifts from C-1 to C-6 of residue H were acquired from the HSQC spectrum on the basis of the C-H pairs. Residue H was postulated to contain the β-D-Galp fragments because the chemical shift of the heterotopic carbon was δ 104.69 ppm. The downfield shifts of C-3 (δ 81.50 ppm) and C-6 (δ 70.76 ppm) confirmed residue H to be →3,6)-β-D-Galp-(1→ [38]. Similarly, residue I was considered to be β-D-Galp-(1→. In residue B, the hydrogen signal at δ 1.14 ppm and the carbon signal at δ 15.31 ppm corresponded to the methyl group in fucose. Combining this information with results of the methylation analysis, residue B was confirmed to be α-L-Fucp-(1→ [39].
The chemical shifts of carbon and hydrogen for all residues are listed in Table 3.  The sequences of the residues in MSEPS were determined from HMBC and NOESY spectra. As shown in the HMBC spectrum ( Figure 5C), the anomeric proton of residue F had a strong cross-peak with its own C-6, indicating the presence of   The sequences of the residues in MSEPS were determined from HMBC and NOESY spectra. As shown in the HMBC spectrum ( Figure 5C), the anomeric proton of residue F had a strong cross-peak with its own C-6, indicating the presence of   The sequences of the residues in MSEPS were determined from HMBC and NOESY spectra. As shown in the HMBC spectrum ( Figure 5C), the anomeric proton of residue F had a strong cross-peak with its own C-6, indicating the presence of  The sequences of the residues in MSEPS were determined from HMBC and NOESY spectra. As shown in the HMBC spectrum ( Figure 5C), the anomeric proton of residue F had a strong cross-peak with its own C-6, indicating the presence of  The sequences of the residues in MSEPS were determined from HMBC and NOESY spectra. As shown in the HMBC spectrum ( Figure 5C

Effect of MSEPS on SGC-7901 Cell Inhibition
The MTT method was used to investigate the in vitro anticancer activity of MSEPS, with HK-2 cells as control. The inhibitory effect of MSEPS on SGC-7901 cell growth became more pronounced with increasing concentrations (Figure 7A). At the highest dose of 1.6 mg/mL, the inhibition rate of SGC-7901 cells for 12 h, 24 h, or 36 h was 42.47%, 47.21%, and 50.65%, respectively. Moreover, the same concentration of MSEPS increased the inhibition rate of SGC-7901 cells at longer treatment times. By contrast, the MSEPS treatment did not significantly affect the viability of HK-2 cells, even after treatment for 36 h ( Figure 7B). Additionally, the viabilities of the SGC-7901 and HK-2 cells were significantly different for treatments with MSEPS concentrations higher than 0.2 mg/mL. Therefore, MSEPS exhibited low toxicity against non-tumor cells, while inhibiting SGC-7901 cell proliferation in a concentration-and time-dependent manner.

Effect of MSEPS on SGC-7901 Cell Inhibition
The MTT method was used to investigate the in vitro anticancer activity of MSEPS, with HK-2 cells as control. The inhibitory effect of MSEPS on SGC-7901 cell growth became more pronounced with increasing concentrations (Figure 7A). At the highest dose of 1.6 mg/mL, the inhibition rate of SGC-7901 cells for 12 h, 24 h, or 36 h was 42.47%, 47.21%, and 50.65%, respectively. Moreover, the same concentration of MSEPS increased the inhibition rate of SGC-7901 cells at longer treatment times. By contrast, the MSEPS treatment did not significantly affect the viability of HK-2 cells, even after treatment for 36 h ( Figure 7B). Additionally, the viabilities of the SGC-7901 and HK-2 cells were significantly different for treatments with MSEPS concentrations higher than 0.2 mg/mL. Therefore, MSEPS exhibited low toxicity against non-tumor cells, while inhibiting SGC-7901 cell proliferation in a concentration-and time-dependent manner.

Effect of MSEPS on SGC-7901 Cell Inhibition
The MTT method was used to investigate the in vitro anticancer activity of MSEPS, with HK-2 cells as control. The inhibitory effect of MSEPS on SGC-7901 cell growth became more pronounced with increasing concentrations (Figure 7A). At the highest dose of 1.6 mg/mL, the inhibition rate of SGC-7901 cells for 12 h, 24 h, or 36 h was 42.47%, 47.21%, and 50.65%, respectively. Moreover, the same concentration of MSEPS increased the inhibition rate of SGC-7901 cells at longer treatment times. By contrast, the MSEPS treatment did not significantly affect the viability of HK-2 cells, even after treatment for 36 h ( Figure 7B). Additionally, the viabilities of the SGC-7901 and HK-2 cells were significantly different for treatments with MSEPS concentrations higher than 0.2 mg/mL. Therefore, MSEPS exhibited low toxicity against non-tumor cells, while inhibiting SGC-7901 cell proliferation in a concentration-and time-dependent manner.

Effect of MSEPS on Morphological Changes of SGC-7901 Cells
The morphological changes of SGC-7901 cells, after treatment with different concentrations of MSEPS for 24 h, were observed under an inverted microscope. As shown in Figure 8A, MSEPS-treated SGC-7901 cells underwent a series of morphological changes, including cell contraction and rounding, arrangement loosening, and a significant decline in the number of attached cells, while the untreated SGC-7901 cells exhibited normal morphology. To investigate the link between the antiproliferative activity of MSEPS and apoptosis induction, the nuclear morphology of SGC-7901 cells was examined after Hoechst 33,258 (nuclear-specific fluorescent dye) staining. As shown in Figure 8B, the untreated cells exhibited evenly distributed nuclei with regular oval shapes, whereas the characteristic apoptotic signs, including karyopyknosis, chromatin compaction, nuclear fragmentation, and bright blue fluorescence were observed upon treating the cells with increasing concentrations of MSEPS [40]. Therefore, MSEPS can trigger apoptosis, contributing to the inhibition of SGC-7901 cell growth. group (12 h); # p < 0.05, ## p < 0.01 compared to the control group (24 h); ΔΔ p < 0.01 compared to the control group (36 h).

Effect of MSEPS on Morphological Changes of SGC-7901 Cells
The morphological changes of SGC-7901 cells, after treatment with different concentrations of MSEPS for 24 h, were observed under an inverted microscope. As shown in Figure 8A, MSEPS-treated SGC-7901 cells underwent a series of morphological changes, including cell contraction and rounding, arrangement loosening, and a significant decline in the number of attached cells, while the untreated SGC-7901 cells exhibited normal morphology. To investigate the link between the antiproliferative activity of MSEPS and apoptosis induction, the nuclear morphology of SGC-7901 cells was examined after Hoechst 33,258 (nuclear-specific fluorescent dye) staining. As shown in Figure 8B, the untreated cells exhibited evenly distributed nuclei with regular oval shapes, whereas the characteristic apoptotic signs, including karyopyknosis, chromatin compaction, nuclear fragmentation, and bright blue fluorescence were observed upon treating the cells with increasing concentrations of MSEPS [40]. Therefore, MSEPS can trigger apoptosis, contributing to the inhibition of SGC-7901 cell growth.

Effect of MSEPS on the Apoptotic Induction of SGC-7901 Cells
Annexin V-FITC/PI staining, combined with flow cytometry, confirmed the apoptotic effects of MSEPS on SGC-7901 cells. As shown in Figure 9, early and late apoptotic cells, as well as normal cells, were detected after 24 h treatment with various concentrations of MSEPS. The exposure of SGC-7901 cells to MSEPS increased the ratios of total apoptotic cells to 7.42%, 15.93%, and 20.93%, in a dose-dependent manner. The untreated group exhibited high cell viability, with 3.82% total apoptosis. Therefore, the growth inhibition of SGC-7901 cells was related to the apoptosis-induction effect.

Effect of MSEPS on the Apoptotic Induction of SGC-7901 Cells
Annexin V-FITC/PI staining, combined with flow cytometry, confirmed the apoptotic effects of MSEPS on SGC-7901 cells. As shown in Figure 9, early and late apoptotic cells, as well as normal cells, were detected after 24 h treatment with various concentrations of MSEPS. The exposure of SGC-7901 cells to MSEPS increased the ratios of total apoptotic cells to 7.42%, 15.93%, and 20.93%, in a dose-dependent manner. The untreated group exhibited high cell viability, with 3.82% total apoptosis. Therefore, the growth inhibition of SGC-7901 cells was related to the apoptosis-induction effect.

Isolation and Purification of EPS from Mucor sp.
Mucor sp. was cultivated in a potato dextrose broth for 6 days at 28 °C and 130 rpm. The fermented broth was centrifuged for 10 min at 5000 rpm. Four volumes of 95% ethanol were added to the resulting supernatant and kept overnight at 4 °C. The precipitate obtained by centrifugation at 8000 rpm for 10 min was subsequently redissolved in deionized water. The precipitate was removed by centrifugation at 8000 rpm for 10 min. The Sevag reagent was used to eliminate the protein from the supernatant [41], followed by the decolorization of the aqueous phase by a macroporous adsorption resin (D301), dialysis, and lyophilization.
The lyophilized powder was prepared as a 10 mg/mL solution and injected into a DEAE-52 column (2.5 × 40 cm) for elution using 0, 0.2, 0.4, 0.8, and 1 M NaCl solutions at 0.7 mL/min flow rate. After collection by an automatic collector, the eluate was analyzed using the anthrone-sulfuric acid method, and an elution curve was plotted, considering tube numbers and absorbance values [42]. The fraction eluted with 0.2 M NaCl was further

Isolation and Purification of EPS from Mucor sp.
Mucor sp. was cultivated in a potato dextrose broth for 6 days at 28 • C and 130 rpm. The fermented broth was centrifuged for 10 min at 5000 rpm. Four volumes of 95% ethanol were added to the resulting supernatant and kept overnight at 4 • C. The precipitate obtained by centrifugation at 8000 rpm for 10 min was subsequently redissolved in deionized water. The precipitate was removed by centrifugation at 8000 rpm for 10 min. The Sevag reagent was used to eliminate the protein from the supernatant [41], followed by the decolorization of the aqueous phase by a macroporous adsorption resin (D301), dialysis, and lyophilization.
The lyophilized powder was prepared as a 10 mg/mL solution and injected into a DEAE-52 column (2.5 × 40 cm) for elution using 0, 0.2, 0.4, 0.8, and 1 M NaCl solutions at 0.7 mL/min flow rate. After collection by an automatic collector, the eluate was analyzed using the anthrone-sulfuric acid method, and an elution curve was plotted, considering tube numbers and absorbance values [42]. The fraction eluted with 0.2 M NaCl was further purified using a Sephadex G-100 column (1.5 × 60 cm), at a 0.3 mL/min flow rate, with ultrapure water. The main fractions were collected according to the elution curve, and the purified polysaccharide, labeled MSEPS, was obtained after dialysis and lyophilization.

Fourier-Transform Infrared (FT-IR) Spectroscopy Analysis
The infrared spectrograms of MSEPS were acquired using an FTIS-8400S spectrometer (Shimadzu, Kyoto, Japan). A mixture of the dried sample (2 mg) and KBr (200 mg) was pressed into a pellet, and FT-IR spectra were recorded in the 4000-500 cm −1 frequency range [43].

Monosaccharide Composition Analysis
Analysis of monosaccharide composition was performed on a high-performance ion exchange chromatography (HPIC) system, equipped with a Dionex™ Carbopac™ PA20 column (ICS5000, 3 × 150 mm, ThermoFisher, MA, USA) [44]. MSEPS was hydrolyzed by 10 mL of 3 M trifluoroacetic acid (TFA) at 120 • C for 3 h. The MSEPS hydrolysates were dried with nitrogen to remove TFA. The obtained sample was dissolved in methanol and dried thrice for the complete removal of residual TFA. Subsequently, the dried hydrolysates were reconstituted in ultrapure water and injected into the HPIC system using water, 15 mM NaOH, and 15 mM NaOH/100 mM NaOAc as the mobile phase at a flow rate of 0.3 mL/min. The injection volume was 5 µL, and the column temperature was kept at 30 • C.

Methylation Analysis
MSEPS was analyzed for methylation by GC-MS using a previously published approach [45]. MSEPS (3 mg) was ultrasonically dissolved in dimethyl sulfoxide (DMSO, 1.0 mL), followed by addition of sodium hydroxide (0.6 mL) in a nitrogen flow. Subsequently, cold CH 3 I (1 mL) was added to the mixture, which was then ultrasonically reacted for 1 h at 30 • C. The addition of ultrapure water (2 mL) terminated the methylation reaction. The methylation product was hydrolyzed by of 2 M TFA (1 mL) at 120 • C for 2 h. Thereafter, the hydrolysis product was dissolved in water and reduced with NABH 4 for 8 h. Acetic anhydride was added to the dried sample and heated at 100 • C for 2 h in a closed chamber. The final acetylation product was dissolved in chloroform (3 mL), and the chloroform layer was washed with water. Shimadzu GC-MS (Shimadzu, Kyoto, Japan), with an RXI-5 SIL MS capillary column (30 m × 0.25 mm × 0.25 m), was used to analyze the methylation of alditol acetates. The temperature was set to rise from 120 to 250 • C at a rate of 3 • C/min, and then maintain 250 • C for 5 min. The temperature of the detector and injector were both set at 250 • C.

Nuclear Magnetic Resonance Spectroscopy (NMR) Analysis
MSEPS (50 mg) was freeze-dried after being dissolved in 0.5 mL D 2 O. This process was carried out thrice for the full replacement of hydrogen, followed by redissolution of the samples in 0.5 mL D 2 O. A Bruker AVANCE 600 MHz spectrometer (Bruker, Karlsruhe, Germany) was used to record the NMR spectra at 25 • C [46].

Cell Lines and Culture
Human gastric adenocarcinoma (SGC-7901) cells were purchased from the Beyotime Institute of Biotechnology (Nanjing, China), and human renal tubular epithelial (HK-2) cells were used as controls. The SGC-7901 and HK-2 cells were cultured in an incubator at 37 • C with 5% CO 2 , using RPMI 1640 and DMEM complete medium containing 10% FBS, respectively.

Measurement of Cell Viability
The viability of SGC-7901 and HK-2 cells were assessed by the MTT method, as reported previously [47]. The SGC-7901 and HK-2 cell suspensions were inoculated at a density of 5 × 10 3 cells/mL into a 96-well plate and cultured for 24 h. MSEPS solutions, at final concentrations of 0, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/mL, were added to the wells and incubated for 12 h, 24 h, or 36 h. Subsequently, 20 µL MTT (5 mg/mL) was added to each well and incubated for 4 h. The supernatants from the wells were carefully aspirated, followed by the addition of DMSO (150 µL) to each well for crystal dissolution. Absorbance values for each well were measured at 490 nm using a full-wavelength microplate reader. All measurements were performed thrice. Cell viability rates were calculated as follows: cell viability rate (%) = OD sample /OD control × 100%, where OD control and OD sample are absorbances of the untreated cells and MSEPS-treated cells, respectively.

Morphologic Observations
SGC-7901 suspensions were inoculated at a density of 2 × 10 5 cells/mL into a six-well plate and cultured for 24 h. Cells were treated with various doses of MSEPS (0, 0.2, 0.4, and 0.8 mg/mL) for 24 h. Changes in cell morphology were observed using an inverted microscope (Olympus, Tokyo, Japan).

Hoechst 33,258 Staining
SGC-7901 suspensions were inoculated at a density of 1 × 10 4 cells/mL into a 24-well plate and cultured for 24 h. After another 24 h of exposure to varied concentrations of MSEPS (0, 0.2, 0.4, and 0.8 mg/mL), cells were fixed in 4% paraformaldehyde for 15 min and then stained with a nuclear dye (Hoechst 33,258) for 20 min, under light-proof conditions. The fixed cells were washed twice with PBS and examined under an inverted fluorescence microscope (Olympus, Tokyo, Japan) to observe changes in the nuclei.

Apoptosis Measurement
An FITC-labeled annexin V probe was used to detect early apoptosis in cells. SGC-7901 suspensions were inoculated at a density of 2 × 10 5 cells/mL into a six-well plate and cultured for 24 h. After being subjected to different concentrations of MSEPS (0, 0.2, 0.4, and 0.8 mg/mL) for 24 h, the cells were collected by centrifugation, after digestion with EDTA-free trypsin. The collected cells were resuspended in a binding solution (500 µL), followed by the addition of Annexin V-FITC (5 µL) and propidium iodide (5 µL). The samples were kept under light-proof conditions for 20 min, before being analyzed using a flow cytometer (FACSVerse, Becton Dickinson, Franklin Lake, NJ, USA).

Statistical Analysis
For data collection, all experiments were repeated at least twice. The results were presented as mean ± SD and statistically analyzed using the Student's t-test and oneway analysis of variance (ANOVA). A p-value < 0.05 was considered to be statistically significant.

Conclusions
A novel heteropolysaccharide named MSEPS, with an M w of 7.78 × 10 4 Da, was obtained from a fermentation broth of Mucor sp. The chemical structure of MSEPS was elucidated by monosaccharide composition, methylation, and NMR spectroscopic analysis. Additionally, MTT assays, morphological observations, and flow cytometry analyses indicated that MSEPS could selectively inhibit SGC-7901 cell growth by inducing apoptosis. This study indicated that MSEPS could be a promising anticancer agent against human gastric cancer cells and could facilitate future research on the influence of Mucor sp. metabolites on human health. Future research may investigate the mechanism of apoptosis induction in SGC-7901 cells to confirm the effects of MSEPS on human gastric cancer cells.