Highlights

• ●Polysaccharides from A. pullulans were extracted, purified, and characterized.
• ●PS_EX, PS_CW, and PS_IN were all identified as β-glucans.
• ●β-Glucans exhibited antibacterial activities against both E. coli and S. aureus.
• ●β-Glucans displayed radical scavenging activity.
• ●β-Glucans protected RAW264.7 macrophages from oxidative stress damage.

Abstract

Polysaccharides from extracellular (PS_EX), cell wall (PS_CW), and intracellular (PS_IN) fractions of Aureobasidium pullulans CGMCC19650 were extracted and purified, respectively. Based on chemical composition analysis and spectral characterization using Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, it was confirmed that the three polysaccharides are β-1,3-1,6-glucans. However, results of molecular weight analysis, Congo red test, X-ray diffraction, and scanning electron microscopy revealed different molecular weight distributions, spatial configurations, and microstructure features. All three β-glucans exhibited potent antibacterial activities against both Escherichia coli and Staphylococcus aureus, but were ineffective against Candida albicans. Moreover, PS_EX, PS_CW, and PS_IN demonstrated robust scavenging activities against 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), hydroxyl, and superoxide anion radicals in vitro. Additionally, these β-glucans protected RAW264.7 macrophages from H₂O₂-induced oxidative stress damage by lowering reactive oxygen species levels, increasing endogenous superoxide dismutase, catalase, and glutathione peroxidase activities, and decreasing intracellular malondialdehyde contents. These findings suggest that β-glucans derived from A. pullulans hold great potential for applications as natural antibacterial and antioxidant agents in food and medicine.

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Keywords

Aureobasidium pullulans

β-Glucan

Polysaccharide

Antimicrobial activity

Antioxidant activity

Oxidative stress

1. Introduction

Microbial polysaccharides, a class of natural macromolecular compounds, are extensively utilized across the food, medical, and cosmetics industries, owing to their unique physical, chemical, and biological activities (Barcelos et al., 2020; Prasad & Purohit, 2023; Wei et al., 2024). Based on their composition, microbial polysaccharides can be categorized into hetero- and homo-polysaccharides, while based on their physiological activities, they can be classified as active and inactive polysaccharides (Wang et al., 2023b). Notably, polysaccharides featuring β-D-glucosidic linkages, known as β-glucan, are abundant in various types of active polysaccharides (Torres et al., 2024). β-Glucans are ubiquitous in algae, cereals, and mushrooms, as well as microorganisms such as brewer’s yeast, Candida albicans, Klebsiella spp., and Aureobasidium pullulans (Murphy et al., 2023; Shui et al., 2021; Timm et al., 2023). In recent years, β-glucans derived from yeasts and bacteria have garnered considerable attention due to their accessibility and favorable physiological activities (Bhosale et al., 2022). However, the biological functions of these microbial β-glucans exhibit significant variation depending on the producing strains and culture conditions, ultimately impacting the scope of their applications (Aimanianda et al., 2009; Lin et al., 2022).

A. pullulans, a yeast-like fungus, has the capacity to produce exopolysaccharides such as pullulan and β-glucan using diverse carbon sources (Prasongsuk et al., 2018; Wang et al., 2020a). Recent studies revealed that β-glucans produced by A. pullulans not only exhibit varying structures (Kono et al., 2020; Liao et al., 2022), but also display a broad spectrum of bioactivities, including antimicrobial, antioxidant, anti-inflammatory, anti-allergic, anti-tumor, and immunomodulatory properties (Guo et al., 2023; Ikewaki et al., 2022; Kono et al., 2023; No et al., 2021; Raghavan et al., 2023; Sato et al., 2012; Shui et al., 2021; Tanioka et al., 2012). In our previous studies, we engineered strains of A. pullulans, optimized culture conditions for β-glucan production, and achieved improved extracellular β-glucan production by disrupting the mal31 gene (Chen et al., 2021, 2022) or adding Triton X-100 and zinc sulfate to the fermentation medium (Wang et al., 2020a; Wang et al., 2020b). Notably, when the mal31 gene was disrupted, both the biomass of A. pullulans CGMCC 19650 and β-glucan production were increased significantly following fermentation medium with identical nitrogen sources (Chen et al., 2021). Microscopy analysis revealed that the mal31 gene disruption mutant was larger in size than the parental strain (Chen et al., 2021). We speculated that polysaccharides may accumulate within the mutant cells, but this is yet to be confirmed experimentally. Moreover, while polysaccharides are also present in the cell wall of A. pullulans (Kataoka-Shirasugi et al., 1994), differences between extracellular (PS_EX), cell wall (PS_CW), and intracellular (PS_IN) polysaccharides remain poorly understood.

In the present study, we extracted polysaccharides from A. pullulans CGMCC 19650 and performed rigorous purification of the extracellular, cell wall, and intracellular polymers. Furthermore, we evaluated the biological properties of PS_EX, PS_CW, and PS_IN by assessing antimicrobial and antioxidant activities. Additionally, we investigated their abilities to protect RAW264.7 macrophages against oxidative stress damage caused by H₂O₂. Our research provides the basis for the application of β-glucans as natural antibacterial and antioxidant agents in functional food and medicine.

2. Materials and methods

2.1. Materials and reagents

A. pullulans CGMCC19650 strain was employed for the production of β-glucans. Potato dextrose broth (PDB) consisting of 20 g/L glucose, 20 % (w/v) potato juice, and natural pH served as seed medium. For β-glucan production, the fermentation medium contained 50 g/L glucose, 3 g/L yeast extract, 0.6 g/L (NH₄)₂SO₄, 2 g/L K₂HPO₄, 1 g/L NaCl, and 0.2 g/L MgSO₄.

Cell counting kit-8 (CCK-8) and bicinchoninic acid (BCA) kits, Dulbecco’s modified Eagle’s medium (DMEM), and trifluoroacetic acid (TFA) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All reagents used were of analytical grade.

2.2. Extraction and purification of polysaccharides

Batch culture of A. pullulans CGMCC19650 was conducted in a 5 L stirred fermenter for 72 h to generate β-glucans. After inactivation at 80 °C for 20 min, the culture broth was centrifuged at 12,000×g and 25 °C for 20 min to separate PS_EX and wet cells. PS_EX was then purified using acetone (Chen et al., 2021).

Wet cells (20 g) were resuspended in 250 mL lysate containing 2 % (w/v) sodium dodecyl sulfate (pH 7.5), heated at 100 °C for 1 h, then centrifuged at 4000×g and 12,000×g at 25 °C for 30 min to collect cell wall and intracellular fractions, respectively (Hao et al., 2016; Mizukoshi et al., 2022). These fractions were separately mixed with papain solution (2 %, w/v) at a ratio of 1:5 (w/v) and incubated at 60 °C and pH 6.5 overnight. After centrifugation at 12,000×g and 25 °C for 30 min, the collected pellets were resuspended in NaOH solution (3 %, w/v) at 60 °C for 4 h, and two volumes of 95 % ethanol were added to precipitate polymers. To remove protein, the polymers were resuspended in 4 % trichloroacetic acid and treated at 4 °C for 24 h. The supernatant was dialyzed (3000 Da) for 48 h, followed by centrifugation at 12,000×g and 25 °C for 30 min to obtain PS_CW and PS_IN. The purified polysaccharides were lyophilized before use.

2.3. Chemical analysis of polysaccharides

2.3.1. Chemical composition determination

The sulfuric acid-phenol method was used to determine the total carbohydrate content with glucose serving as a standard. The sulfuric acid-carbazole method was used to measure uronic acid content (Wang et al., 2021). Commercial BCA kits were used to quantify the protein content followed by the manufacturer’s protocol, and the absorbance at 562 nm was measured.

2.3.2. Ultraviolet (UV) analysis

Polysaccharide solutions (0.5 mg/mL, 1.0 mL) were scanned using a UV-3600 UV–VIS–NIR spectrophotometer (Shimadzu, Japan), and the absorbance ranging from 200 to 400 nm was measured at 25 °C (Liao et al., 2022).

2.3.3. Monosaccharide composition analysis

Polysaccharides (10 mg) were hydrolyzed with 5 mL of 3 mol/L TFA in sealed tubes at 120 °C for 6 h, and TFA was removed using a vacuum rotary evaporator. The hydrolysates were dissolved in 10 mL deionized water for detection of monosaccharides by high-performance liquid chromatography (HPLC) using an Aminex HPX-87H column (7.8 × 300 mm; Bio-Rad, USA). The mobile phase was 5 mmol/L H₂SO₄ and detection was conducted at 30 °C (He et al., 2023).

2.3.4. Molecular weight assessment

High-performance gel permeation chromatography (HPGPC) was employed to assess the weight-average molecular weight (Mw) of polysaccharides at 35 °C (Nie et al., 2005). An Ultrahydrogel 500 column (7.8 × 300 mm; Waters, USA) and a RefractoMax521 RI detector were used for HPGPC, with 0.02 mol/L phosphate buffer (pH 6.5) used as the mobile phase.

2.3.5. Scanning electron microscopy (SEM) analysis

SEM was used to investigate the morphological characteristics of freeze-dried polysaccharides at an accelerating voltage of 15 kV using an S-4800 instrument (Hitachi, Japan) after spraying gold on the surface of polysaccharides (Liao et al., 2022). Images were recorded at 2000 × and 4500 × magnification.

2.4. Structural characterization of polysaccharides

2.4.1. X-ray diffraction (XRD) analysis

XRD analysis of polysaccharides was conducted using a D8 ADVANCE powder XRD instrument (Bruker, Germany). Data were recorded in the 2θ range of 5–60° at a tube pressure of 30 kV and a tube flow of 10 mA (Kono et al., 2023).

2.4.2. Congo red test

Polysaccharide solutions (0.5 mg/mL, 2 mL) were mixed with 2 mL of Congo red reagent (80 μmol/L), and 1 mol/L NaOH was added to final NaOH concentrations ranging from 0 to 0.5 mol/L (Liao et al., 2022). Mixtures were incubated in the dark for 10 min. The maximum absorption wavelength (λ_max) was recorded at 400–700 nm using a spectrophotometer.

2.4.3. Fourier-transform infrared (FT-IR) analysis

Polysaccharides (2 mg) mixed with 100 mg KBr were analyzed using a VERTEX 70 FT-IR spectroscopy instrument (Bruker, Germany) over a wavenumber range of 400–4000 cm⁻¹ (Liao et al., 2022).

2.4.4. Nuclear magnetic resonance (NMR) spectroscopy analysis

Polysaccharides (20 mg) were dissolved in 0.6 mL d₆-DMSO for NMR analysis (Liao et al., 2022). ¹³C NMR and ¹H NMR spectra were recorded at 25 °C using an ADVANCE NEO 400 instrument (Bruker, Germany).

2.5. Antimicrobial activity evaluation

Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, and Candida albicans ATCC 10231 were selected for antimicrobial activity evaluation. The plate count method was used to measure the antimicrobial activities of polysaccharides against these three pathogenic microorganisms (Meng et al., 2021). The bacteria were cultured in LB medium (tryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L), and C. albicans was cultured in YPD medium (glucose 20 g/L, yeast extract 10 g/L, and peptone 20 g/L). The bacterial suspension containing about 1 × 10⁶ colony forming units (CFU) was employed for the antimicrobial test. 100 μL of bacterial suspension was spread onto agar plates containing LB/YPD medium mixed with 0–5 mg/mL polysaccharide, and incubated at 37 °C for 48 h.

The plates containing no polysaccharide were designed as the positive control. The number of colonies was counted, and the inhibition rate of polysaccharides was calculated as: inhibition rate (%) = (1 − CFU_sample/CFU_control) × 100 %.

The minimum inhibitory concentrations (MICs) of polysaccharides against the three test strains were detected using the serial dilution method as described by Shao et al. (2017), with minor modifications. In brief, 100 μL of polysaccharide solutions with concentrations ranging from 0, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0–16.0 mg/mL) were mixed with 100 μL of bacterial suspension (∼1 × 10⁶ CFU/mL) in a 96-well plates. The plate was then incubated at 37 °C for 24 h. The well without bacterial suspension served as the positive control. The absorbance of each well was measured at 600 nm using a microplate reader (Techcomp Ltd., China).

2.6. Radicals scavenging activity assay

The scavenging activities of polysaccharides against the 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl, and superoxide anion radicals were assayed, and polysaccharide solution ranging from 0 to 2 mg/mL was used as the sample (Shao et al., 2017; Zheng et al., 2024).

The DPPH radicals scavenging activity was assayed by mixing 1 mL sample with 1 mL DPPH (0.2 mmol/L) at 37 °C in the dark for 30 min, the absorbance of the mixture was determined at 517 nm. The ABTS radicals scavenging activity was assayed by mixing 1 mL sample with 3 mL ABTS (7 mmol/L) at 25 °C in the dark for 2 h, the absorbance of the mixture was determined at 734 nm. The hydroxyl radicals scavenging activity was assayed by mixing 1 mL sample with 1 mL FeSO₄ (1.5 mmol/L), 0.7 mL H₂O₂ (3 %), and 0.3 mL salicylic acid (20 mmol/L), and incubated at 37 °C for 30 min, the absorbance of the mixture was determined at 517 nm. The superoxide anion radicals scavenging activity was assayed by mixing 0.1 mL sample with 0.1 mL Tris-HCl buffer (50 mmol/L, pH 8.2), reacted with 0.2 mL pyrogallol (7 mmol/L) at 25 °C for 4 min, then 1 mL HCl (10 mol/L) was added to terminate the reaction, the absorbance of the mixture was determined at 560 nm.

The ascorbic acid (V_C) ranging from 0 to 2 mg/mL was used as a positive control. The distilled water replaced reaction reagents for the control (A_control), while distilled water replaced the sample solution for the blank (A_blank). The scavenging rate of radicals was calculated as:

2.7. Protective effects of polysaccharides against H₂O₂-induced oxidative damage

2.7.1. Macrophage culture and viability evaluation

RAW264.7 macrophages purchased from Aptbiotech Co., Ltd. (Shanghai, China) were cultured in DMEM containing 10 % (v/v) fetal bovine serum at 37 °C and 5 % CO₂. Cells were inoculated in 96-well plates (1 × 10⁵ cells/mL). After cells were attached to the plates, the medium was replaced with fresh DMEM containing polysaccharides (0–200 μg/mL). A blank control with no polysaccharide addition was included. Cell viability was measured using a CCK-8 kit followed by the manufacturer’s protocol (Wang et al., 2024). The absence at 450 nm was measured using a microplate reader.

2.7.2. Determination of the oxidizing damage concentration of H₂O₂

RAW264.7 cells were prepared according to section 2.7.1. After removing the culture medium, DMEM containing different concentrations of H₂O₂ (0–500 μmol/L) was added to wells and incubated for 20 h. The group with cell viability ∼60 % was selected as the cell model (Cheng et al., 2015).

2.7.3. Protective effects of polysaccharides against H₂O₂

RAW264.7 cells were prepared according to section 2.7.1. After removing the culture medium, DMEM containing different concentrations of polysaccharides (0–200 μg/mL) was added to wells and incubated for 24 h. Cells without supernatant were exposed to DMEM containing H₂O₂ (150 μmol/L) and incubated at 37 °C for 20 h. Intracellular ROS levels were determined using ROS detection kits followed by the manufacturer’s protocol. The fluorescence data were acquired using a microplate reader (Techcomp Ltd., China) with an excitation wavelength set at 488 nm and an emission wavelength recorded at 525 nm (Lv et al., 2025).

2.7.4. Intracellular SOD, CAT, GSH-Px, and MDA assays

RAW264.7 cells were washed twice with phosphate buffer, then lysed using cell lysate (100 μL/well) to collect the supernatant. SOD, CAT, and GSH-Px activities, and MDA contents were determined using corresponding commercial kits, and the absorbance values at 550, 405, 412, and 532 nm, respectively, were measured using spectrophotometry (He et al., 2024).

2.8. Statistical analysis

All results were averaged from three parallel experiments, and expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and t-test were carried out using SPSS 26 (SPSS Inc., Chicago, IL, USA) to assess the significance of differences. Differences were considered at p < 0.05.

3. Results

3.1. Physicochemical properties of polysaccharides derived from A. pullulans

Polysaccharides of PS_EX, PS_CW, and PS_IN derived from A. pullulans CGMCC 19650 were extracted and purified, >98 % of carbohydrate and <1.5 % of uronic acid were present in these three polysaccharides, with no protein detection (Table S1). UV spectra analysis revealed no peaks in the range of 260–280 nm (Fig. S1a), confirming the absence of proteins and nucleic acids in PS_EX, PS_CW, and PS_IN. Mw analysis using HPGPC revealed distinct Mw distributions of PS_EX, PS_CW, and PS_IN. Based on the standard curve (logMw = −0.254 t + 10.235, R² = 0.9994) and the retention times of polysaccharide peaks (Fig. S1b), the weight-average Mw was calculated. PS_EX exhibited Mw distributions of 6.94 × 10³, 92.15, and 2.79 kDa; PS_CW showed Mw distributions of 1.99 × 10³ and 2.48 kDa; and PS_IN had Mw distributions of 2.69 × 10³ and 2.44 kDa (Table S2). Monosaccharide composition analysis by HPLC showing only glucose in the hydrolysates (Fig. S1c), indicated that PS_EX, PS_CW, and PS_IN were all glucans.

The surface morphology of PS_EX, PS_CW, and PS_IN powders was examined using SEM and showed distinct differences in microstructure of polysaccharides. As shown in Fig. 1, PS_EX powder comprises rough particles of various sizes, with some elongated portions and dense, rough edges; PS_CW powder contains long ellipsoidal particles with loose surfaces and numerous wrinkles; PS_IN powder has smooth cluster structure with small particles adhering to the surface, along with a few elongated particles.

3.2. Structure characterization of polysaccharides

The structural characteristics of PS_EX, PS_CW, and PS_IN were firstly analyzed using XRD spectra. As shown in Fig. 2a, the presence of a diffuse peak at 2θ = 6.5° indicates the triple-helical structure of β-glucan. In addition, peaks observed at 2θ = 13.5° for PS_CW and PS_IN imply the presence of lattice-type microgels in both polysaccharides. The bread-like peaks observed at 2θ = 20° indicate that all three polysaccharides are primarily composed of amorphous components (Kono et al., 2023).

Congo red tests were further carried out to investigate whether the polysaccharides adopt a triple helix conformation by observing the red shift in the maximum absorption wavelength (λ_max) (Wang et al., 2021). As shown in Fig. 2b, the λ_max of Congo red-polysaccharide complexes formed in the presence of PS_EX, PS_CW, and PS_IN exhibited a red shift when the NaOH concentration ranged from 0 to 0.2 mol/L. However, a blue shift was observed when the NaOH concentration was further increased. By contrast, in the blank group without polysaccharide, the presence of NaOH only caused a blue shift in λ_max. Therefore, it can be concluded that PS_EX, PS_CW, and PS_IN all possess triple-helical structures, which is consistent with the results of XRD analysis.

In addition, PS_EX, PS_CW, and PS_IN were analyzed by FT-IR spectroscopy. As shown in Fig. 2c, all FT-IR spectra exhibited distinctive absorption peaks associated with polysaccharides. The absorption peak at 3410 cm⁻¹ was primarily attributed to the O–H stretching vibration, while the peak at 2920 cm⁻¹ corresponding to the C–H stretching vibration (Liao et al., 2022). The absorption peak at 1650 cm⁻¹ was assigned to the asymmetric stretching vibration of the carboxyl group (C=O), and the peak at 1370 cm⁻¹ was related to the CH– bending vibration (Liao et al., 2022). The prominent absorption peak at 1040 cm⁻¹ was generated by the stretching vibration of the C–O–C bond in the pyranose ring, resulting from the presence of multiple glycosidic bonds in polysaccharides (Dong et al., 2021). The characteristic absorption peak at 890 cm⁻¹ was attributed to the C–H bending vibration of β-D-glucopyranose (Boutros et al., 2022). Based on these findings, it was concluded that PS_EX, PS_CW, and PS_IN are β-glucans.

Furthermore, ¹³C NMR and ¹H NMR analyses of PS_EX, PS_CW, and PS_IN were performed to analyze the main chemical bonding of polysaccharides. In the ¹³C NMR spectrum (Fig. 3a), six anomeric carbon signals were identified at 103.75, 86.75, 68.89, 61.37, 73.30, and 77.02 ppm for the three polysaccharides. The signal of C-1 at 103.75 ppm is characteristic of β-glucans, while the signal at 86.75 ppm produced by C-3 indicates the presence of β-1,3 glycosidic bonds. The signal at 61.37 ppm derived from C-6 corresponds to β-1,6 glycosidic bonds. The chemical shifts at 73.30, 68.89, and 77.02 ppm are attributed to C-2, C-4, and C-5, respectively (Qiao et al., 2022). In the ¹H NMR spectra (Fig. 3b), peaks at 4.52 ppm and 3.80 ppm represent anomalous proton H-1 and H-6, respectively. The peak at 3.69 ppm corresponds to H-3/H-6, and peaks at 3.27–3.44 ppm are associated with H-2/H-5/H-4, indicating the presence of β-1,3 glycosidic linkages. The peak at 4.64 ppm reflects the presence of β-1,6 glycosidic bonds in the side chain. The peak at 10.21 ppm in PS_EX suggests the formation of hydrogen bonds (Liao et al., 2022). Based on the analysis of ¹³C NMR and ¹H NMR spectra, PS_EX, PS_CW, and PS_IN were defined as β-1,3-1,6-glucans.

3.3. Antimicrobial activities of β-glucans

Owing to their special structure, polysaccharides always exert antibacterial activities by trapping metal ions and affecting the nutrient uptake of bacteria (Qin et al., 2021). To assess the antimicrobial activities of PS_EX, PS_CW, and PS_IN, a Gram-negative bacterium E. coli ATCC 25922, a Gram-positive bacterium S. aureus ATCC6538, and a pathogenic fungus C. albicans ATCC10231 were selected as test strains. As shown in Fig. 4, the medium containing β-glucans (1–5 mg/mL) exhibited bacteriostatic effects against both E. coli and S. aureus, with inhibition rates increasing as the concentration of β-glucans increased. However, minimal bacteriostatic effects (<5 %) were observed against C. albicans, and the number of colonies on plates containing β-glucans showed no significant differences from controls without β-glucans.

Moreover, PS_EX, PS_CW, and PS_IN had different influences on the bacterial strains. In the presence of 5 mg/mL β-glucans, the inhibition rates of PS_EX, PS_CW, and PS_IN on E. coli were 39.7 %, 31.7 %, and 27.2 %, respectively, while on S. aureus they were 42.0 %, 27.7 %, and 51.6 %, respectively. According to the MIC analysis, the MIC values for PS_EX, PS_CW, and PS_IN against E. coli were determined to be 2.0, 8.0, and 8.0 mg/mL, respectively. Similarly, their MIC values against S. aureus were calculated as 4.0, 8.0, and 2.0 mg/mL, respectively. These results suggest that E. coli is more susceptible to PS_EX, while S. aureus is more sensitive to PS_IN. Therefore, β-glucans derived from A. pullulans CGMCC19650 hold potential as antibacterial agents.

3.4. Radical scavenging activities of β-glucans

Most polysaccharides have good antioxidant properties because they contain carbonyl, carboxyl, hydroxyl, and ester groups (Mirzadeh et al., 2020). To examine antioxidant activities of PS_EX, PS_CW, and PS_IN, their activities to scavenge DPPH, ABTS, hydroxyl, and superoxide anion radicals were investigated in vitro. As shown in Fig. 5a, the scavenging rate of DPPH radicals increased with the concentration of β-glucans in the range of 0.4–2.0 mg/mL. At 2.0 mg/mL, the DPPH radicals scavenging rate was 56.3 %, 21.9 %, and 17.8 % for PS_EX, PS_CW, and PS_IN, respectively. Similarly, the scavenging rate of ABTS radicals also increased with the concentration of β-glucans. At 2.0 mg/mL, the scavenging rate of ABTS radicals was 79.9 %, 36.2 %, and 31.1 % for PS_EX, PS_CW, and PS_IN, respectively (Fig. 5b). As shown in Fig. 5c, the scavenging rate of hydroxyl radicals exhibited a positive correlation with the concentration of β-glucans in the range of 0.4–2.0 mg/mL. At 2.0 mg/mL, the scavenging rate of hydroxyl radicals was 42.3 %, 28.6 %, and 22.0 % for PS_EX, PS_CW, and PS_IN, respectively. The scavenging rate of superoxide anion radicals was shown in Fig. 5d. At 2.0 mg/mL, PS_EX, PS_CW, and PS_IN were able to scavenge 95.0 %, 89.3 %, and 82.5 % of superoxide anion radicals, respectively.

In addition, the IC₅₀ values for PS_EX, PS_CW, and PS_IN against the four types of free radicals mentioned above were determined. Specifically, their IC₅₀ values against DPPH radicals were 1.76, 4.36, and 5.40 mg/mL, respectively; those against ABTS radicals were 1.13, 2.73, and 3.09 mg/mL, respectively. Similarly, their IC₅₀ values for hydroxyl radicals were 2.31, 3.59, and 4.69 mg/mL, while for superoxide anion radicals, the values were 0.07, 0.72, and 0.80 mg/mL, respectively. Based on these findings, PS_EX demonstrated superior ability to scavenge radicals compared with PS_CW and PS_IN. Additionally, all three β-glucans exhibited better scavenging abilities toward superoxide anion radicals than other radicals. Therefore, the β-glucans produced by A. pullulans CGMCC19650 are promising candidates for use as antioxidants to scavenge free radicals.

3.5. Effects of β-glucans on H₂O₂-induced oxidative stress in RAW264.7 macrophages

3.5.1. Cell viability and oxidative stress modeling

RAW264.7 macrophages were cultured in DMEM supplemented with PS_EX, PS_CW, or PS_IN at different concentrations. As shown in Fig. 6a, cell proliferation rates increased in the presence of β-glucans in the concentration range of 50–200 μg/mL. This indicates that the three β-glucans were not toxic to RAW264.7 cells, and instead promoted cell proliferation.

To establish an oxidative stress model, RAW264.7 cells were treated with H₂O₂ (100–600 μmol/L). The optimal level of H₂O₂ for modeling oxidative stress was defined when the cell viability reached ∼60 % (Cheng et al., 2015). Cell viability was 62.4 % in the presence of 150 μmol/L H₂O₂ (Fig. 6b). Therefore, H₂O₂ at 150 μmol/L was use for subsequent cell experiments involving the oxidative stress model.

3.5.2. Protective effect of β-glucans on oxidative stress

The protective effects of PS_EX, PS_CW, and PS_IN against oxidative damage induced by H₂O₂ in RAW264.7 macrophages was investigated. As shown in Fig. 7a, the cell viabilities of groups supplemented with β-glucans were higher than those of the model group (61.5 %), indicating that PS_EX, PS_CW, and PS_IN exerted protective effects against oxidative stress caused by H₂O₂. In addition, an increase in cell viability was observed in the presence of β-glucan at concentrations ranging from 50 to 200 μg/mL. Furthermore, PS_EX demonstrated higher cell viabilities than PS_CW and PS_IN at the same concentration, suggesting that PS_EX had the best protective effect against H₂O₂-induced oxidative stress among the three β-glucans.

Intracellular ROS levels in RAW264.7 macrophages induced by H₂O₂ were measured. As shown in Fig. 7b, the model group exhibited excessive ROS accumulation (88.8 % increase) when incubated with 150 μmol/L of H₂O₂. However, ROS levels in macrophages were significantly decreased by β-glucans in a dose-dependent manner. Notably, PS_EX demonstrated superior activity compared with PS_CW and PS_IN in reducing ROS induced by H₂O₂. In the presence of 200 μg/mL PS_EX, ROS levels approached 105.2 % relative to the control group, indicating the effective elimination of ROS.

3.5.3. Effects of β-glucans on the cellular enzymatic antioxidant system

The activities of SOD, CAT, and GSH-Px, as well as MDA contents, were measured in RAW264.7 cells. As shown in Fig. 8, the model group incubated with H₂O₂ exhibited significantly reduced SOD, CAT, and GSH-Px activities, along with increased MDA contents. However, when supplemented with PS_EX, PS_CW, or PS_IN, the activities of these three enzymes were restored, and MDA contents were decreased. Specially, when 200 μg/mL β-glucans was supplemented, the activities of SOD, CAT, and GSH-Px were recovered averagely to 87.3 %, 92.9 %, and 79.7 % of control group levels, respectively, while MDA contents were decreased to levels similar to the control group. These results indicate that β-glucans exerted strong effects on the cellular enzymatic antioxidant system.

4. Discussion

A. pullulans is an important fungus with immense potential in industrial applications (Prasongsuk et al., 2018). Among the various polysaccharides produced by different strains, β-glucans synthesized by A. pullulans are particularly appealing due to their favorable bioactivities (Suzuki et al., 2021). In this study, polysaccharides (PS_EX, PS_CW, and PS_IN) derived from A. pullulans CGMCC19650 were extracted, purified, characterized, and their antimicrobial and antioxidant activities were investigated.

First of all, we have investigated the physiochemical properties of PS_EX, PS_CW, and PS_IN produced by A. pullulans CGMCC19650. According to chemical components determination, we identified that the three polysaccharides are all glucans. Additionally, we have characterized chemical structure of PS_EX, PS_CW, and PS_IN by FT-IR, ¹³C NMR, and ¹H NMR, spectra indicated that the three polysaccharides are all β-1,3-1,6-glucans. However, PS_EX, PS_CW, and PS_IN exhibited distinct differences in terms of Mw distributions and SEM analysis. Variations in spatial configuration and microstructures were also distinct among PS_EX, PS_CW, and PS_IN. These differences in structural properties among the three polysaccharides undoubtedly contribute to their different biological activities and potential applications (Liao et al., 2022; Qiao et al., 2022).

Despite species differences, fungal β-glucans are generally composed of β-1,3/1,6 glycosidic bonds and often exhibit a triple helix structure known to be the beneficial in biomedical and clinical applications (Boutros et al., 2022; Caruso et al., 2022). For example, a triple-helical β-glucan derived from yeast displayed immunomodulatory activity and the ability to alleviate dextran sulfate induced colitis in mice by downregulating inflammatory genes (Qiao et al., 2022). In this study, results of XRD analysis and Congo red test both confirmed that all the PS_EX, PS_CW, and PS_IN possessed a triple helix structure, signifying their potential for use in biomedicine.

Many microbial polysaccharides have antibacterial activities (Meng et al., 2021). For instance, a polysaccharide produced by Chaetomium globosum CGMCC 6882 was found to possess antibacterial activities against S. aureus and E. coli, with stronger bacteriostatic activity against S. aureus than E. coli (Wang et al., 2023a). Intracellular zinc polysaccharides produced by Grifola frondosa SH-05 exhibited potential antibacterial effects against E. coli, S. aureus, Bacillus megaterium, and Listeria monocytogenes (Zhang et al., 2017). The antibacterial activities of polysaccharides usually depend on molecular weight, monosaccharide composition, branching properties, and chemical modification (Han et al., 2016). The mechanism underlying antibacterial effects of polysaccharides mainly include changing cell membrane permeability, altering cell membrane polarity, inhibiting the uptake of nutrients and oxygen, trapping metal ions involved in enzymatic catalysis, and lowering the entry and exit of substances across the cell wall (Shao et al., 2017; Wang et al., 2023a). In the present study, β-glucans of PS_EX, PS_CW, and PS_IN demonstrated bacteriostatic effects against E. coli and S. aureus, but not against C. albicans. Moreover, according to MIC values of the three polysaccharides, PS_EX exhibited better antibacterial effects against E. coli, while PS_IN was more effective against S. aureus. These differences in antibacterial activity may be attributed to the ability of β-glucans to form mucinous colloids and cause damage to the bacterial cell wall (Liu et al., 2018). Nevertheless, further in-depth research is needed to elucidate the antibacterial mechanism of these three polysaccharides.

In addition, β-glucans produced by A. pullulans ATCC 20524 exhibited scavenging activity against hydroxyl, organic, and sulfate radicals (Kono et al., 2023). In the present study, we examined the antioxidant activities of β-glucans derived from A. pullulans CGMCC19650 through free radicals scavenging assays. When 2 mg/mL PS_EX, PS_CW, or PS_IN was present, the β-glucans had partial scavenging effects on DPPH, ABTS, hydroxyl, and superoxide anion radicals. In particular, all three β-glucans displayed higher scavenging rates for superoxide anion radicals than other radicals, with high antioxidant activities in vitro. The IC₅₀ results for PS_EX, PS_CW, and PS_IN against radicals also indicate that these three β-glucans exhibit a significantly enhanced ability to scavenge superoxide anion radicals, with values notably lower than those reported in previous studies (Hu et al., 2019; Wang et al., 2023a; Zhao et al., 2019).

Moreover, polysaccharides with complex Mw distributions are known to possess better free radical scavenging abilities than those with a single Mw distribution (Yuan et al., 2015). Therefore, the remarkable radical scavenging abilities of PS_EX, PS_CW, and PS_IN may be attributed to their complex Mw distributions.

Furthermore, we conducted cell experiments to investigate the antioxidant activities of PS_EX, PS_CW, and PS_IN. RAW264.7 macrophages is usually used to construct a H₂O₂-induced oxidative stress model, therefore we have established the model in RAW264.7 with a cell viability of ∼60 % (Cheng et al., 2015; Li et al., 2021). All three β-glucans exhibited distinct activities in eliminating ROS generated by H₂O₂. When supplemented at 200 μg/mL, PS_EX, PS_CW, and PS_IN were able to restore the activities of SOD, CAT, and GSH-Px, while reducing the intracellular MDA contents. These findings indicate that the β-glucans effectively activated endogenous antioxidant defense enzymes in RAW264.7 cells, thereby protecting cells from oxidative stress damage. Meanwhile, β-glucan from Saccharomyces cerevisiae can also regulate lipopolysaccharides-induced oxidative stress in RAW264.7 cells, increased activities of SOD, CAT, GSH-Px, and decreased the production of ROS and MDA (Yu et al., 2021). Based on the results of both free radical scavenging and the endogenous antioxidant defense system, the three β-glucans produced by A. pullulans CGMCC19650 have significant potential in antioxidant applications.

In the end, it should be mentioned that the antibacterial and antioxidant effects of PS_EX seemed to be superior to those of PS_CW and PS_IN, the mechanism underlying these differences should be revealed through the structure-activity relationship of the three polysaccharides (Zhang et al., 2024). Hence, the detailed chemical structure of PS_EX, PS_CW, and PS_IN will be elucidated using methylation analysis and other modification methods in future, providing more information on the structure difference between three β-glucans.

5. Conclusions

Polysaccharides of PS_EX, PS_CW, and PS_IN produced by A. pullulans CGMCC19650 were extracted, purified, and identified as β-glucans. These β-glucans had different physiochemical properties and structure characteristics. PS_EX, PS_CW, and PS_IN exhibited antibacterial activities against both E. coli and S. aureus. Additionally, they displayed antioxidant activities by scavenging DPPH, ABTS, hydroxyl, and superoxide anion radicals and protecting RAW264.7 cells against H₂O₂-induced oxidative stress damage. Therefore, these β-glucans have great potential as natural antibacterial and antioxidant agents used in food and medicine.

CRediT authorship contribution statement

Junqiu Ao: Writing – original draft, Investigation, Data curation. Yuqing Liu: Investigation. Yanyu Wei: Investigation. Dahui Wang: Methodology, Funding acquisition. Chonglong Wang: Validation, Methodology. Lin Wei: Writing – review & editing, Supervision. Gongyuan Wei: Writing – review & editing, Supervision, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

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

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21776189), the Natural Science Foundation of Jiangsu Province (BK20181440), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Appendix A. Supplementary data

The following is the Supplementary data to this article.Download: Download Word document (122KB)

Multimedia component 1.

Data availability

Data will be made available on request.

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