Purification, characterization and anticancer activities of exopolysaccharide produced by Rhodococcus erythropolis HX-2

Journal Pre-proof Purification, characterization and anticancer activities of exopolysaccharide produced by Rhodococcus erythropolis HX-2

Xin Hu, Dahui Li, Yue Qiao, Xiaohua Wang, Qi Zhang, Wei Zhao, Lei Huang PII:

S0141-8130(19)39132-9

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.228

Reference:

BIOMAC 14256

To appear in:

International Journal of Biological Macromolecules

Received date:

8 November 2019

Revised date:

10 December 2019

Accepted date:

24 December 2019

Please cite this article as: X. Hu, D. Li, Y. Qiao, et al., Purification, characterization and anticancer activities of exopolysaccharide produced by Rhodococcus erythropolis HX-2, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.12.228

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© 2019 Published by Elsevier.

Journal Pre-proof

Purification, characterization and anticancer activities of exopolysaccharide produced by Rhodococcus erythropolis HX-2

Xin Hu, Dahui Li, Yue Qiao, Xiaohua Wang, Qi Zhang, Wei Zhao, Lei Huang*

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AFFILIATIONS:

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College of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic Solar Cells and

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University of Technology, Tianjin 300384, China

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Photochemical Conversion, Tianjin Key Laboratory of Drug Targeting and Bioimaging, Tianjin

*Corresponding Author: College of Chemistry and Chemical Engineering, Tianjin University of

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[email protected] (L. Huang)

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Technology, Binshui West Road 391, Tianjin 300384, China. Tel.: 86-22-60214259; E-mail address:

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Highlights

The purified EPS fractions (HPS) were obtained from Rhodococcus erythropolis HX-2. The HPS production reached 8.957 g/L in MSM medium through response surface method (RSM). HPS is a heteropolysaccharide of glucose, galactose, fucose, mannose and glucuronic acid and a reticular structure was observed by SEM. This is the first report of exopolysaccharide showed the higher viscosity and anticancer activity by Rhodococcus sp.

Abstract In the present study, an exopolysaccharide (EPS) producer Rhodococcus erythropolis HX-2 was isolated from Xinjiang oil field, China. The HX-2 EPS (name HPS) production reached 8.957 g/L by RSM in MSM medium. The HPS was purified by ethanol precipitation and fractionation by

Journal Pre-proof DEAE-Cellulose and Sepharose column, the yield of HPS was 3.736 g/L. HPS composed by glucose, galactose, fucose, mannose and glucuronic acid. FT-IR spectroscopy indicated the presence of a large amount of hydroxyl groups. NMR spectroscopy indicated the existence of both α and β-configuration for sugar moieties present in HPS. The degradation temperature (255.4 °C) of the HPS were determined by thermogravimetric analysis (TGA). A reticular structure of HPS was observed by SEM and the AFM analysis of the HPS revealed straight chains line. Meanwhile, the WSI and WHC of HPS were 92.15 ± 3.05% and 189.45 ± 5.65%, respectively. Finally, In vitro anticancer activity purified EPS was evaluated on L929 normal cells, A549 cancer cells, SMMC-7721 liver cancer cells and Hela cervical

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cancer cell. HPS inhibited the growth of cancer cells in a certain concentration without damage to

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normal cells. These characteristics indicate that its potential application value in food, industry and

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pharmaceutical application.

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Graphical abstract

Keywords: Rhodococcus sp.; Exopolysaccharide; Characterization; Anticancer activities

1.Introduction Exopolysaccharide (EPS) are polymers produced by the metabolic processes of microorganisms that accumulate on the cell surface. EPS is a high molecular weight, biodegradable polymers in nature with significant ecological and physiological functions [1]. In addition, the production of EPS depends on the growth of bacteria that the maximum EPS yield can be observed during the exponential and

Journal Pre-proof stationary phases of bacterial growth in batch mode [2]. Furthermore, the composition of EPS is strongly influenced by various external factors such as pH, temperature, salinity, carbon and nitrogen sources [3-6]. EPS has attracted extensive attention due to the environmentally characters and applications of EPS, such as non-toxic, anti-oxidant, anti-tumor, stable, emulsified, viscosified, anti-biofilm and anti-microbial [7-10]. EPS can also protects cells from antibiotics, toxic metals, phagocytosis and other unfavorable factors [11, 12].

In recent years, various microbial sources of EPS have been explored and studied [13]. Their structure

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is different between diverse species, resulting in various properties and applications [14]. Typically,

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EPS consists primarily of glucose, galactose, fucose, xylose, arabinose, rhamnose, mannose, fructose

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and/or sugar derivatives. However, organic or inorganic substituents are usually present in EPS, which affects its physiological and biological properties[15]. Rapp et al. studied the formation of

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polysaccharides from Rhodococcus sp. by using low-grade mono-, di- and trihydric alcohols, sugars

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and n-alkanes as carbon sources [16]. Neu et al. shown that the polysaccharide by Rhodococcus sp. PS-33 consisted of rhamnose, galactose, glucose and glucuronic acid in molar ratios of 2:1:1:1 [17].

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Chemical analysis of EPS in Rhodococcus opacus by Czemierska et al. showed that the concentrations of reducing sugar, uronic acid and amino sugar were 184.79 μg/mg, 117.6 μg/mg and 9.23 μg/mg,

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respectively [18].

Due to the practical application value of EPS, it has attracted extensive attention in these fields, such as pharmacology, nutraceuticals, useful foods, cosmeceuticals, herbicides and insecticides [19, 20], while prospects incorporates utilizes as anticancer and immunomodulation [21]. EPS has a wide range of biological and pharmacological activities, many studies have reported the ability of polysaccharides to induce apoptosis in cancer cells, while the cytotoxicity to normal cells is small [22, 23]. Many cancer patients benefit from commercial products of polysaccharides and polysaccharide combinations, such as grifolan, PSP (polysaccharide–peptide complex) and krestin (polysaccharide–protein complex) [24, 25].

Therefore, an EPS-producing strain Rhodococcus erythropolis HX-2 was isolated from Xinjiang Oil Field, China. The yield of EPS is optimized by response surface methodology (RSM). And purification

Journal Pre-proof of crude EPS was conducted by DEAE-Cellulose and Sepharose column. Then, the chemical structure and physicochemical property of the purified EPS were characterized by High Performance Liquid Chromatography (HPLC), ultraviolet (UV), fourier transform infrared spectroscopy (FT-IR), one dimensional 1H and

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C nuclear magnetic resonance (NMR), thermogram analysis (TGA), scanning

electron microscopy (SEM), atomic force micrograph (AFM), water holding capacity (WHC) and water solubility index (WSI). These properties clarify the relationship between their structure and biological activity and their potential value in industrial applications. Finally, the anticancer activities

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were investigated using MTT-assay that prove its function in clinical applications.

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2. Materials and methods 2.1. Chemical reagents, medium and strain

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Molecular biology reagents were purchased from Beijing Tiangen biochemical technology company, China. And other reagents were analytically pure. All cells were purchased from the Shanghai Cell

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Bank of the chinese academy of sciences, China.

LB medium contained (g/L): peptone 10, yeast powder 5, NaCl 5, pH 7.0–7.2.

pH 7.2.

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Minimal salt medium (g/L): Na2HPO4 1.5, KH2PO4 3.48, (NH4)2SO4 4, MgSO4 0.7, yeast powder 0.01,

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The strain was a Rhodococcus sp. isolated from the laboratory [26].

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2.2. Extraction and purification of EPS The crude EPS was extracted according to the ethanol precipitation methods of Liu et al. [27], with some modifications. In short, the stain HX-2 was inoculated in 2 L minimal salt medium broth with 2% (v/v) sodium citrate culture and cultured for 72 h at 25 °C. After fermentation, the culture was heated at 90 °C for 15 min to inactivate enzyme and centrifuged to remove cells at 10,000 ×g at 4 °C for 15 min, and then 70% (w/v) trichloroacetic acid (TCA) was added to the cell-free supernatant with a final concentration 15% (w/v), kept at 4 °C overnight and centrifuged at 11,000 ×g at 4 °C for 20 min to remove protein. Afterwards, the supernatant was precipitated by mixing with three times volume of pre-chilled 95% (v/v) ethanol and kept at 4 °C for 12 h, and the crude EPS was collected with centrifugation at 11,000 ×g at 4 °C for 20 min. The obtained crude EPS was dissolved in ultrapure water and dialyzed (with MW cut off 14 kDa) at 4 °C for 2 d and then lyophilized.

Journal Pre-proof The freeze-dried crude EPS was fractionated with an anion exchange chromatography on the DEAE-Cellulose DE-52 column (1.6 × 30 cm), eluted with gradient concentration of NaCl solutions (0, 3, 6, 9 and 12 %) at a flow rate of 1mL/min. Five mL per tube was collected and determined for carbohydrate content by the phenol sulfuric acid method [28]. The fractions containing EPS were concentrated, dialyzed and lyophilized. Further purification of the EPS was performed by gel filtration using a Sepharose CL-6B column (1.6×80 cm) and eluted with ultrapure water at a flow rate of 0.5 mL/min. The major fraction was pooled, dialyzed and lyophilized for further analysis.

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2.3. Optimization of EPS production

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Three independent variables: pH (7-9), sodium citrate concentration (1.5-2.5 %) and sodium nitrate

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concentration (0.5-1.5 %) were analyzed in this study. The experiments were designed by using a design expert software (Design Expert, version 8.0.6). The subsequent EPS production rate was

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evaluated by coefficient of determination (R2), ANOVA and response surface plots. A second-order

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3 2

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polynomial equation was developed to fit the data from the experimental investigations [29, 30]:

𝑖<𝑗=1

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𝑖=1

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Y = 𝑎0 + ∑ 𝑎𝑖𝑖 𝑋𝑖 + ∑ ∑ 𝑎𝑖𝑖 𝑋𝑖 𝑋𝑗 Eq(1)

where Xi and Xj are independent variables; a0,ai, aii and aij are regression coefficients for intercept, linear, quadratic and interaction terms, respectively.

2.4. Characterization of EPS

2.4.1. Estimation of molecular weight, carbohydrate, protein and lipid content of EPS As described by López-Ortega et al. [31], molecular weight (Mw) of EPS was determined using by high-performance size-exclusion chromatography (GPC, Malvern-VISCOTEK TDAmax system, UK) equipped with a refractive index detector (RID) and Waters Ultra-hydrogel 500 column (7.8 mm × 300 mm, 6 μm particles, USA). The mobile phase was NaNO3 0.1 M, NaH2PO4 0.01 M (pH = 7) with sodium azide 0.02% (w/v) as a bactericidal agent. The EPS sample was dissolved in the mobile phase (1 mg/ml) and filtered through 0.45 μm membranes. The injection volume was 100 μL and the flow rate of the mobile phase was 1 mL/min.

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The total carbohydrate content was determined using the phenol-sulfuric acid method in procedure 2.2. 500 µL of 80% phenol solution was mixed with 100 µL of sample (EPS: 2.5 mg/mL) followed by vigorous mixing and stream addition of 2 ml H 2SO4. After 15 min of incubation at room temperature, OD was recorded using distilled water as blank at 490 nm. The obtained results were compared against a glucose standard curve. Bovine Serum Albumin (BSA) standard was prepared and the protein estimation was done using Bradford method [32]. During estimation, Bradford reagent (800 µL) was added and mixed well with 200 µL sample (EPS: 5 mg/mL) followed by 30 min dark incubation at

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room temperature; a colour change due to protein binding was observed and absorbance was taken at

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595 nm. To deduce the lipid content, chloroform:methanol (3:1) extraction was followed as detailed by

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Zhang et al. [33].

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2.4.2. Compositional analysis by high performance liquid chromatography (HPLC)

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Monosaccharide composition of the purified EPS fractions was determined as previously described by Sran et al. [34], with some modifications. The purified EPS sample (50 mg) was hydrolyzed with 4

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mol/L trifluoroacetic acid (TFA) at 120 °C for 2 h and blowed dry with nitrogen. Addition 1 mL of 0.5 mol/L PMP(1-phenyl-3-methyl-5-pyrazolone)-methanol solution and 0.5 mL of 0.3 mol/L NaOH

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solution to the blown sample, and cool in a 70 °C water bath for 60 min. After adding 0.5 mL of 0.3 mol/L HCl solution and 0.5 mL of chloroform, shaking the solution and stand for 20 minutes. The lower layer was discarded, repeated extraction three times, and the aqueous layer was taken through the membrane. The aqueous layer (hydrolysates) were analyzed by HPLC equipped with an Agilent1200 (Column: SHISEIDO C18; 4.6mm × 250 mm, 5 μm). 0.1 mol/L KH2PO4 (pH 6.8) was mixed with acetonitrile as mobile phase (mixing ratio was 82:18) with a flow rate of 1 mL/min at 25 °C. Injection volume 10 μL and wavelength at 245 nm. In addition, several monosaccharides, such as rhamnose, arabinose, galactose, glucose, mannose and fructose were chosen as standards.

2.4.3. Ultraviolet (UV) and Fourier transform infrared spectroscopy (FT-IR) spectrum analysis The purified EPS was dissolved in ultrapure water (1 mg/mL) and the UV spectrum of the EPS solution was obtained using a UV-visible spectrophotometer (U-3900H, HITACHI, Japan) scanning in a wavelength range of 190-350 nm.

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The functional groups present in the EPS were determined by using Fourier transform infrared spectroscopy (FT-IR) spectroscopy (NICOLET iS10, Thermo Scientific, USA). An infrared spectrum of the EPS was recorded using the KBr method in the range of 4000-400cm-1. A metal mold was obtained by mixing the powder of the dried EPS (1 mg) with dried KBr (100 mg) and then pressing the mixture. Finally, the FT-IR spectrum was obtained by scanning the metal mold.

2.4.4. Nuclear magnetic resonance (NMR) spectroscopy analysis

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Nuclear magnetic resonance experiments of HPS were performed using a 600MHz Bruker Avance

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IIIHD NMR spectrometer operating at 14.1 Tesla (600.13 MHz for 1H) equipped with a 5 mm inverse probe (QXI) with gradient on the Zaxis. 1H and 13C NMR analyses were performed at 70 °C in D 2O.

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Chemical shifts are expressed in δ(ppm) relative to acetone (δ 24.63 for

C signal and 4.80 for 1H

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signal) used as internal standard.

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2.4.5. Thermogravimetric (TG)analysis

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The pyrolysis property of the purified EPS was studied in thermal analyzer operating at atmospheric pressure (TG 209 F3, NETZSCH, Germany). The purified EPS sample (10 mg) was placed in an Al2O3

atmosphere.

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crucible and heated at a rate of 10 °C/min over a temperature range from 40 to 700 °C in air

2.4.6. Scanning electron microscopy (SEM) and Atomic Force Microscope (AFM) analysis The surface morphology and microstructure of the purified EPS were investigated using a SEM and AFM as described by Zhang et al. [35] and Zhao et al. [36]. The purified EPS (10 mg) was glued onto the SEM stubs and gold-coated, and then SEM image was conducted at an accelerating voltage of 2.0 kV by SEM instrument (FEI-Verios 460L, USA).

The purified EPS solution (1 mg/mL) for AFM analysis was prepared using ultrapurewater and continuously diluted to the final concentration of 10 mg/L. About 10 μL of EPS solution (10 mg/L) was dropped on the surface of a mica sample carrier and dried at room temperature (24 h). The AFM images were obtained using a Dimension® Iconinstrument (Bruker Instruments Co., Germany) in

Journal Pre-proof tapping mode.

2.4.7. Determination of water holding capacity (WHC) and water solubility index (WSI) The WHC and WSI of EPS were evaluated as per the method [37], with a few modifications. The EPS sample (50 mg) was dissolved in 10 mL of distilled water and kept in a water bath at 50 °C for 30 min to obtain a solution, which was then centrifuged at 11,000 ×g for 30 min and the supernatant was saved for further analysis. The EPS particles were filtered on pre-weighed filter paper to completely remove

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moisture. Then the filter paper was weighed and recorded for WHC.

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For WSI, the collected supernatant was poured into a petri dish and dried at 110 °C for 8 h, and the dry

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solid weight was obtained. The percentages of WHC and WSI were estimated according to the

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑦 𝑠𝑜𝑙𝑖𝑑𝑠 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 × 100% 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑦 𝑠𝑎𝑚𝑝𝑙𝑒

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WSI (%) =

𝑇𝑜𝑡𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑎𝑓𝑡𝑒𝑟 𝑤𝑎𝑡𝑒𝑟 𝑎𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 × 100% 𝑇𝑜𝑡𝑎𝑙 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑠𝑎𝑚𝑝𝑙𝑒

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WHC (%) =

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following equations [38]:

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2.4.8.Determination of solution viscosity

Using the Sanzan gum, Guar gum, Xanthan gum and purified HPS product, solution with concentrations of 1mg/ml was prepared. The viscosity of different solution was determined using NDJ-8S digital viscometer (Shanghai Jingke Industrial Co., Ltd., CHN) at 25 °C and 30 rpm. Each sample was tested three times.

2.5. MTT assay MTT-assay was done for cytotoxicity analysis on L929 normal cells, A549 cancer cells, SMMC-7721 liver cancer cells and Hela cervical cancer cell in 96-well plates at a density of 1 × 105 cells/well in a humidified 5% CO2 incubator at 37 °C. After 24 h of incubation, the prepared culture by RPMI 1640 media was replaced by 100 µL of media with different concentrations of the EPS (0, 50, 100, 200, 400, 600 and 800 µg/mL) [39, 40]. Then, the cells were incubated for a period of 24 and 48 h. After

Journal Pre-proof completion of the exposure time, the media was carefully removed and replaced with 25 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution for 4 h [41]. Subsequent to incubation, media was aspirated, DMSO (100 µL) added for solubilization of formazan crystals and optical density read at 570 nm. Cell viability was expressed as the relative cell viability related to vehicle control wells. The vehicle control were the wells which has cells in RPMI 1640 media but no test samples but has equal volume of DMSO as test sample wells. The cell livability could be evaluated as follows[42]:

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Cell livability (%) = [(𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑡𝑒𝑠𝑡 ) − (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑙𝑎𝑛𝑘 )] × 100%

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Where Acontrol is the absorbance value of the untreated cells at 570 nm; Atest is the absorbance value of the treated cells at 570 nm; Ablank is the absorbance value of untreated cells added to the same amount

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of PBS solution at 570 nm as the test cells.

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2.6. Statistical analysis

The SPSS 19.0 statistical software programwas used for all data analyses. Results are expressed as the

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mean ± SD. Single factor analysis for variance was performed by using the ANOVA program, followed

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by the LSD test. P < 0.05 indicates a significant value.

3. Results and discussion

3.1. Isolation and purification of EPS The crude EPS produced by strain HX-2 was obtained from minimal salt medium through a series of steps including cell removal, deproteinization, alcohol precipitation and dialysis [43]. The crude EPS yield was determined to be 6.365 g/L. As shown in Fig. S1, the crude exopolysaccharide produced by the strain HX-2 has a high viscosity and crystal clear.

The crude EPS was further purified via a DEAE Cellulode DE-52 column. Three fractions were eluted, the first peak was intense, indicating that there were main fractions in the EPS (Fig. 1A). Following this, the second fraction was pooled, dialyzed, freeze-dried and further purified via Sephadex G-100 column. As a result, a single fraction was obtained and named as HPS (Fig. 1B), which was collected,

Journal Pre-proof dialyzed and freeze-dried for further analysis.

3.2. Response surface optimization of EPS production and ANNOVA The effect of abiotic factors, such as pH (7–9), sodium citrate concentration (1.5–2.5 %), sodium nitrate concentration (0.5–1.5 %) and EPS yield (g/L) was analyzed using BBD. Based on the experimental design, the maximum EPS yield (8.957g/L) was obtained at pH 8, 2 % sodium citrate concentration, and 1 % sodium nitrate concentration. Xing et al. reported that the addition of carbon source into the growth medium enhanced the production of EPS by Leuconostoc mesenteroides [44]. Costa et al.

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showed that the changes in pH and nitrogen source concentration affect EPS production [43]. The

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optimization value showed that the sodium citrate and sodium nitrate concentration in the MSM

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medium significantly affect the EPS production.

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The BBD experimental design and results of the EPS production are showed in Table S1. An ANOVA

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test for the EPS production was performed to make sure the significance of the model terms. The test result for the quadratic model was highly significant,as evident from the Fisher's F-test (P < 0.005).

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The predicted R2 and adjusted R2 values were close to 1.0, indicating the model well fitted the experimental data. In addition, sequential model sum of squares, lack of fit tests and model summary

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statistics further supported the significance and adequacy of the model (Table S2). 3D plots provided in Fig. S2 vividly show the regression equations and were used to visualize the relationship between the response and experimental levels of each variable to determine optimum EPS production.

The relationship between experimental variables and response values can be verified by a quadratic model, which can be proved to be Eq(1) by actual factors:

Y = 8.87 + 0.073 × A − 0.06 × B − 0.31 × C − 0.15 × A × B + 0.16 × A × C + 0.23 × B × C − 0.60 × 𝐴2 − 0.39 × 𝐵2 − 0.94 × 𝐶 2

where Y is the EPS yield (g/L); A, B and C are pH, sodium citrate concentration (%) and sodium nitrate concentration (%), respectively.

Journal Pre-proof Through a series of purification methods, the yield of purified HPS was 3.736 g/L with 58.7% purification rate.

3.3. Characterization of HPS 3.3.1. Chemical characterization (molecular weight, carbohydrate, protein and lipid content) The Mw of the HPS were measured by GPC as shown in Table S3. The HPS exhibited higher Mw, which is 1.04 × 106 Da. Among the reported EPS from Rhodococcus sp., it much lower than EPS from Rhodococcus erythropolis [45], while higher than that of EPS from Rhodococcus opacus [18]. This

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suggests that HPS may have a higher viscosity and can be applied to food as well as industry.

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The total carbohydrate (eg: a combination of mono, di, oligo and polysaccharides), protein and lipid content in the HPS was found to be 79.24 %, 5.204 % and 8.45 % w/w according to the standard curve

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(Fig. S3) which were within limits (carbohydrates 40–9 5%, proteins 0–60 %, lipids 1–10 %) as

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reported by More et al. [46]. In recent years, few reports have reported the content of each component in the extracellular polymer produced by Rhodococcus sp. Czemierska et al. founded that the

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extracellular polymer produced by Rhodococcus opacus contained 64.6 % polysaccharide and 9.44 % protein [18]. Compared with the above literature, HPS has more polysaccharide components and less

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protein, which seems to lay the foundation for its application in food additives and cosmetics.

3.3.2.Monosaccharide composition analysis of HPS Monosaccharide analysis was carried out by HPLC after acid hydrolysis by comparing their retention time with that of the monosaccharide standards (Fig. 2). A standard curve was drawn using the area normalization method (data not showen) to calculate the content of each monosaccharide component in HPS (Table 1). The result revealed that HPS was a heteropolysaccharide, which was composed of glucose, galactose, fucose, mannose and glucuronic acid with a mass ratio of 27.29 %, 24.83 %, 4.79 %, 26.66 % and 15.84 %, respectively. In recent years, few authors have reported the monosaccharide composition of EPS in Rhodococcus sp. According to the literature, chemical analysis showed the presence of reducing sugars, uronic acids, and amino sugars at concentrations of 184.79 μg/mg, 117.6 μg/mg and 9.23μg/mg in Rhodococcus opacus, respectively [18]. Therefore, the different composition and ratio of the HPS in our work may be explained by that the monosaccharide

Journal Pre-proof composition of EPS produced by Rhodococcus sp. This also laid the foundation for exploring the composition of polysaccharides in Rhodococcus sp.

3.4.3. UV and FT-IR spectrum analysis of HPS UV spectra of the EPS showed only a single peak in the range of 190-210 nm, which is a common characteristic of carbohydrates [47]. No absorbance at 260 and 280 nm by ultraviolet spectra assay was detected, which revealed the EPS contained no or little nucleic acid and proteins, with a high purity.

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In the present study, the functional groups of HPS were characterized via FT-IR analysis (as

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shown in Fig. 3). The broad absorption peak at 3448 cm−1 suggested that the sample contained massive hydroxyl groups (-OH) stretching vibration, which was a characteristic of polysaccharide

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and further confirmed that the substance was polysaccharide [48-50]. A peak at 2931 cm−1 was

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observed in HPS spectra which corresponds to the methyl groups as well as the C-H stretching -1

vibration [51] and C-H bending was detected at 1411 cm-1 [52, 53]. A broad band at 1629 cm was

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due to the bound water [54]. The strong peaks at 1064 cm−1and 1153 cm−1 were also the characteristic

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absorptions of C-O-C and C-O-H of pyranose ring [36].

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3.4.4. NMR spectra analysis of HPS

Further investigation of the HPS structure was carried out by the nuclear magnetic resonance (NMR). 1

H NMR is widely used in analyzing the configuration of glycosidic bonds in EPS (Fig. 4A), mainly

containing C2-C6 ring proton regions (δ 3.1-4.5 ppm) and anomeric proton regions (δ 4.5-5.5 ppm) [55, 56]. The 1H NMR spectrum showed five anomeric proton signals appeared at 5.10, 5.05, 5.00, 4.53 and 4.45 ppm in a relative integral of nearly 1.00: 1.31: 3.44:0.82: 1.39, respectively. The anomeric signals appeared at the regions between 4.35 and 5.11 ppm indicated that HPS contained both α and β-configuration sugar residues [57]. In the

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C NMR spectrum (Fig. 4B), the signals in 95-101 ppm

were related to α-anomeric carbons, and the signals in 101–105 ppm were due to β-anomeric carbons, which were consisted with the results of 1H NMR. The peaks in the range of 75-85 ppm indicated that there were branched chains in HPS. What's more, the multitudes of signals at δH 3.2-4.1 and δC 60-80 were attributed to atoms H2-H6 and C2-C6, respectively. The absence of furanosidic residues was evident from the lack of carbon signals in the region δ 82.00-84.00 [58]. The lack of carbon signals in

Journal Pre-proof the region of 175 ppm proved the absence of carboxylic carbon.

3.4.5. TG analysis of HPS Fig. 5 shown that the thermal property characterization of HPS.A degradation temperature (Td) was determined to be 255.4°C on the basis of TGA curve. HPS experienced three phase of weight loss with increasing temperature. An initial weight loss (18.02 %) was observed between around 40 and 180°C owing to the loss of moisture, indicating that the HPS may have a number of carboxyl groups (-OH), thus increasing its water holding capacity and moisture-retlining capacity [45]. The second phase saw a

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distinct loss of approximate 68.59 % and the weight loss reached the maximum at around 601.2 °C,

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which was due to degradation of EPS accompanied by the cleavage of chemical bond such as C-O and

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C-C bonds [59]. Finally, the weight loss gradually reached equilibrium with only 11.23 % of the residue remained at 685.9 °C. The DTG curve confirmed the thermal stability of the HPS where a mass

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loss was observed between 210-250°C similar to the TGA curve. Nevertheless, our works showed that

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HPS has a highlevel of thermal stability that suggests its potential to be used in high thermal processes.

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3.4.6. SEM and AFM analysis of HPS

Scanning electron microscopy (SEM) analysis of the macromolecules can be a mighty tool to

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understand the microstructural properties of these materials. So we determined the surface morphology and the microstructure of HPS by SEM analysis (Fig.6A, B). As can be seen that the HPS image presented a three-dimensional spider web with porous entanglement. At the same time, it was observed that the details of the microstructure of HPS were inner filaments composed of irregularly shaped particles when magnified 10,000 times (Fig. 6B). The viscosity, filmforming properties and water retention performance was usually stronger for this net structure. There were many literatures on surface morphology of EPS or other bacterial exopolysaccharides. For example, the exopolysaccharide produced by the strains B. longum subsp. inantis CCUG 52486 and B. infantis NCIMB 702205 all had a highly branched entangled porous structure [60]. The EPS produced by Bacillus cereus KMS3-1 showed to be an irregular highly porous web-like structure and had an uneven surface [61]. EPS produced by Enterococcus faecalis EJRM152 revealed a dispersive structure with irregular lumps of different sizes [62]. Compared with the SEM of the above strains, the HPS SEM exhibited a serried porous network structure and a more uniform pore size distribution in this study. And this work also

Journal Pre-proof reported for the first time that EPS exhibited a network structure in Rhodococcus sp. Due to its small pore size (<1 μm) and dense distribution, the exopolysaccharide may retain more moisture, which was an perfect characteristic for excipients in the food or pharmaceutical industry [63]. Therefore the HPS has the potential as agelling agent, thickener, emulsifier, stabilizer, water binder and the like.

In recent years, AFM became another powerful tool to show microstructure of exopolysaccharide. Fig. 6C and D shows a topographical AFM image of the HPS deposited onto the mica surface from a 10 mg/L solution and air-dried prior to imaging. The surface of HPS was rough and uneven and there were

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irregular protrusions with a height ranging from 1.6 ± 0.2 nm. Its length of several hundreds of

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nanometers revealed that polysaccharide chains were also aggregated. Some areas formed a fiber

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network, while others were relatively sparse, indicating that the HPS may be a large and complex network structure, which was consistent with the microstructure morphology from its SEM observation.

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Also, the HPS exhibited similar molecular structures with that the EPS produced by Alteromonas

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infernus GY785, indicating this kind of EPS showed that can regulate various biological activities through their interactions with growth factors[64]. The HPS also exhibited characteristics of straight

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chains and polyhydroxylation (FT-IR), which accounts for its strong inter/intra-molecular hydrogen bonds. Polysaccharides chains crosslink with each other through hydrogen bonding to form aggregates

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[65]. The stereoscopic network formed by this aggregation of polysaccharides plays an important role in the retention of water molecules [66].

3.4.7. WSI and WHC of HPS

The WSI and WHC of HPS were 92.15 ± 3.05 % and 189.45 ± 5.65 %, respectively. These dates show that HPS may have good hydrophilicity and the potential to hold a mass of water through hydrogen bonding [67]. The surface morphology of HPS also confirmed this conclusion. Carbohydrates can also improve the textural and rheological properties of products as a result of physico-chemical properties such as water holding capacity, solubility and viscosity [68]. The WSI and WHC of EPS produced by Leuconostoc lactis KC117496 were found to be 14.2 % and 117 % by Saravanan et al. [69]. Vinothini et al. shown the WHC and WSI of the EPS from S. griseorubens GD5 were found to be 20.5 % and 134 % [38]. Compared to the above literature, HPS has better water solubility and water holding capacity. In recent years, the WHC and WSI of exopolysaccharide produced by Rhodococcus

Journal Pre-proof erythropolis have not been reported. Based on these physical and chemical properties, HPS could be potentially used in the food industry as a bio-thickener, an additive, a stabilizer or as a viscosifying.

3.4.7. Solution viscosity with different sample As shown in Fig. S4, the solution viscosity of HPS was 18 mPa·s in 1 mg/ml and 30 rpm, which was lower than the viscosity of xanthan gum (61 mPa·s) and Sanzangum (69 mPa·s). However, it was about 2 times than the viscosity of Guar gum (10 mPa·s). This is also the first report that exopolysaccharide produced by Rhodococcus sp. show higher viscosity. Zhu et al. reported that the exopolysaccharide

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produced by Bacillus atrophaeus WYZ strain shown the viscosity of 5 mPa·s in 2g/L [70]. HPS has a

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higher viscosity at the same concentration compared to the above report and the already

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commercialized Guar gum. Having a high viscosity indicates a thickening capacity, which was more

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beneficial to the application of excipients to the food and medicine industry.

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3.5.The effect of HPS on different cells

There are many reports that exopolysaccharide produced by microorganisms have a function of

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inhibiting cell proliferation. Lu et al. shown that the TPS of Trichoderma kanganensis could inhibit the proliferation of the mouse colon cell lines (CT26) [71]. Kokoulin et al. reported that anticancer activity

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in vitro of the sulfated lipopolysaccharide isolated from the marine bacterium Cobetia litoralis KMM 3880T [72]. Chen et al. investigated the anticancer activity of exopolysaccharide (PEP) of Antarctic bacterium Pseudoaltermonas sp. S-5 [73]. Huang et al. researched a novel exopolysaccharide (EPS) was isolated from the fermentation broth of Trichoderma pseudokoningii and studied anticancer activities on human leukemia K562 cells [74]. Ramamoorthy et al. shown extracellular polysaccharides (EPS) producing bacterium Bacillus thuringiensis RSK CAS4 was isolated from ascidian didemnum granulatum and evaluated on HEp-2 cells, A549 and Vero cell lines [75]. However, some drugs that inhibit cancer cells also affect normal cells [71]. Thus, we used L929 cells as the control to examine the inhibitory effect of HPS on cancer cells. Figure 7 demonstrated that HPS did not exhibit significant inhibitory effects on the L929 cells at 24 or 48h (cell viability dropped by 10 %), which indicated that HPS had no cytotoxicity in normal cells. On the other hand, unlike L929 cells, HPS inhibited the proliferation of A549 cells, SMMC-7721 and Hela cell in a concentration-dependent manner. Furthermore, the cell viability decreased significantly, reaching 21.86 %, 31.24 % and 37.65 % at a

Journal Pre-proof HPS concentration of 800 μg/ml in 24 h, respectively (Fig. 7A). And the inhibitory effect of HPS on cancer cells increased by 4.01 %, 3.21 % and 2.04 % at 48 h than at 24 h (Fig. 7B). Obviously, HPS can rapidly inhibit the proliferation of cancer cells on the first day and achieve a relatively high inhibition effect. The above results implied that HPS could inhibit cancer cells without affecting the normal cells.

4. Conclusions

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In this paper, we have studied the yield and properties of EPS (HPS) produced by Rhodococcus erythropolis. The yield of HPS reached 8.957 g/L by response surface optimization and the product was

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further purified. The Mw of HPS is 1.04×106 Da. The total carbohydrate, protein and lipid content in the

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HPS was found to be 79.24 %, 5.204 % and 8.45 % (w/w). And HPS was a heteropolysaccharide, which was composed of glucose, galactose, fucose, mannose and glucuronic acid, with a mass ratio of

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27.29 %, 24.83 %, 4.79 %, 26.66 % and 15.84 %, respectively. The FT-IR analysis confirmed the

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presence of the functional groups in HPS (-OH, C-O-C and C-O-H). Sugar moieties present in HPS exist both α and β-configuration by NMR spectroscopy. Degradation temperature (Td) was determined

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to be 255.4 °C on the basis of TGA curve.The HPS image presented a three-dimensional spider web with porous entanglement and straight chains by SEM and AFM. HPS also has high water holding

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capacity (189.45 ± 5.65%) and water solubility index (92.15 ± 3.05 %). The solution viscosity of HPS is 18 mPa·s in 1mg/ml and 30 rpm. Ultimately, MTT test reveal that HPS could inhibit cancer cells without affecting the normal cells.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21777113), the Natural Science Foundation of Tianjin City (Grant No. 15JCQNJC08800), the National Undergraduate Training Programs for Innovation and Entrepreneurship (No. 201810060147 and 201910060142) and the Training Project of the Innovation Team of Colleges and Universities in Tianjin.

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Conflict of interest

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All authors declared that there is no conflict of interest.

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References [1] Y.R. Saadat, A.Y. Khosroushahi, B.P. Gargari, A comprehensive review of anticancer, immunomodulatory and health beneficial effects of the lactic acid bacteria exopolysaccharides, Carbohydr. Polym. 217 (2019) 79-89. https://doi.org/10.1016/j.carbpol.2019.04.025. [2] C. Caruso, C. Rizzo, S. Mangano, A. Poli, P. D. Donato, I. Finore, Production and biotechnological potential of extracellular polymeric substances from sponge-associated Antarctic bacteria, Appl. Environ. Microbiol. 84 (2018) e01624-01617. https://doi.org/10.1128/aem.01624-17.

of

[3] S.W. Hassan, H.A. Ibrahim, Production, characterization and valuable applications of exopolysaccharides from marine Bacillus subtilis SH1, Pol. J. Microbiol. 66 (2017) 449-461.

ro

https://doi.org/10.5604/01.3001.0010.7001.

-p

[4] R.J. Roll, J.D. Vieira, J.V.S. Rosa, R.M. Castro, R.H. Tresbach, V.M. Stefenon, Exopolysaccharides

https://doi.org/10.21694/2379-7959.17003.

re

production by Mesorhizobium loti: effect of carbon source and pH, Am. Res. J. Biosc. 3 (2017) 1-5.

lP

[5] C. Delattre, G. Pierre, C. Laroche, P. Michaud, Production, extraction and characterization of microalgal and cyanobacterial exopolysaccharides, Biotechnol. Adv. 34 (2016) 1159-1179.

na

https://doi.org/10.1016/j.biotechadv.2016.08.001.

[6] J. Liu, X. Wang, H. Pu, S. Liu, J. Kan, C. Jin, Recent advances in endophytic exopolysaccharides:

Jo ur

Production, structural characterization, physiological role and biological activity, Carbohydr. Polym. 157 (2017) 1113-1124. https://doi.org/10.1016/j.carbpol.2016.10.084. [7] R. Xiao, Y. Zheng, Overview of microalgal extracellular polymeric substances (EPS) and their applications, Biotechnol. Adv. 34 (2016) 1225-1244. https://doi.org/10.1016/j.biotechadv.2016.08.004. [8] S.I. Shofia, K. Jayakumar, A. Mukherjee, N. Chandrasekaran, Efficiency of brown seaweed (Sargassum longifolium) polysaccharides encapsulated in nanoemulsion and nanostructured lipid carrier against colon cancer cell lines HCT 116, RSC Adv. 8 (2018) 15973-15984. https://doi.org/10.1039/c8ra02616e. [9] Y.C. Chen, Y.J. Wu, C.Y. Hu, Monosaccharide composition influence and immunomodulatory effects

of

probiotic

exopolysaccharides,

Int.

J.

Biol.

Macromol.

133

(2019)

575-582.

https://doi.org/10.1016/j.ijbiomac.2019.04.109. [10] S. Singh, K.S. Sran, A.K. Pinnaka, A. Roy Choudhury, Purification, characterization and

Journal Pre-proof functional properties of exopolysaccharide from a novel halophilic Natronotalea sambharensis sp. nov, Int. J. Biol. Macromol. 136 (2019) 547-558. https://doi.org/10.1016/j.ijbiomac.2019.06.080. [11] P. Kanmani, R.S. kumar, N. Yuvaraj, K.A. Paari, V. Pattukumar, V. Arul, Production and purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus phocae PI80 and its functional

characteristics

activity

in

vitro,

Bioresour.

Technol.

102

(2011)

4827-4833.

https://doi.org/10.1016/j.biortech.2010.12.118. [12] P. Gupta, B. Diwan, Bacterial Exopolysaccharide mediated heavy metal removal: A Review on biosynthesis, mechanism and remediation strategies, Biotechnol. Rep. (Amst), 13 (2017) 58-71.

of

https://doi.org/10.1016/j.btre.2016.12.006.

ro

[13] J. Schmid, Recent insights in microbial exopolysaccharide biosynthesis and engineering strategies,

-p

Curr. Opin. Biotechnol. 53 (2018) 130-136. https://doi.org/10.1016/j.copbio.2018.01.005. [14] V. Gopinath, S. Saravanan, A.R. Al-Maleki, M. Ramesh, J. Vadivelu, A review of natural

re

polysaccharides for drug delivery applications: Special focus on cellulose, starch and glycogen,

lP

Biomed. Pharmacother. 107 (2018) 96-108. https://doi.org/10.1016/j.biopha.2018.07.136. [15] Z. Xu, Q. Guo, H. Zhang, Y. Wu, X. Hang, L. Ai, Exopolysaccharide produced by Streptococcus

na

thermophiles S-3: Molecular, partial structural and rheological properties, Carbohydr. Polym. 194 (2018) 132-138. https://doi.org/10.1016/j.carbpol.2018.04.014.

Jo ur

[16] P. Rapp, C.H. Beck, F. Wagner, Formation of exopolysaccharides by Rhodococcus erythropolis and partial characterization of a heteropolysaccharide of high molecular weight, Eur. J. Appl. Microbiol. Biotechnol. 7 (1979) 67-78. https://doi.org/10.1007/bf00522480. [17] T. Neu, T. Dengler, B. Jann, K. Poralla, Structural studies of an emulsion-stabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain, Microbiology. 138 (1992) 2531-2537. https://doi.org/10.1099/00221287-138-12-2531. [18] M. Czemierska, A. Szcześ, A. Pawlik, A. Wiater, A. Jarosz-Wilkołazka, Production and characterisation of exopolymer from Rhodococcus opacus, Biochem. Eng. J. 112 (2016) 143-152. https://doi.org/10.1016/j.bej.2016.04.015. [19] M.F. Raposo, R.M. de Morais, A.M. Bernardo de Morais, Bioactivity and applications of sulphated

polysaccharides

from

marine

microalgae,

Mar. Drugs.

11

(2013)

233-252.

https://doi.org/10.3390/md11010233. [20] A.M. Mahgoub, M.G. Mahmoud, M.S. Selim, E.L.A. ME, Exopolysaccharide from marine

Journal Pre-proof Bacillus velezensis MHM3 induces apoptosis of human breast cancer MCF-7 cells through a mitochondrial

pathway,

Asian

Pac.

J.

Cancer

Prev.

19

(2018)

1957-1963.

10.22034/APJCP.2018.19.7.1957. https://doi.org/10.22034/APJCP.2018.19.7.1957. [21] M. Hamidi, R. Mirzaei, C. Delattre, K. Khanaki, G. Pierre, C. Gardarin, Characterization of a new exopolysaccharide produced by Halorubrum sp. TBZ112 and evaluation of its anti-proliferative effect on gastric cancer cells, 3 Biotech, 9 (2019) 1. https://doi.org/10.1007/s13205-018-1515-5. [22] Y. Mei, H. Zhu, Q. Hu, Y. Liu, S. Zhao, N. Peng, Y. Liang, A novel polysaccharide from mycelia of cultured Phellinus linteus displays antitumor activity through apoptosis, Carbohydr. Polym. 124

of

(2015) 90-97. https://doi.org/10.1016/j.carbpol.2015.02.009.

ro

[23] L. Ren, C. Perera, Y. Hemar, Antitumor activity of mushroom polysaccharides: a review, Food

-p

Funct. 3 (2012) 1118-1130. https://doi.org/10.1039/c2fo10279j.

[24] K. Ina, T. Kataoka, T. Ando, The use of lentinan for treating gastric cancer, Anti-Cancer Agents

re

Med. Chem. 13 (2013) 681-688. https://doi.org/10.2174/1871520611313050002.

lP

[25] W. Yu, G. Chen, P. Zhang, K. Chen, Purification, partial characterization and antitumor effect of an exopolysaccharide from Rhizopus nigricans, Int. J. Biol. Macromol. 82 (2016) 299-307.

na

https://doi.org/10.1016/j.ijbiomac.2015.10.005.

[26] X. Hu, Y. Qiao, L.Q. Chen, J.F. Du, Y.Y. Fu, S. Wu, L. Huang, Enhancement of solubilization and

Jo ur

biodegradation of petroleum by biosurfactant from Rhodococcus erythropolis HX-2, Geomicrobiol. J. (2019) 1-11. https://doi.org/10.1080/01490451.2019.1678702. [27] T. Liu, K. Zhou, S. Yin, S. Liu, Y. Zhu, Y. Yang, C. Wang, Purification and characterization of an exopolysaccharide produced by Lactobacillus plantarum HY isolated from home-made Sichuan Pickle, Int. J. Biol. Macromol. 134 (2019) 516-526. https://doi.org/10.1016/j.ijbiomac.2019.05.010. [28] M. Dubois, K.A. Gilles, J.K. Hamilton, P.t. Rebers, F. Smith, Colorimetric method for determination

of

sugars

and

related

substances,

Anal.

Chem.

28

(1956)

350-356.

https://doi.org/10.1021/ac60111a017. [29] C. Wang, H. Wang, G. Gu, Ultrasound-assisted xanthation of cellulose from lignocellulosic biomass optimized by response surface methodology for Pb(II) sorption, Carbohydr. Polym. 182 (2018) 21-28. https://doi.org/10.1016/j.carbpol.2017.11.004. [30] M. Govarthanan, S. Kamala-Kannan, T. Selvankumar, R. Mythili, P. Srinivasan, H. Kim, Effect of blue light on growth and exopolysaccharides production in phototrophic Rhodobacter sp. BT18

Journal Pre-proof isolated

from

brackish

water,

Int.

J.

Biol.

Macromol.

131

(2019)

74-80.

https://doi.org/10.1016/j.ijbiomac.2019.03.049. [31] M.A. Lopez-Ortega, A.I. Rodriguez-Hernandez, R.M. Camacho-Ruiz, J. Cordova, M. Del Rocio Lopez-Cuellar, N. Chavarria-Hernandez, Y. Gonzalez-Garcia, Physicochemical characterization and emulsifying properties of a novel exopolysaccharide produced by haloarchaeon Haloferax mucosum, Int. J. Biol. Macromol. (2019). https://doi.org/10.1016/j.ijbiomac.2019.09.087 [32] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-254.

of

https://doi.org/10.1006/abio.1976.9999.

oils

production,

Appl.

Energy.

135

(2014)

192-201.

-p

microbial

ro

[33] X. Zhang, S. Yan, R.D. Tyagi, R. Surampalli, J.R. Valéro, Wastewater sludge as raw material for

https://doi.org/10.1016/j.apenergy.2014.08.078.

re

[34] K.S. Sran, S.S. Sundharam, S. Krishnamurthi, A. Roy Choudhury, Production, characterization

Rhodobacter

johrii

CDR-SL

lP

and bio-emulsifying activity of a novel thermostable exopolysaccharide produced by a marine strain of 7Cii,

Int.

J.

Biol.

Macromol.

127

(2019)

240-249.

na

https://doi.org/10.1016/j.ijbiomac.2019.01.045.

[35] J. Zhang, L. Liu, Y. Ren, F. Chen, Characterization of exopolysaccharides produced by microalgae

Jo ur

with antitumor activity on human colon cancer cells, Int. J. Biol. Macromol. 128 (2019) 761-767. https://doi.org/10.1016/j.ijbiomac.2019.02.009. [36] D. Zhao, J. Jiang, R. Du, S. Guo, W. Ping, H. Ling, J. Ge, Purification and characterization of an exopolysaccharide from Leuconostoc lactis L2, Int. J. Biol. Macromol. 139 (2019) 1224-1231. https://doi.org/10.1016/j.ijbiomac.2019.08.114. [37] C. Saravanan, P.K.H. Shetty, Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter, Int. J. Biol. Macromol., 90 (2016) 100-106. https://doi.org/10.1016/j.ijbiomac.2015.02.007. [38] G. Vinothini, S. Latha, M. Arulmozhi, D. Dhanasekaran, Statistical optimization, physio-chemical and bio-functional attributes of a novel exopolysaccharide from probiotic Streptomyces griseorubens GD5, Int. J. Biol. Macromol. 134 (2019) 575-587. https://doi.org/10.1016/j.ijbiomac.2019.05.011. [39] M. Shirzad, J. Hamedi, E. Motevaseli, M.H. Modarressi, Anti-elastase and anti-collagenase potential of Lactobacilli exopolysaccharides on human fibroblast, Artif. Cells, Nanomed., Biotechnol.

Journal Pre-proof 46 (2018) 1051-1061. https://doi.org/10.1080/21691401.2018.1443274. [40] S. Kianpour, A. Ebrahiminezhad, M. Negahdaripour, M. Mohkam, F. Mohammadi, S.V. Niknezhad, Y. Ghasemi, Characterization of biogenic Fe (III)-binding exopolysaccharide nanoparticles produced

by

Ralstonia

sp.

SK03,

Biotechnol.

Prog.

34

(2018)

1167-1176.

https://doi.org/10.1002/btpr.2660. [41] M. Kumari, P. Ghosh, Swati, I.S. Thakur, Microcosmic study of endosulfan degradation by Paenibacillus sp. ISTP10 and its toxicological evaluation using mammalian cell line, Int. Biodeterior. Biodegrad. 96 (2014) 33-40. https://doi.org/10.1016/j.ibiod.2014.08.003.

of

[42] X. Hu, X. Pang, P.G. Wang, M. Chen, Isolation and characterization of an antioxidant

ro

exopolysaccharide produced by Bacillus sp. S-1 from Sichuan Pickles, Carbohydr. Polym. 204 (2019)

-p

9-16. https://doi.org/10.1016/j.carbpol.2018.09.069.

[43] C. Costa, R.A. Menolli, E.F. Osaku, R. Tramontina, R.H. de Melo, A.E. do Amaral,

re

Exopolysaccharides from Aspergillus terreus: Production, chemical elucidation and immunoactivity,

lP

Int. J. Biol. Macromol. 139 (2019) 654-664. https://doi.org/10.1016/j.ijbiomac.2019.08.039. [44] H. Xing, R. Du, F. Zhao, Y. Han, H. Xiao, Z. Zhou, Optimization, chain conformation and

na

characterization of exopolysaccharide isolated from Leuconostoc mesenteroides DRP105, Int. J. Biol. Macromol. 112 (2018) 1208-1216. https://doi.org/10.1016/j.ijbiomac.2018.02.068.

Jo ur

[45] P. Rapp, C.H. Beck, F. Wagner, Formation of exopolysaccharides by Rhodococcus erythropolis and partial characterization of a heteropolysaccharide of high molecular weight, Eur. J. Appl. Microbiol. Biotechnol. 7.1 (1979): 67-78. https://doi.org/10.1007/bf00522480. [46] T.T. More, J.S. Yadav, S. Yan, R.D. Tyagi, R.Y. Surampalli, Extracellular polymeric substances of bacteria and their potential environmental applications, J. Environ. Manage. 144 (2014) 1-25. https://doi.org/10.1016/j.jenvman.2014.05.010. [47] H.W. Kwak, M.K. Kim, J.Y. Lee, H. Yun, M.H. Kim, Y.H. Park, K.H. Lee, Preparation of bead-type biosorbent from water-soluble Spirulina platensis extracts for chromium (VI) removal, Algal Res. 7 (2015) 92-99. https://doi.org/10.1016/j.algal.2014.12.006. [48] G. Vinothini, S. Latha, M. Arulmozhi, D. Dhanasekaran, Statistical optimization, physio-chemical and bio-functional attributes of a novel exopolysaccharide from probiotic Streptomyces griseorubens GD5, Int. J. Biol. Macromol. 134 (2019) 575-587. https://doi.org/10.1016/j.ijbiomac.2019.05.011. [49] O. Taylan, M.T. Yilmaz, E. Dertli, Partial characterization of a levan type exopolysaccharide (EPS)

Journal Pre-proof produced by Leuconostoc mesenteroides showing immunostimulatory and antioxidant activities, Int. J. Biol. Macromol. 136 (2019) 436-444. https://doi.org/10.1016/j.ijbiomac.2019.06.078. [50] S. Singh, K.S. Sran, A.K. Pinnaka, A. Roy Choudhury, Purification, characterization and functional properties of exopolysaccharide from a novel halophilic Natronotalea sambharensis sp. nov, Int. J. Biol. Macromol. 136 (2019) 547-558. https://doi.org/10.1016/j.ijbiomac.2019.06.080. [51] O. Taylan, M.T. Yilmaz, E. Dertli, Partial characterization of a levan type exopolysaccharide (EPS) produced by Leuconostoc mesenteroides showing immunostimulatory and antioxidant activities, Int. J. Biol. Macromol. 136 (2019) 436-444. https://doi.org/10.1016/j.ijbiomac.2019.06.078.

of

[52] Y. He, M. Ye, Z. Du, H. Wang, Y. Wu, L. Yang, Purification, characterization and promoting effect

-p

169-176. https://doi.org/10.1016/j.carbpol.2014.01.080.

ro

on wound healing of an exopolysaccharide from Lachnum YM405, Carbohydr. Polym. 105 (2014)

[53] G.k. Liu, N. Li, S.-y. Song, Y.j. Zhang, J.r. Wang, Three exopolysaccharides from the liquid

re

fermentation of Polyporus umbellatus and their bioactivities, Int. J. Biol. Macromol. 132 (2019)

lP

629-640. https://doi.org/10.1016/j.ijbiomac.2019.03.057.

[54] X. Xu, C. Gao, Z. Liu, J. Wu, J. Han, M. Yan, Z. Wu, Characterization of the levan produced by

na

Paenibacillus bovis sp. nov BD3526 and its immunological activity, Carbohydr. Polym. 144 (2016) 178-186. https://doi.org/10.1016/j.carbpol.2016.02.049.

Jo ur

[55] Y. Liu, B. Zhang, S.A. Ibrahim, S.S. Gao, H. Yang, W. Huang, Purification, characterization and antioxidant activity of polysaccharides from Flammulina velutipes residue, Carbohydr. Polym. 145 (2016) 71-77. https://doi.org/10.1016/j.carbpol.2016.03.020. [56] J. Zhang, L. Liu, F. Chen, Production and characterization of exopolysaccharides from Chlorella zofingiensis and Chlorella vulgaris with anti-colorectal cancer activity, Int. J. Biol. Macromol. 134 (2019) 976-983. https://doi.org/10.1016/j.ijbiomac.2019.05.117. [57] P. P. Yue, G. Q. Fu, Y. J. Hu, J. Bian, M.F. Li, Z.J. Shi, Changes of chemical composition and hemicelluloses structure in differently aged bamboo (neosinocalamus affinis) Culms, J. Agric. Food Chem. 66 (2018) 9199-9208. https://doi.org/10.1021/acs.jafc.8b03516. [58] N. Bergström, G.B. Nair, A. Weintraub, P. E. Jansson, Structure of the O-polysaccharide from the lipopolysaccharide

from

Vibrio

cholerae

O6,

Carbohydr.

Res.

337

(2002)

813-817.

https://doi.org/10.1016/s0008-6215(02)00056-3. [59] M. Miao, Y. Ma, C. Huang, B. Jiang, S.W. Cui, T. Zhang, Physicochemical properties of a water

Journal Pre-proof soluble extracellular homopolysaccharide from Lactobacillus reuteri SK24.003, Carbohydr. Polym. 131 (2015) 377-383. https://doi.org/10.1016/j.carbpol.2015.05.066. [60] P.H. Prasanna, A. Bell, A.S. Grandison, D. Charalampopoulos, Emulsifying, rheological and physicochemical properties of exopolysaccharide produced by Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205, Carbohydr. Polym. 90 (2012) 533-540. https://doi.org/10.1016/j.carbpol.2012.05.075. [61] M. Krishnamurthy, C.J. Uthaya, M. Thangavel, V. Annadurai, R. Rajendran, A. Gurusamy, Optimization, compositional analysis, and characterization of exopolysaccharides produced by resistant

Bacillus

cereus

KMS3-1,

Carbohydr.

Polym.

of

multi-metal

(2020)

115369.

ro

https://doi.org/10.1016/j.carbpol.2019.115369.

227

-p

[62] W. Kansandee, D. Moonmangmee, S. Moonmangmee, P. Itsaranuwat, Characterization and Bifidobacterium sp. growth stimulation of exopolysaccharide produced by Enterococcus faecalis isolated

from

human

breast

milk,

Carbohydr.

Polym.

206

(2019)

102-109.

re

EJRM152

lP

https://doi.org/10.1016/j.carbpol.2018.10.117.

[63] W. Zhu, Y. Wang, F. Yan, R. Song, Z. Li, Y. Li, B. Song, Physical and chemical properties,

na

percutaneous absorption-promoting effects of exopolysaccharide produced by Bacillus atrophaeus WYZ strain, Carbohydr. Polym. 192 (2018) 52-60. https://doi.org/10.1016/j.carbpol.2018.03.063.

Jo ur

[64] A. Zykwinska, M. Marquis, C. Sinquin, L. Marchand, S. Colliec-Jouault, S. Cuenot, Investigation of interactions between the marine GY785 exopolysaccharide and transforming growth factor-beta1 by atomic

force

microscopy,

Carbohydr.

Polym.

202

(2018)

56-63.

https://doi.org/10.1016/j.carbpol.2018.08.104. [65] Y. Chi, H. Ye, H. Li, Y. Li, H. Guan, H. Mou, P. Wang, Structure and molecular morphology of a novel moisturizing exopolysaccharide produced by Phyllobacterium sp. 921F, Int. J. Biol. Macromol. 135 (2019) 998-1005. https://doi.org/10.1016/j.ijbiomac.2019.06.019. [66] E. Shaw, D.R. Hill, N. Brittain, D.J. Wright, U. Tauber, H. Marand, Unusual water flux in the extracellular polysaccharide of the cyanobacterium nostoc commune, Appl. Environ. Microbiol. 69 (2003) 5679-5684. https://doi.org/10.1128/aem.69.9.5679-5684.2003. [67] Y. Yang, F. Feng, Q. Zhou, F. Zhao, R. Du, Z. Zhou, Y. Han, Isolation, purification, and characterization of exopolysaccharide produced by Leuconostoc Citreum N21 from dried milk cake, trans. Tianjin Univ. 25.2 (2019): 161-168. https://doi.org/10.1007/s12209-018-0143-9.

Journal Pre-proof [68] D. Mudgil, S. Barak, Composition, properties and health benefits of indigestible carbohydrate polymers

as

dietary

fiber:

a

review,

Int.

J.

Biol.

Macromol.

61

(2013)

1-6.

https://doi.org/10.1016/j.ijbiomac.2013.06.044. [69] C. Saravanan, P.K. Shetty, Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter, Int. J. Biol. Macromol. 90 (2016) 100-106. https://doi.org/10.1016/j.ijbiomac.2015.02.007. [70] F. Zhao, C. Guo, Q. Cui, Q. Hao, J. Xiu, S. Han, Y. Zhang, Exopolysaccharide production by an indigenous isolate Pseudomonas stutzeri XP1 and its application potential in enhanced oil recovery,

of

Carbohydr. Polym. 199 (2018) 375-381. https://doi.org/10.1016/j.carbpol.2018.07.038.

ro

[71] Y. Lu, L. Xu, Y. Cong, G. Song, J. Han, G. Wang, Structural characteristics and

-p

anticancer/antioxidant activities of a novel polysaccharide from Trichoderma kanganensis, Carbohydr. Polym. 205 (2019) 63-71. https://doi.org/10.1016/j.carbpol.2018.09.068.

re

[72] M.S. Kokoulin, A.S. Kuzmich, A.I. Kalinovsky, S.V. Tomshich, L.A. Romanenko, V.V. Mikhailov,

lP

Structure and anticancer activity of sulfated O-polysaccharide from marine bacterium Cobetia litoralis KMM 3880(T), Carbohydr. Polym. 154 (2016) 55-61. https://doi.org/10.1016/j.carbpol.2016.08.036.

na

[73] G. Chen, W. Qian, J. Li, Y. Xu, K. Chen, Exopolysaccharide of Antarctic bacterium Pseudoaltermonas sp. S-5 induces apoptosis in K562 cells, Carbohydr. Polym. 121 (2015) 107-114.

Jo ur

https://doi.org/10.1016/j.carbpol.2014.12.045. [74] T. Huang, J. Lin, J. Cao, P. Zhang, Y. Bai, G. Chen, K. Chen, An exopolysaccharide from Trichoderma pseudokoningii and its apoptotic activity on human leukemia K562 cells, Carbohydr. Polym. 89 (2012) 701-708. https://doi.org/10.1016/j.carbpol.2012.03.079. [75] S. Ramamoorthy, A. Gnanakan, S.S. Lakshmana, M. Meivelu, A. Jeganathan, Structural characterization and anticancer activity of extracellular polysaccharides from ascidian symbiotic bacterium

Bacillus

thuringiensis,

https://doi.org/10.1016/j.carbpol.2018.02.047.

Carbohydr.

Polym.

190

(2018)

113-120.

Journal Pre-proof List of Figures and tables Fig. 1. Elution profile of the EPS from Rhodococcus sp. HX-2 on DEAE Cellulode DE-52 (A) and Sephadex G-100 (B). Fig. 2. HPLC chromatography of standard monosaccharide (A) and acid hydrolyzed EPS (B) from Rhodococcus sp. HX-2. Fig. 3. The FT-IR spectra of HPS. Fig. 4. 1H (A) and 13C (B) NMR spectra of the purified EPS from HPS. Fig. 5. The TGA curves of HPS.

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Fig. 6. The SEM (Magnification A: 1000×, B: 10000×) and AFM planar (C) and cubic(D) images of

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purified HPS.

Fig. 7. Effects of HPS on the viability of L929, A549, SMMC and Hela cells. (A) 24h (B) 48h. All data

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are expressed as mean ± S.D (*P<0.05, **P<0.01, ***P<0.001).

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Table 1. Monosaccharide component.

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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Fig. 7.

Journal Pre-proof Table 1 monosaccharide component. Run

Monosaccharide

Retention time

Content (mg/kg)

Proportion (%)

(min) Glucose

29.59

272951.96

27.29

2

Galactose

33.91

248200.49

24.83

3

Glucuronic acid

22.15

158424.50

15.84

4

Rhamnose

18.80

1055.28

0.10

5

Galacturonic acid

25.78

27.04

0.02

6

Fucose

42.02

47939.71

7

Xylose

35.30

7.42

0.02

8

Mannose

14.01

9

Arabinose

10

Ribose

4.79

266507.10

26.66

37.00

3692.08

0.38

18.00

653.46

0.06

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Total

99.95

Journal Pre-proof Author contributions: Xin Hu and Dahui Li contributed the conception and design of the study. Yue Qiao and Xiaohua Wang organized the database and analysis EPS information. Qi Zhang and Wei Zhao carried out the study. Xin Hu performed the statistical analysis and wrote section of the manuscript. Xin Hu wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the

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submitted version.