International Journal of Biological Macromolecules 139 (2019) 252–261
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Physicochemical characteristics and in vitro and in vivo antioxidant activity of a cell-bound exopolysaccharide produced by Lactobacillus fermentum S1 Kun Wang a,b,1, Mengmeng Niu a,1, Di Yao a, Jing Zhao a, Yue Wu a, Baoxin Lu a,b,⁎, Xiqun Zheng a,b,⁎ a b
College of Food science, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China National Coarse Cereals Engineering Research Center, Daqing, PR China
a r t i c l e
i n f o
Article history: Received 20 May 2019 Received in revised form 22 July 2019 Accepted 29 July 2019 Available online 30 July 2019 Keywords: Cell-bound exopolysaccharide NaCl solution Antioxidant Caenorhabditis elegans Superoxide dismutase Malondialdehyde
a b s t r a c t A cell-bound exopolysaccharide (c-EPS) from Lactobacillus fermentum S1 was isolated and purified to near homogeneity by anion exchange and gel filtration chromatography. The c-EPS is a homogeneous heteropolysaccharide with an average molecular weight of 7.19 × 105 Da and comprises mainly mannose, rhamnose, glucose, and galactose. Fourier transform infrared spectroscopy spectrum of the c-EPS exhibited typical characteristic absorption peaks of polysaccharides. Methylation and NMR analyses showed that the c-EPS had a backbone of α-D-Galp-(1 → 3), α-L-Rhap-(1 → 2), α-D-Glcp-(1 → 3), β-D-Galp-(1 → 3), β-D-Glclp-(1 → 2), and β-L-Rhap-(1 → 3,4) residues, terminated with α-D-Manp-(1 → residue. The advanced structure study indicated the c-EPS not to have a triple-helical conformation, while the microstructural study revealed a hollow porous structure for c-EPS. Further, the thermal analysis showed that the degradation temperature for the c-EPS was 288.0 °C; its peak temperature was 89.4 °C with an enthalpy value of 273.1 J/g. Moreover, the c-EPS exhibited potent DPPH, hydroxyl, and ABTS+ radicals scavenging activities, as well as FRAP in a dose-dependent manner, which could significantly enhance the T-AOC and SOD activity and reduce MDA level in Caenorhabditis elegans. Therefore, this c-EPS could be utilized as a promising natural antioxidant for application in functional foods. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Lactic acid bacteria (LAB) are a class of gram-positive and spore-free bacteria that produce large amounts of lactic acid as metabolic end products of carbohydrate fermentation. For a long time, the LAB has been an important source of nutrition, and consuming cultured milk products were considered to have health benefits and probiotic effects [1]. Exopolysaccharide (EPS), one of the most important metabolites produced by LAB, have attracted much attention in recent years due to their physicochemical properties and extensive beneficial effects in pharmacological, nutraceutical, and cosmeceutical aspects, among others [2,3]. Many studies have reported that the utilization of EPSAbbreviations: c-EPS, Cell-bound exopolysaccharide; Mw, Molecular weight; ELSD, Evaporative light-scattering detector; PMP, 1-phenyl-3-methyl-5-pyrazolone; UV, Ultraviolet; FTIR, Fourier-transform infrared spectroscopy; GC–MS, Gas chromatography-mass spectrometer; FE-SEM, Field emission scanning electronmicroscopy; DSC, Differential scanning calorimetry; TGA, Thermogram analysis; DPPH, 1,1-diphenyl-2-picrylhydrazyl; Vc, Ascorbic acid; NMR, Nuclear magnetic resonance; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); FRAP, Ferric reducing antioxidant power; T-AOC, Total antioxidant capacity; SOD, Superoxide dismutase; MDA, Malondialdehyde. ⁎ Corresponding authors at: College of Food science, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China. E-mail addresses:
[email protected] (B. Lu),
[email protected] (X. Zheng). 1 These authors contributed equally to this study.
https://doi.org/10.1016/j.ijbiomac.2019.07.200 0141-8130/© 2019 Elsevier B.V. All rights reserved.
producing LAB could improve the rheology, texture, and mouthfeel properties of various food products [3–5]. Moreover, the EPS of LAB have various functional properties, including antitumor, antioxidant, antibiofilm, cholesterol-lowering, immunostimulatory, and probiotic activities [1,6,7]. The EPS generally exist in two forms: a cell-bound exopolysaccharide (c-EPS) that firmly binds to the bacterial surface and a released exopolysaccharide (r-EPS) that is released into the surrounding medium [5]. Most of the previous studies were conducted on r-EPS, whereas only a few studies have documented c-EPS [3–6,8–10]. Besides, a systematic and comprehensive study on the physicochemical and biological activity of c-EPS from LAB is still lacking. In consideration of the important role in microbial cells protection and the play of probiotic function [4], further studies are needed to investigate c-EPS from LAB. Unregulated accumulation of free radicals could damage biomacromolecules (for example, DNA and proteins), thus accelerating the aging process and increasing the risk of chronic disorders such as cardiovascular diseases, diabetes, arthritis, cancer, rheumatoid arthritis, and atherosclerosis [8,11,12]. Even though currently used synthetic antioxidants such as butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) have proven to exert potent antioxidant effects, their safety and potential side effects remain plausible [8,12]. EPS from LAB might, therefore, be a promising safe substitute for the synthetic agents. Further studies using in vitro and in vivo models could broaden our
K. Wang et al. / International Journal of Biological Macromolecules 139 (2019) 252–261
knowledge in evaluating the antioxidant potential of LAB polysaccharides. Approximately 40% of the genes in Caenorhabditis elegans are homologous to those in humans, and the cellular processes of aging are similar between nematodes and mammals, including humans [13,14]. Its simple structure, easy mode of cultivation, and a clear genetic background make C. elegans an excellent model for in vivo study on antioxidants [15]. The present study aimed to characterize the physicochemical properties of a c-EPS from Lactobacillus fermentum S1 and evaluate its potential applications as an antioxidant in functional foods. We purified the cEPS and analyzed its molecular weight, monosaccharide composition, glycosidic bonds, advanced structure, apparent morphology, and thermal property. The antioxidant activities of the c-EPS were also investigated in vitro and in vivo.
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Sonxi Ultrasonic Instrument Co., Ltd., Shanghai, China) at 45 W, 4 °C for 5 min with an interval of 4 s On and 2 s Off. The fluid was then centrifugated, concentrated, and precipitated with 75% ethanol. The precipitate was collected, redissolved, dialyzed, lyophilized, and deproteinized following the Sevage method [17] to finally obtain a crude c-EPS. The c-EPS was purified through two steps. Crude c-EPS solution (15 mg/mL) was first loaded onto a DEAE-52 anion exchange column (26 mm × 300 mm) and eluted sequentially with deionized water, 0.1 M, and 0.3 M NaCl at a flow rate of 1 mL/min. The fractions were collected and monitored with a phenol sulfuric acid method [3]. Fractions were then subsequently loaded onto a gel filtration chromatography column (Sephadex G-100, 16 mm × 600 mm) and eluted with deionized water. The fractions were collected, concentrated, dialyzed, and finally lyophilized to give rise to purified c-EPS.
2. Materials and methods 2.4. Estimation of homogeneity and molecular weight (Mw) 2.1. Chemicals, microorganisms, and culture conditions DEAE-cellulose-52 was purchased from Waterman Co. Ltd. (Springfield Mill, UK). Sephadex G-100 was purchased from Pharmacia Biotech Co., Ltd. (Uppsala, Sweden). Fucose, rhamnose, arabinose, mannose, glucose, galactose, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5fluorouracil (5-FU), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma Chemical Co., Ltd. (St. Louis, USA). 1-phenyl-3-methyl-5-pyrazolone (PMP) was from Aladdin Chemical Co., Ltd. (Shanghai, China). Dialysis membranes (Mw cut-off 8000–14,000 Da) were from Solarbio Co., Ltd. (Beijing, China). All other reagents were of analytical grade. Lactobacillus fermentum S1 (GenBank accession no. MK226442) was isolated from Fuyuan pickle and cultured in a growth medium (1000 mL) containing 20 g of glucose, 5 g of ammonium citrate, 10 g of soya peptone, 6 g of yeast extract, 0.05 g of MnSO4, 0.04 g of FeSO4, 0.2 g of MgSO4 and 1 mL of Tween 80. The growth of the organisms was carried out at 33 °C for 24 h after the addition of 3% (v/v) inoculum. Caenorhabditis elegans (wild type N2) and Escherichia coli OP50 were obtained from the C. elegans Genetics Center (CGC, University of Minnesota). Nematodes were maintained at 23 °C on nematode growth medium (NGM) agar plates seeded with a lawn of E. coli OP50 as the food source, according to the conditions described previously by Vayndorf et al. [16].
The homogeneity and Mw of c-EPS were evaluated by an Agilent 1100 series system (Wilmington, DE) equipped with a TSK-GEL G4000SWXL column (300 mm × 7.8 mm, Tosoh Co., Tokyo, Japan) and an evaporative light-scattering detector (ELSD). Fifty microliters of sample solution were injected in the column and eluted with deionized water at 30 °C at a flow rate of 0.6 mL/min. The sample Mw was calculated by using the calibration curve of the dextran standards (1 × 104 to 80 × 104 Da). 2.5. Basic components and monosaccharide composition analysis The contents of total carbohydrate, protein, sulfate group, and uronic acid were determined by a phenol sulfuric acid method, the Bradford method, a barium chloride-gelatin method, and m-hydroxybiphenyl colorimetric method, respectively [3]. Monosaccharide composition was analyzed by 1-phenyl-3-methyl5-pyrazolone (PMP) derivative method [18]. The analysis was performed on an Agilent 1260 Infinity II HPLC with a Hypersil ODS-C18 column (250 mm × 4.6 mm, 5 μm; Dalian Elite Analytical Instruments, Dalian, China). The chromatography conditions were as follows: moving phase, a mixture of acetonitrile and 0.1 M phosphate buffer (pH 6.7) in a ratio of 20:80; flow rate, 1.0 mL/min; oven temperature, 25 °C and detection wavelength, 245 nm. The monosaccharide standards were used for quantitative and qualitative analysis.
2.2. Observation of bacterial capsules 2.6. Ultraviolet (UV) and Fourier-transform infrared spectroscopy (FTIR) After bacterial growth, L. fermentum S1 was stained using a negative staining method as described previously by Li et al. [1]. Briefly, the fresh culture was spread, air-dried, stained with crystal violet, and washed with copper sulfate solution. The bacterial capsules were viewed under a Leica DM750 optical microscope (Leica Microsystems GmbH, Solms, Germany) using an oil-immersion lens. To further confirm the bacterial capsules by electron microscopy, a JEM-2100 Plus TEM (JEOL, Tokyo, Japan) operating at 120 kV [5], we prepared the samples by mounting the thallus onto a copper grid, followed by immersing in 2% phosphotungstic acid, and then drying at ambient temperature. 2.3. Isolation and purification of c-EPS L. fermentum S1 was inoculated as described above. After two successive transfers and activation at 33 °C for 24 h and 18 h, respectively, 3% (v/v) inoculum of activated culture was again added to the growth media and incubated for another 24 h. The cultures were then centrifuged at 11,000g at 4 °C for 10 min. The extraction of c-EPS was performed using a method described by Wang et al. [4] with slight modifications. Briefly, the viscous cell precipitates were washed twice with 0.85% NaCl solution, resuspended in the same solution, and homogenized using a sonicator, FS-450 N ultrasonic processor (Shanghai
Purified c-EPS was dissolved in distilled water at 1 mg/mL and scanned by a Specord 210 Plus UV/VIS spectrometer (Analytik Jena AG, Germany) between a range of 190 nm to 400 nm. FTIR was recorded by a Bruker Tensor-27 FTIR spectrophotometer from 400 to 4, 000 cm−1 (Bruker Co., Ettlingen, Germany). 2.7. Methylation and nuclear magnetic resonance (NMR) analysis The polysaccharide was completely methylated with methyl iodide and sodium hydroxide in dimethylsulfoxide (DMSO) as described previously by Cao et al. [19]. After hydrolysis with 2 M trifluoracetic acid (TFA) at 121 °C for 90 min, the partially methylated monosaccharides were reduced with sodium borodeuteride (NaBD4) and acetylated for gas chromatography-mass spectroscopy (GC–MS) analysis using an Agilent 6890–5975 GC–MS (Palo Alto, CA, USA) equipped with a HP5MS capillary column. The initial column temperature was kept at 140 °C for 2 min, then gradually increased to 230 °C at 3 °C/min and maintained for 3 min. The structure of c-EPS was further analyzed by a Bruker Avance AV-600 NMR spectrometer (Bruker Group, Fällanden, Switzerland). To increase the solubility and reduce the viscosity, 20 mg samples were dissolved and analyzed in 500 μL D2O-CF3COOD
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(19:1, v/v). Both of 1H and 13C NMR were recorded at 313 K, and the chemical shifts (δ) were expressed as parts per million (ppm).
3. Results and discussions 3.1. Visualization of capsules
2.8. Congo red test The Congo red test was performed according to the procedure described by Li et al. [1]. Briefly, the c-EPS was dissolved at 1 mg/mL in different concentrations of NaOH solutions (0–0.4 M) containing 40 μM of Congo red. Spectra were recorded with a Specord 210 Plus UV/VIS spectrometer (Analytik Jena AG, Germany). 2.9. Field emission scanning electron microscopy (FE-SEM) The c-EPS sample was glued to aluminum stubs and gold-sputtered. The surface morphology was observed by an FE-SEM (Hitachi S-4800, Japan) at an accelerating voltage of 5 kV. 2.10. Differential scanning calorimeter (DSC) and thermogram (TG) analysis The DSC analysis was performed using a 200F3 DSC (Netzsch, Germany). A 4.5 mg of c-EPS was placed in a sealed aluminum pan and heated at a temperature range 30-300 °C with a linear heating rate of 10 °C/min. TG analysis was performed with a TG209F3 thermogravimetric analyzer (Netzsch, Germany). The c-EPS (10 mg) was placed in an Al2O3 crucible and heated at a linear heating rate of 10 °C/min over a temperature range 30-800 °C. The experiments were performed in a nitrogen atmosphere at a flow rate of 20 mL/min. 2.11. Analysis of antioxidant activity in vitro The radical scavenging activities of DPPH radical, hydroxyl radical, ABTS radical cation (ABTS+), as well as the ferric reducing antioxidant power (FRAP), were analyzed by the methods reported by Liu et al. [20] and Xiao et al. [21]. Ascorbic acid (Vc) was used as a positive control. 2.12. Analysis of antioxidant activity in vivo using C. elegans Synchronization of nematodes was achieved by preparing eggs from gravid adults with sodium hypochlorite sodium hydroxide solution [22]. Eggs were washed with M9 buffer, transferred onto fresh NGM plates covered with lawns of E. coli OP50, and incubated at 23 °C for 48 h to allow them to reach the L4 stage. Then the nematodes were transferred to NGM agar plates covered with lawns of E. coli OP50 and supplemented with 50 μM of 5-fluorouracil and increasing amounts of c-EPS (0, 25, 50, 100 or 200 μg/mL). After four consecutive days of a daily transfer, the treated nematodes were harvested, washed, and homogenized to prepare cell lysates for assaying the in vivo antioxidant activity. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were analyzed by the enzyme activity test kits commercially available from Nanjing Jiancheng Biology Engineering Institute (Nanjing, China). Data were normalized to the soluble protein of the tissue homogenate. Three independent replicates were carried out with each amount of polysaccharide. 2.13. Statistical analysis All data are expressed as mean ± standard deviation. The statistical analysis was performed by one-way ANOVA with the Duncan method (SPSS 18.0), and p b 0.05 was considered to be significant.
L. fermentum S1 was stained by the negative staining method and then viewed under an optical microscope with an oil-immersion lens. The capsules appeared as reddish halos surrounding the L. fermentum S1 cells against a dull purple background, whereas the bacterial cells stained distinctly violet (Fig. 1A). The transmission electron microscopy (TEM) analysis revealed a visible colloidal material wrapped tightly on the bacterial surface, further confirming the presence of capsules (Fig. 1B). 3.2. Isolation and composition of c-EPS The yield of crude c-EPS was 25.4 mg/g cell (dry weight). Anion exchange chromatography (DEAE-52) elution resulted in one main fraction and two trace fractions (Fig. 2A). The main fraction was further purified by a Sephadex G-100 column (Fig. 2B). A single symmetrical peak of the eluate suggested the presence of the purified c-EPS as a homogeneous polysaccharide. Further analysis by HPLC showed only one single, sharp, and symmetrical peak, confirming that the c-EPS was a homogeneous polysaccharide (Fig. 2C). According to the elution curve of the dextran standards, the average Mw of the c-EPS was estimated to be 7.19 × 105 Da, which was larger than that of the c-EPS produced by L. plantarum 70,810 (approximately 1.69 × 105 Da) [4]. The c-EPS MW was also higher than those of c-EPS from L. helveticus MB2–1 (1.83 × 105 Da), L. rhamnosus JAAS8 (5.5 × 105 Da), and Bifidobacterium animalis subsp. lactis LKM512 (4 × 105 Da), but lower than that of the c-EPS from L. plantarum EP56 (8.50 × 105 Da) [1,5,10,23]. Basic component analysis indicated that total carbohydrate, protein, and sulfate group contents of the c-EPS were 93.80%, 0.32%, and 1.05%, respectively. The presence of protein in the c-EPS was also demonstrated by the UV spectrum which showed a weak absorption peak at about 280 nm (data not shown), suggesting that the c-EPS might be a protein-bound polysaccharide [6]. However, there was no uronic acid found in the c-EPS. Monosaccharide analysis showed that the c-EPS of L. fermentum S1 comprised mainly mannose, rhamnose, glucose, and galactose at a molar ratio of 1.00: 3.81: 7.96: 5.23 (Fig. 3A and B). These data were consistent with the observations reported by Li et al. [1] which indicated that the c-EPS of L. helveticus MB2–1 comprised mainly mannose, rhamnose, glucose, galactose, and arabinose in a molar ratio of 1.01: 0.18: 3.12: 1.00: 0.16. Uemura and Matsumoto [23] also reported that the cEPS from B. animalis subsp. lactis LKM512 comprised glucose, galactose, and rhamnose in a molar ratio of 1: 2: 3. However, the c-EPS isolated from L. plantarum 70,810 was a branched homopolysaccharide consisting of only galactose residues [4]. Moreover, Nacetylglucosamine, N-acetylgalactosamine, and phosphate were also found in the c-EPS produced by various LAB [5,10]. These studies suggest that monosaccharide compositions and their molar ratios in c-EPS produced by LAB may be affected by the types of strain. 3.3. Structure of purified c-EPS The FTIR spectra were recorded with frequencies between 4000 and 400 cm−1 (wavenumbers) at the absorbance mode to investigate the functional groups of c-EPS (Fig. 3C). A broad absorption peak at 3297.37 cm−1 corresponds to the hydroxyl group (O\\H) stretching frequency [9]. The weak absorption peak at 2924.66 cm−1 is assigned to C\\H and C\\O groups, and the peaks near 1645 cm−1 and 1030 cm−1 indicate the presence of C\\O bond [8,24,25]. All of these are typical absorption peaks for polysaccharides. Moreover, a slight peak at about 1547 cm−1 corresponding to the stretching vibrations of amide groups suggest the presence of protein in the c-EPS [26]. The weak absorption at 1387 cm−1 is possibly due to the symmetric CH3
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Fig. 1. Micrographs of L. fermentum S1 capsules observed by optical microscopy (A) and TEM (B).
bending [27]. Another small peak at around 1233 cm−1 may be attributed to the S_O stretching vibrations, suggesting the possible existence of sulfate groups in the c-EPS [28]. Moreover, the peak at about 918 cm−1 is the stretching frequency of glucosyl residue in the β-
A 1.2
pyranose form [1]. All these data are in agreement with the results of basic composition analysis. The fully methylated c-EPS was hydrolyzed, reduced, acetylated, and analyzed by GC–MS. The results of analysis (Table 1) revealed that the
B 1.5
0.4
OD490nm
OD490nm
Concentration of NaCl (mol/L)
1.0
0.6
0.2
0.4 0.1 0.2 0.0
0
10
20
30
40
50
OD490nm
OD490nm
0.8
Concentration of NaCl (mol/L)
0.3
0.0
1.2
0.9
0.6
0.3
0.0 0
10
20
Tube number (10 mL/tube)
C
30
Number of tube
40
50
mV
Response (mV)
400 300 200 100 0
0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0 min
Retention time Fig. 2. DEAE-cellulose-52 anion exchange chromatogram (A), Sephadex G-100 gel filtration chromatogram (B), and HPLC chromatogram (C) of c-EPS.
60
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Fig. 3. Chromatograms of standard sugar (A) and hydrolyzed c-EPS (B), and FTIR spectrum of c-EPS (C).
3,4,6-Me3Rhap, 2,3,4,6-Me4Manp, 2,6-Me2Rhap, 3,4,6-Me3Glcp, 2,4,6Me3Glcp and 2,4,6-Me3Galp were the main methylated sugar derivatives with a molar ratio of 0.94: 1.19: 1.00: 1.35: 1.47: 2.35. Therefore, the c-EPS of L. fermentum S1 comprises mainly (1 → 2)-linked-L-Rhap, (1 → 3,4)-linked-L-Rhap, (1 → 2)-linked-D-Glcp, (1 → 3)-linked-DGlcp, (1 → 3)-linked-D-Galp, and tail (1→)-linked-D-Manp residues.
Further investigation of the c-EPS structure was carried out by the nuclear magnetic resonance (NMR). The signals of c-EPS were in the regions ranging from 3.0 to 5.2 ppm (1H NMR, Fig. 4A) and 60–105 ppm (13C NMR, Fig. 4B), which are consistent with the typical characteristic distribution of NMR signals for polysaccharides [29]. The 1H NMR spectrum showed seven anomeric proton signals appeared at 5.10, 5.00,
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Table 1 Methylation analysis data of c-EPS from L. fermentum S1. Retention time (min)
Methylated sugar
Primary mass fragments (m/z)
11.757 12.994 13.886 15.636 15.868 16.549
3,4,6-Me3Rhap 2,3,4,6-Me4Manp 2,6-Me2Rhap 3,4,6-Me3Glcp 2,4,6-Me3Glcp 2,4,6-Me3Galp
43, 72, 89, 100, 115, 131, 190 43, 71, 87, 102, 118, 129,145, 162, 205 43, 59, 87, 99, 118, 129, 160 43, 71, 88, 101, 130, 145, 161, 190, 205, 234 43, 87, 99, 101, 118, 129, 161, 174, 203, 234 43, 71, 87, 101, 118, 129, 161, 174, 203, 234
4.94, 4.76, 4.67, 4.41 and 4.37 ppm in a relative integral of nearly 1.00:0.91:1.09:0.94:0.94:1.10:0.95, and were labeled as a, b, c, d, e, f, and g, respectively. The anomeric signals appeared at the regions between 4.35 and 5.11 ppm indicated that c-EPS contained both α and β-configuration sugar residues. Based on the results of methylation analysis as well as the data reported before [1,30–34], different anomeric proton and carbon signals could be assigned. The anomeric signals at 5.10/92.28 ppm and 5.00/99.83 ppm belonged to →3)-α-DGalp-(1 → (residue a) and →2)-α-L-Rhap-(1 → (residue b), respectively [30,31]. The signals at 4.94/95.31 ppm and 4.76/102.18 ppm were attributed to →3)-α-D-Glcp-(1 → (residue c) and α-D-Manp-(1 → (residue d), respectively [31,32]. The rest of anomeric signals appeared at 4.67/97.89 ppm, 4.41/104.12 ppm and 4.37/102.67 ppm could be assigned to →3)-β-D-Galp-(1 → (residue e), →2)-β-D-Glcp-(1 → (residue f), and →3,4)-β-L-Rhap-(1 → (residue g), respectively [1,33,34]. The appearance of β-configuration glucose residues further corroborated the results of FTIR analysis. Also, the signals at 0.99/17.03 ppm and 0.94/16.56 ppm corresponded to the –CH3 groups of Rhamnose residues from →3,4)-β-L-Rhap-(1 → and →2)-α-L-Rhap-(1→, respectively [31,34]. These results suggested that c-EPS had a backbone mainly composed of α-D-Galp-(1 → 3), α-L-Rhap-(1 → 2), α-D-Glcp-(1 → 3), β-DGalp-(1 → 3), β-D-Glcp-(1 → 2), and β-L-Rhap-(1 → 3,4) residues. The tail ends were composed of α-D-Manp-(1 → residue. A possible structure of c-EPS monomer was predicted and summarized in Fig. 4C. 3.4. Conformational and morphological characterization The maximum absorption wavelength (λmax) of a triple-helical polysaccharide-Congo red-complex in diluted aqueous NaOH solution showed a redshift phenomenon compared with that of Congo red [7]. Therefore, the Congo red test was used to determine whether the cEPS exists in a triple helix conformation in this experiment. As a result, the same absorption pattern was observed for both polysaccharideCongo red solution and Congo red alone with the increasing concentrations of NaOH, suggesting that c-EPS of L. fermentum S1 exists as random coil rather than a triple-helical conformation (Fig. 4D). The FE-SEM images of S1 c-EPS (Fig. 4E and F) revealed that the surface of c-EPS was uneven and filled up with circular holes when viewed under a microscope at 2000× magnification. However, at a higher magnification of 5000×, the surface of the polysaccharide fragment appeared as a hollow porous structure covered with potholes. The high porosity structure exposes a large number of free hydroxyl groups within polysaccharides, which enhances hydration and allows good water retention capacity, thus favoring the application of c-EPS in the food industry as a texturing agent.
Molar ratio 0.94 1.19 1.00 1.35 1.47 2.35
Deduced linkage →2-L-Rhap-(1→ T-D-Manp-(1→ →3,4-L-Rhap-(1→ →2-D-Glcp-(1→ →3-D-Glcp-(1→ →3-D-Galp-(1→
of C\\C and C\\O bonds in the sugar rings [36]. The degradation temperature (Td) of 288.0 °C for c-EPS was higher than those of EPS from L. plantarum KF5, L. plantarum YW11, and L. plantarum YW32, which might be due to their different molecular compositions [27,35,37]. It is of particular note that the c-EPS (approximately 90%) was stable up to 265 °C, and therefore, the c-EPS should not be utilized near or above 265 °C, in order to protect the structural integrity of the material. Finally, the weight loss gradually decreased to leave a final residue of 21.27% of the initial weight. A relatively higher Td suggests that the c-EPS would be suitable for its applications in the food industry. Fig. 5B shows the DSC curve of the c-EPS from L. fermentum S1 in two distinct stages. The first stage ranged from 30 °C to 280 °C, including two endothermic peaks. The first endothermic peak at 89.4 °C corresponds to its melting point, and the enthalpy change needed to melt 1 g of cEPS is about 273.1 J. Different results were reported previously for other EPS produced by LAB. For example, Kanmani et al. [9] reported that the EPS produced by S. phocae PI80 had a peak melting point of 120.09 °C, and the enthalpy change needed to melt 1 g of EPS was about 404.6 J. Further, Ahmed et al. [26] mentioned that the peak temperature and the enthalpy change of L. kefiranofaciens ZW3 EPS were about 97.38 °C, and 249.7 J/g, respectively. However, when the EPS were produced by coculturing the L. kefiranofaciens with S. thermophilus, the resultant EPS have a lower peak temperature and enthalpy change, as compared to L. kefiranofaciens ZW3 EPS. The differences in thermal property might be likely due to the different molecular configurations of the polymer [36]. The following small endothermic peak started at 260.8 °C and peaked at 281.01 °C, which might be associated with the removal of proteins covalently bound with polysaccharide [38]. At the second stage, a prominent exothermic peak appeared at 296.98 °C, which was possibly due to the degradation of the polysaccharide sample. Our findings are consistent with the previous results of TG analysis. 3.6. Antioxidant activity in vitro
3.5. Thermal properties
3.6.1. Hydroxyl radical scavenging activity As shown in Fig. 6A, the scavenging activity of c-EPS was weak at a low concentration (0.25 mg/mL of the c-EPS), with only 7.06% ± 1.83%, but it significantly increased with the increasing concentration of c-EPS. The maximum scavenging activity of 68.93% ± 3.41% was observed with 4.0 mg/mL of c-EPS, which was higher than those of polysaccharides from Brassica rapa L. and L. helveticus MB2–1 at the similar concentration [39,40]. The higher scavenging capacity of c-EPS might be associated with its lower content of uronic acid, which could form inter- and intra-molecular hydrogen bonds thereby inhibiting the reactivity of hydroxyl groups in sugar chains [40]. However, the scavenging capacity of the c-EPS was not as strong as ascorbic acid (Vc).
The results of TG analysis were presented in Fig. 5A. An initial weight loss of an approximate 6% was observed between 30 °C and 110 °C, which might be due to the loss of moisture, thus suggesting that the cEPS was not completely anhydrous [35]. As the temperature increases, a dramatic weight loss (about 58.72%) occurred between 220 °C and 380 °C, which suggested the depolymerization of c-EPS due to breakage
3.6.2. DPPH radical scavenging activity As shown in Fig. 6B, c-EPS exhibited the DPPH radical scavenging effect in a dose-dependent manner ranged from 0.25 to 4.0 mg/mL. At 4.0 mg/mL, the scavenging effect of c-EPS was 83.05% ± 1.65%. It was postulated that the antioxidant activity of polysaccharides was inversely correlated with their Mw, indicating that small Mw
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Fig. 4. 1H NMR (A) and 13C NMR (B) spectrograms of c-EPS; (C) A predicted structure of c-EPS monomer; (D) The maximum absorption wavelength of Congo red-polysaccharide complex at various concentrations of NaOH solution; FE-SEM micrographs of c-EPS (E, 2000×; F, 5000×).
K. Wang et al. / International Journal of Biological Macromolecules 139 (2019) 252–261
polysaccharide should have stronger antioxidant activity [8]. However, the DPPH scavenging activity of EPS (Mw, 2.0 × 105 Da) from L. helveticus MB2–1 was lower than those of EPS (Mw, 1.15 × 10 6 Da) from L. plantarum C88, and c-EPS (Mw, 7.19 × 105 Da) from L. fermentum S1 [11,39]. These results suggested that the antioxidant capacity of polysaccharides was not the result of any single factor but a combination of several factors [41]. In addition to the Mw of the EPS, other factors such as monosaccharide composition and glycosidic linkage types may also affect the free radical scavenging activity of polysaccharides [39]. 3.6.3. FRAP assay The FRAP of c-EPS is shown in Fig. 6C and compared with Vc as a positive control. The c-EPS exhibited a positive correlation between the concentration and reducing capacity of the c-EPS in converting TPTZFe (III) complex to TPTZ-Fe (II) complex. The FRAP of c-EPS varied from 43.96 ± 2.04 μM FeSO4 equivalent to 118.70 ± 2.02 μM FeSO4
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equivalent, and that of Vc from 444.97 ± 0.54 μM FeSO4 equivalent to 459.25 ± 0.38 μM FeSO4 equivalent at the concentrations ranged from 0.25 mg/mL to 4.0 mg/mL. The FRAP of c-EPS was lower than that of Vc, indicating the FRAP of c-EPS was relatively weak. A previous study suggested that the FRAP activity of polysaccharides is affected by the protein and uronic acid contents but not by neutral sugar [42]. Therefore, the low contents of protein and uronic acid in c-EPS might be the reason for its weak FRAP activity. 3.6.4. ABTS+ radical scavenging activity As shown in Fig. 6D, a similar trend in the scavenging activity of the c-EPS was observed when compared with other radical models, and the ABTS+ radical scavenging effect of c-EPS was dose-dependent. The scavenging activity of c-EPS increased from 27.78% ± 4.01% to 99.53% ± 0.52% at the concentration range of 0.25–4.0 mg/mL. The ABTS+ scavenging activity at 4.0 mg/mL was the same as that of Vc, which showed that the c-EPS had a strong ABTS+ radical scavenging activity. The
A
B
Fig. 5. TG (A) and DSC (B) curves of c-EPS.
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Scavenging of hydroxyl free radical (%)
A
60
ABTS+ radical scavenging activity of L. fermentum S1 c-EPS was higher than those of polysaccharides (Mw, 8.38 × 105–1.51 × 106 Da) from Brassica rapa L, but lower than that of EPS (Mw, 3.87 × 104 Da) from L. plantarum KX041, suggesting that molecular weight of polymer affects the ABTS+ radical scavenging activity of polysaccharides [38,40]. Furthermore, the contents of protein and uronic acid in polysaccharides may also influence their ABTS+ radical scavenging activity [42].
40
3.7. Antioxidant activity in vivo using C. elegans
c-EPS
Ascorbic acid
100
80
20
0 0.25
0.5
1.0
2.0
4.0
Concentrations of samples (mg/mL) 100
Scavenging of DPPH free radical (%)
B
c-EPS
Ascorbic acid
80
60
40
20
0 0.25
0.5
1.0
2.0
4.0
Concentrations of samples (mg/mL)
C
500
c-EPS
Ascorbic acid
FeSO 4 equivalent (µM)
400 300 200 120
4. Conclusions
80 40
0 0.25
0.5
1.0
2.0
4.0
Concentrations of samples (mg/mL)
D
c-EPS
Ascorbic acid
100
The results of our study confirm that the c-EPS obtained from L. fermentum S1 is a branched heteropolysaccharide with an average Mw of 7.19 × 105 Da; composed of mannose, rhamnose, glucose, and galactose, and adopted a random coil conformation. The unique surface morphology and high degradation temperature would support its application in the food industry. Furthermore, the c-EPS exhibited strong antioxidant activity in vitro and could improve the T-AOC and SOD activity and decrease MDA level in Caenorhabditis elegans. The c-EPS from L. fermentum S1 might, therefore, have potential applications in functional foods as a natural agent. Acknowledgments
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This work was co-financed by Natural Science Foundation of Heilongjiang Province, China (Grant No. QC2016032), Program for Young Scholars with Creative Talents in Heilongjiang BaYi Agricultural University (No. CXRC2016-10; 2016-KYYWF-0170), Dr. Startup project in Heilongjiang BaYi Agricultural University (No. XDB201606), and was also supported by University Nursing Program of Young Scholars with Creative Talents in Heilongjiang Province (No. UNPYSCT-2018082).
+
Scavenging of ABTS free radical (%)
The effects of various concentrations of c-EPS on T-AOC, MDA levels, and T-SOD activities in C. elegans were analyzed. As shown in Table 2, the T-AOC levels were 5.73 ± 0.839, 10.27 ± 1.134, 11.07 ± 0.906, 9.79 ± 1.168 and 6.97 ± 1.049 U/mg protein, when treated with 0, 25, 50, 100, and 200 μg/mL of c-EPS, respectively. The highest T-AOC level was 0.93 times greater than that of the untreated group. It is worth noting that although the T-AOC levels of the treated group remained greater than that of the untreated group, the T-AOC levels showed a trend to increase first and then decrease as concentrations of c-EPS increased. Similarly, the T-SOD activities also showed a tendency to increase first and then decrease. The highest T-SOD activity of 71.51 ± 1.534 U/mg protein was about 1.54 times greater in the cEPS-treated group (50 μg/mL) than in the untreated group. MDA is often used as a marker of the lipid peroxidation, and an indicator of oxidative damage in cell membranes [11]. Different from the T-AOC levels and T-SOD activities, the MDA levels in C. elegans treated with different concentrations of c-EPS were significantly decreased (p b 0.05) when compared with the untreated group. The MDA levels decreased along with the increasing doses of c-EPS, and the highest dose of c-EPS resulted in the lowest MDA level. The maximum decrease amplitude reached to 85.84%. These results were consistent with a previous report, which indicated that the polysaccharide from Panax notoginseng could decrease the MDA levels in C. elegans [43]. EPS produced by LAB has been considered as one of the most potential candidates for the search for effective and safe substances with free radical scavenging and antioxidant activities [3]. Our results demonstrated that the c-EPS of L. fermentum S1 possessed significant antioxidant activities in vitro and in vivo, thereby indicating that the c-EPS could be developed as a promising safe and effective antioxidant for its future use in foods or drugs.
60
40
20 0.25
0.5
1.0
2.0
Concentrations of samples (mg/mL)
4.0
Fig. 6. Antioxidant capacity of c-EPS in vitro: Hydroxyl radical (A), DPPH radical (B) and ABTS+ radical (D) scavenging activity and FRAP (C). All values were expressed as means ± standard deviation (SD) of three replications.
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Table 2 The effects of c-EPS on T-SOD activity, T-AOC and MDA levels in C. elegans. Concentrations (μg/mL) 0 25 50 100 200
T-AOC (U/mg prot) 5.73 ± 0.839a 10.27 ± 1.134b 11.07 ± 0.906b 9.79 ± 1.168b 6.97 ± 1.049a
Growth rates (%) – 79.23 93.19 70.86 21.64
T-SOD (U/mg prot) 46.40 ± 1.036a 65.01 ± 1.242b 71.51 ± 1.534c 65.18 ± 1.693b 65.07 ± 1.945b
Growth rates (%)
MDA (nmol/mg prot)
Growth rates (%)
– 40.11 54.12 40.47 40.23
9.04 ± 1.051a 2.25 ± 0.347bc 2.62 ± 0.273b 1.50 ± 0.200bc 1.28 ± 0.131c
– −75.11 −71.02 −83.41 −85.84
Notes: data in the same line with different lowercase letters indicate significant differences (p b 0.05).
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