Accepted Manuscript

Title: Structural characterization and antioxidant property of

released exopolysaccharides from Lactobacillus delbrueckii
ssp. bulgaricus SRFM-1

Authors: Weizhi Tang, Mingsheng Dong, Weilu Wang, Shuo

Han, Xin Rui, Xiaohong Chen, Mei Jiang, Qiuqin Zhang,
Junjun Wu, Wei Li

PII: S0144-8617(17)30676-8
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2017.06.039
Reference: CARP 12427

To appear in:

Received date: 5-5-2017

Revised date: 5-6-2017
Accepted date: 9-6-2017

Please cite this article as: Tang, Weizhi., Dong, Mingsheng., Wang, Weilu., Han, Shuo.,
Rui, Xin., Chen, Xiaohong., Jiang, Mei., Zhang, Qiuqin., Wu, Junjun., & Li, Wei.,
Structural characterization and antioxidant property of released exopolysaccharides
from Lactobacillus delbrueckii ssp.bulgaricus SRFM-1.Carbohydrate Polymers
http://dx.doi.org/10.1016/j.carbpol.2017.06.039

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Structural characterization and antioxidant property of released exopolysaccharides

from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1

Weizhi Tang a,b, Mingsheng Dong a, Weilu Wang c, Shuo Hana, Xin Rui a, Xiaohong Chen a, Mei

Jiang a, Qiuqin Zhang a, Junjun Wu a, Wei Li a,b,*

a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,

P.R. China.

b
Glycomics and Glycan Bioengineering Research Center (GGBRC), Nanjing Agricultural University,

Nanjing, Jiangsu 210095, P.R. China.

c
Waigaoqiao Entry-Exit Inpection and Quarantine Bureau, Shanghai 200137, P.R. China.

*Corresponding author:

Dr Wei Li*

College of Food Science and Technology

Nanjing Agricultural University

1 Weigang Road, Nanjing, Jiangsu, P.R. China

Tel: +86 25 84396989

Fax: +86 25 84399090

E-mail address: lw1981@njau.edu.cn

 1 Highlights:

2  Three heteropolysaccharide fractions derived from L. delbrueckii ssp. bulgaricus SRFM-1;

3  Three r-EPS fractions had similar molecular mass (Mw) distributions;
4  Their possible structures were elucidated by methylation analysis, GC–MS and NMR;
5  Three r-EPS fractions exhibited high antioxidant and metal ion chelating activities;
6  Antioxidant activities were correlated with structure of different r-EPS fractions.

7 Abstract

8 Three released exopolysaccharide fractions (r-EPS1, r-EPS2 and r-EPS3) were isolated from the

9 fermented milk of Lactobacillus delbrueckii ssp. bulgaricus SRFM-1 and purified by anion exchange

10 chromatography, and characterizations of the structures were conducted. The r-EPS1 and r-EPS2 were

11 homogenous with the average molecular weights of 3.97 × 105 Da and 3.86 × 105 Da, respectively.

12 Three r-EPS fractions were composed of galactose and glucose with a molar ratio of 1.23: 1.00, 1.33:

13 1.00 and 1.00: 1.34, respectively. Structural characterization indicated that the r-EPS1 contained a

14 backbone of →6--D-Galp-(1→4)--D-Glcp-(1→4)--D-Galp-(1→4)--D-Galp-(1→6)--D-Galp-

15 (1→4)--D-Glcp-(1→4)--D-Galp-(1→4)--D-Galp-(1→4)--D-Glcp-(1→, and had three branching

16 points which existed in terminal with D-Glcp residues with /-D-(1→6) linkages. The r-EPS2 was

17 composed of →6--D-Galp-(1→4)--D-Glcp-(1→6)--D-Galp-(1→ as the backbone chain with a

18 branching point which also existed in terminal D-Glcp residue with -(1→6) linkage. In addition, three

19 r-EPS fractions exhibited strong scavenging activities on superoxide radical, hydroxyl radical, DPPH

20 radical and chelating activity on ferrous ion, and their antioxidant activities decreased in the order of r-

21 EPS1 > r-EPS2 > r-EPS3.

22 Keywords: Lactobacillus delbrueckii ssp. bulgaricus SRFM-1; Released exopolysaccharide (r-EPS);

23 Isolation; Purification; Characterization; Antioxidant activity.

2
24 1. Introduction

25 As we all know, lactic acid bacteria (LAB) are gram-positive, acid tolerant, cocci or rods bacteria and

26 are capable of producing lactic acid during heterofermentative or homofermentative metabolism (Patel,

27 Majumder, & Goyal, 2012). LAB are known through the ages for their extensive use in food, biological

28 and pharmaceutical industries. In addition, the LAB can convert sugars, proteins and fats into the

29 flavour compents and functional ingredients (Suresh Kumar, Mody & Jha, 2007). The polysaccharide is

30 a kind of long-chain polymer produced mainly by plants, animals and microorganisms with a variety of

31 biological activities (Wang, Zhao, Yang, Wang, & Kuang, 2016; Liu et al., 2017). Several LAB can

32 excrete several types of polysaccharides that are classified according to their position relative to the

33 bacteria. Those that are secreted outside the cell wall are called exopolysaccharides (EPS). The EPS

34 usually have two forms depending on their locations: cell-bound exopolysaccharides (c-EPS) which

35 closely conglutinate to the bacterial surface and released exopolysaccharides (r-EPS) that release into

36 the surrounding medium (Badel, Bernardi & Michaud, 2011).

37 Capacity of LAB to secrete a diffusely range of EPS with functionality and variable composition is

38 extending their industrial applications. EPS derived from LAB play significant role in improving the

39 texture, rheology, mouth feel of fermented food. Food associated LAB are granted the status of

40 ‘generally recognised as safe’ (GRAS) and are discovered to be the suitable candidates for the

41 production of functional EPS. At present, the development of functional foods containing LAB is an

42 expanding market with both health and economic benefits. In fact, some health benefits have been

43 attributed to some of these EPS, such as antioxidant activity (Qi et al., 2006; Li et al., 2014a),

44 antitumour activity (Li et al., 2015) and immunomodulating activity (Liu et al., 2011). Furthermore,

45 growing evidences show that biological activities of various EPS are dependent on their

3
46 monosaccharide composition, type of linkages present, molecular weight (Mw) and degree of

47 branching (Coda, Lanera, Trani, Gobbetti & Cagno, 2012; Zhang et al., 2010).

48 It is well known that Lactobacillus delbrueckii ssp. bulgaricus is one of the widely applicable LAB

49 in fermented milk products, especially yogurt and acidophilus milk, which is EPS producer and closely

50 related to human health. According to the previous published reports, the EPS production is ranging

51 from 55 to 150 mg/L for L. delbrueckii ssp. bulgaricus in fermented milk or MRS agar medium

52 (Bouzar, Cerning & Desmazeaud, 1997; Marshall and Rawson, 1999). There are a few researches

53 focused on the biosynthesis, purification and structural characterization of EPS secreted by L.

54 delbrueckii ssp. bulgaricus and also considered in some food applications (Van Calateren, Gagnon,

55 Nishimura & Makino, 2015; Bouzar, Cerning & Desmazeaud, 1996). However, there are few studies

56 on the physiological activities of EPS from L. delbrueckii ssp. bulgaricus. Since the EPS-producing L.

57 delbrueckii ssp. bulgaricus have been widely used in functional dairy products, the better

58 understanding of the structure-function relationship of EPS in these fermented products is crucial to

59 further technological applications of these biopolymers.

60 Recently, we have isolated an EPS-producing strain L. delbrueckii ssp. bulgaricus SRFM-1 from the

61 traditional Sayram ropy fermented milk (SRFM) in southern Xinjiang of China. Its real origin is

62 unknown, but it has spread from family to family. It is usually made from local yellow cattle’s milk

63 fermented by its normal microbiota. SRFM is made by a set-style fermentation process and is

64 consumed directly without maturation (Li, Mutuvulla, Chen, Jiang & Dong, 2012). In this study,

65 we described the production, isolation, purification and characterization r-EPS from L. delbrueckii ssp.

66 bulgaricus SRFM-1 by ultraviolet (UV), fourier-transform infrared spectroscopy (FT-IR), gas

67 chromatography (GC), methylation, gas chromatography-mass spectrometer (GC-MS) and nuclear

4
68 magnetic resonance (NMR) spectroscopy including one- and two-dimensional NMR (1D and 2D

69 NMR). Accordingly, the in vitro antioxidant activities of r-EPS derived from L. delbrueckii ssp.

70 bulgaricus SRFM-1 were also evaluated.

71 2. Material and methods

72 2.1. Microorganism and chemicals

73 L. delbrueckii ssp. bulgaricus SRFM-1 was isolated from traditional SRFM, which was collected

74 from southern Xinjiang province, China. It was identified according to its morphological characteristics

75 and 16S rDNA sequences analysis (Supporting Information Fig. S1). DEAE-cellulose-52 was

76 purchased from Waterman Co. Ltd. (Springfield Mill, UK). Dialysis membrane (Mw cut-off 8,000-

77 14,000 Da) was obtained from Solarbio Co., Ltd. (Beijing, China). Phenazine methosulfate (PMS), 1,1-

78 diphenyl-2-picrylhydrazyl (DPPH), vitamin C (Vc), 1,10-phenanthroline, streptomycin (100 mg/L) and

79 penicillin (100 U/mL), nitro blue tetrazolium (NBT), arabinose, mannose, rhamnose, fucose, glucose,

80 galactose, xylose and inositol were purchased from Sigma Chemical Co., Ltd. (St. Louis, Mo, USA). T-

81 series dextrans (T-500, T-200, T-100, T-50 and T-10) were obtained from Pharmacia Co., Ltd.

82 (Uppsala, Sweden). All other reagents used were of analytical grade.

83 2.2. Analysis of pH, titration acidity (TA), cell growth and r-EPS production

84 Cultivations of L. delbrueckii ssp. bulgaricus SRFM-1 for r-EPS production were performed as

85 batch cultures with inoculum concentration of 4 % (v/v) in milk at 40 °C in 5 L capacity fermenters.

86 Samples (approximately 100 mL) were collected at different time intervals from 0 to 48 h. A Schott pH

87 Meter (model, Sartorius PB-10, Germany) was used to determine the pH values during fermentation of

88 48 h. The TA (oT, expressed as Thorner degree, oT× 0.009 = lactic acid %) of the different samples was

5
 89 accomplished according to AOAC (Horwitz, 2000). The active bacteria number was evaluated by

90 different dilution plating incubated at 37 °C for 48 h with MRS agar medium. r-EPS was isolated from

91 fermentation broth as described in Section 2.3. r-EPS yields were measured based on the process

92 descripted by our previous method (Li et al., 2014b).

93 2.3. Isolation and purification of r-EPS

94 r-EPS was isolated according to our previous method (Wang et al., 2014a). In brief, fermentation

95 broth was kept at 4 °C for 12 h followed by centrifugation (4 °C, 12,000 rpm, 15 min) to remove cells

96 and denatured proteins. The protein in supernatant was further removed by adding 4% (w/v)

97 trichloroacetic acid (TCA) and kept at 4 °C for 6-8 h followed by centrifugation. The r-EPS then was

98 precipitated with 98% ethanol precipitation (1: 3 volumetric ratio) for overnight. After centrifugation,

99 the precipitate was suspended in deionized water and dialyzed using dialysis bag (Mw cut-off: 8,000-

100 14,000 Da) against distilled water for 2 days. After centrifugation, the retentate was lyophilized to

101 collect as crude r-EPS.

102 The further purification of crude r-EPS (10 mL, 10 mg/mL) was subjected to a DEAE-52 anion

103 exchange column (2.6 × 30 cm) and eluted with deionized water, 0.1 and 0.3 M NaCl at 1 mL/min flow

104 rate. Each 10 mL of elution was collected automatically and the total sugar content was measured by

105 phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956). For further study, the

106 obtained three fractions were named r-EPS1, r-EPS2 and r-EPS3, respectively, after dialysis and

107 lyophilization.

108 2.4. Chemical composition analysis

6
109 Total sugar content was measured by the phenol–sulfuric acid method as mentioned above. Protein

110 content was evaluated by Bradford method (Bensadoun & Weinstein, 1976). Uronic acid content was

111 determined by spectrophotometry with m-hydroxybiphenyl (Blumenkrantz & Asboe-Hansen, 1973).

112 Sulfate group content was analyzed with barium chloride-gelatin method according to Silvestri,

113 Simpson, Hurst and Settine (1982).

114 2.5. Homogeneity and Mw determination

115 Homogeneity and Mw were determined by high performance liquid chromatography (HPLC)

116 equipped with a TSK GEL G4000 PW XL column (300 × 7.8 mm, Tosoh Corp., Tokyo, Japan) on an

117 Agilent 1100 system and an evaporative light-scattering detector (ELSD). Sample solution (50 L) was

118 injected and eluted with distilled water at a flow rate of 0.6 mL/min with the temperature of 30 °C. The

119 linear curve was calibrated with standard T-series dextrans.

120 2.6. UV and FT-IR spectra analysis

121 Crude r-EPS, r-EPS1, r-EPS2 and r-EPS3 (1 mg/mL) were dissolved with distilled water, and

122 scanned on a Shimadzu UV-1603 spectrophotometer (Shimadzu Co., Kyoto, Japan) from 190 nm to

123 500 nm. FT-IR spectra analysis of three purified r-EPS r-EPS fractions were performed by the

124 potassium bromide (KBr) pellet method with a Bruker Tensor-27 FT-IR spectrophotometer (Bruker

125 Co, Ettlingen, Gremany) from 400 to 4000 cm−1.

126 2.7. Analysis of monosaccharide composition

127 Monosaccharide composition of three r-EPS fractions was determined by GC as described by Wang et

128 al. (2014b). Briefly, 5 mg of purified r-EPS sample was hydrolyzed with 2 mL trifluoroacetic acid

129 (TFA, 2 M) at 120 °C for 2 h. Then the hydrolysates were subjected to aldononitrile acetate precolumn-

7
130 derivatization GC. After cooling, derivatives were analyzed on Agilent HP 6890N GC (Palo Alto, CA,

131 USA) fitted with flame ionization detector (FID) and a HP-5 silica capillary column (30 m × 0.32 mm

132 × 0.25 mm, J&W Scientific Inc., Folsom, CA, USA). The operating conditions were followed used: N 2

133 as carrier gas at a flow rate of 1 mL/min; the detector and injector temperature were set at 280 °C and

134 250 °C, respectively; initial column temperature was held at 120 °C for 3 min, then increased at a rate

135 of 15 °C/min to 210 °C and maintained there for 4 min. Seven standard rhamnose, fucose, arabinose,

136 mannose, xylose, galactose, glucose and internal standard inositol were treated with the same way.

137 Calculation of the molar ratio of the monosaccharide composition was completed on the basis of each

138 peak area.

139 2.8. Methylation and GC-MS analysis for r-EPS

140 Methylation and GC-MS analysis of three r-EPS fractions were performed by using our previously

141 reported method (Li et al., 2014a). Complete methylation was confirmed by the disappearance of O-H

142 absorption band (3100–3700 cm-1) in FT-IR spectrum. Briefly, the r-EPS was hydrolyzed with 2 M

143 TFA (120 °C, 2 h). Then the methylated r-EPS fractions were reduced with NaBH4 for 2 h and

144 acetylated with pyridine-acetic anhydride (1:1, v/v) at 100 °C for 1 h. The resulting methylated alditol

145 acetate derivatives were analyzed by an Agilent 5975MSD-6890 GC-MS (Agilent Technologies, CA,

146 USA) equipped with a HP-5 capillary column used with He as carrier gas. The temperature was

147 programmed from initially 100 °C to 250 °C at 6 °C/min and maintained there for 5 min.

148 2.9. 1D and 2D NMR spectra analysis

149 The structure of r-EPS1 and r-EPS2 was performed by 1H NMR, 13C NMR and 2D NMR spectra.

150 Each sample (40 mg/mL) was dissolved in D2O (99.9 at.% D, Cambridge Isotope Laboratories, Inc.,

8
151 Andover, MA, USA) at 323 K with a Bruker AVANCE AV-500 spectrometer (Bruker Group,

152 Fällanden, Switzerland) using the residual solvent (D2O) signal as internal standard. The chemical

153 shifts (δ) are expressed in parts per million (ppm). The 2D correlated spectroscopy (COSY), total

154 correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), heteronuclear

155 multiple quantum coherence (HMBC) and nuclear Overhauser effect spectroscopy (NOESY)

156 measurements were used to assign signals and to determine the sequence of sugar residues.

157 2.10 Assay of antioxidant activity

158 The hydroxyl radical scavenging activity of different r-EPS fractions was determined using the

159 method mentioned by Li, et al. (2014a). The scavenging activity on hydroxyl radical (%) = (A sample –

160 Ablank)/(A0 – Ablank) × 100%, where A0 was the absorbance of the deionized water instead of sample and

161 H2O2 in the system. Ascorbic acid and deionized water were used as the positive and blank control,

162 respectively.

163 The superoxide radical scavenging activity was measured according to Qi, et al. (2006) with slight

164 modifications. The scavenging activity on superoxide radical (%) = (1 – Asample/Ablank) × 100%.

165 Ascorbic acid and deionized water were used as the positive and blank control, respectively.

166 The DPPH free radicals scavenging activity was measured according to the approach reported by

167 Qiao, et al. (2009) with a little modification. The scavenging activity on DPPH radical (%) = [1 –

168 (Asample – A0)/Ablank] × 100%, where A0 is the absorbance of the sample under identical condition as

169 Asample with water instead of DPPH solution. Ascorbic acid and deionized water were used as the

170 positive and blank control, respectively.

9
171 The chelating ability of metal ion was also measured according to the method reported by Qiao, et al.

172 (2009). The chelating ability on ferrousion (%) = [Ablank – (Asample – A0)]/Ablank × 100%, where A0 is the

173 absorbance of the sample under identical conditions as Asample with water instead of FeCl2 solution.

174 EDTA-2Na and deionized water were used as the positive and blank control, respectively.

175 2.11. Statistical analysis

176 Statistical analysis was performed using SPSS for windows, version 16.0. One-way analysis of

177 variance (ANOVA) and the Fisher's least significant difference (LSD) test were used to determine

178 significant differences for different samples. Differences were considered to be significant when P <

179 0.05. Data were obtained from three independent experiments and each sample was analyzed in

180 triplicates.

181 3. Results and Discussion

182 3.1. Time course of bacterial growth and r-EPS production

183 The growth of L. delbrueckii ssp. bulgaricus SRFM-1, TA of the medium, pH value and the r-EPS
184 production at different fermentation times are shown in Fig. 1. The viable count reached peak value at
185 24 h, whereas the pH values decreased throughout all fermentation periods in contrast to the TA values
186 which increased with time until of 48 h. The final pH and TA values were 3.65 and 110.31°T,
187 respectively. The amount of r-EPS increased rapidly during the first 20 h and then reached a maximum
188 value of 141.63 mg/L after the cell growth (9.35 log CFU/mL) reached the stationary phase in 28 h,
189 however the yield dropped slowly with further fermentation. The cell growth was demonstrated closely
190 related to the r-EPS production. Our results were similar to some previous reports (Zhang et al., 2013;
191 Wang et al., 2014b) which researched differences in the amount of r-EPS produced by different EPS-
192 producing Lactobacillus strains. They certified that the amount of dissimilarity was probably due to the
193 accumulation of glycohydrolases in the fermentation medium that favored the degradation of r-EPS in
194 the medium. These results indicated the amount of r-EPS largely was influenced by the LAB strains
195 and fermentation time. Therefore, r-EPS obtained at 28 h was extracted and used for further analysis.

196 3.2. Basic properties of three r-EPS fractions

10
197 The crude r-EPS (72.45%) containing small amounts of uronic acid (2.65%), sulfate (1.84%) and protein

198 (6.98%) was loaded to an DEAE-52 cellulose anion exchange column, and the corresponding

199 chromatogram showed three peaks (Fig. 2a). The obtained three fractions were named r-EPS1, r-EPS2

200 and r-EPS3, respectively. The sugar contents in r-EPS1, r-EPS2 and r-EPS3 were 96.75%, 93.54% and

201 70.92%, respectively. The relative higher contents of uronic acid (1.47% and 2.48%) and sulfate (0.51%

202 and 0.88%) in r-EPS2 and r-EPS3 (Table 1). The relatively high content of protein (0.87%) for EPS-3

203 suggested that it might be protein-bound r-EPS (Qiao et al., 2009). A further HPLC analysis of each r-

204 EPS fraction confirmed its homogeneity (Fig. 2b). Both r-EPS1 and r-EPS2 showed only one single,

205 sharp and symmetrical peak on HPLC chromatograms, indicating they should be the homogeneous r-

206 EPS fractions. In addition, their average Mw was estimated to be 3.97 × 10 5 Da and 3.86 × 105 Da based

207 on a calibration curve (Log Mw = −2.4743TR + 25.181, R2 = 0.9994, where Mw represented the molecular

208 weight, TR represented retention time) obtained from various standard T-series dextrans with different

209 Mw. In contrast, r-EPS3 was eluted as two peaks, indicating it was mainly comprised of two

210 polysaccharides with different Mw distributions. Harding et al (2005) reported that L. delbrueckii subsp.

211 bulgaricus NCFB2074 grown in skimmed milk secretes a highly branched r-EPS, which its Mw was

212 greater than 1.8 × 106 Da; Van Calsteren, Gagnon, Nishimura and Makino (2015) studied the

213 characteristic of the neutral EPS from L. delbrueckii subsp. bulgaricus OLL1073R-1. The results

214 indicated an average Mw was 5.0 × 106 Da. Both of them were comparatively larger than the r-EPS

215 fractions obtained in our study.

216 The monosaccharide composition of r-EPS1, r-EPS2 and r-EPS3 was analyzed by GC by comparing

217 the retention time with the reference sugar standards (Fig. 2c). The results indicated that all the L.

218 delbrueckii ssp. bulgaricus SRFM-1 r-EPS fractions were composed of glucose and galactose. The

11
219 molar ratios of glucose: galactose in r-EPS1, r-EPS2 and r-EPS3 were 1.00: 1.23, 1.00: 1.33 and 1.34:

220 1.00, respectively. The presences of different sugar moieties suggested that three r-EPS fractions are

221 heteropolysaccharides. The r-EPS produced by L. delbrueckii ssp. bulgaricus strain of fermented milk

222 was also found to contain galactose and glucose, but in a different molar ratio (Nagai, Makino,

223 Ikegami, Itoh, & Yamada, 2011), while the EPS produced by L. delbrueckii ssp. bulgaricus

224 NCFB2074, grown in skim milk, contained glucose and galactose in a molar ratio of approximately 3:

225 4 (Harding et al., 2005). This difference might be related to the different medium compositions, as well

226 as the type of strains and fermentation time.

227 * Not detected.

228 3.3. UV and FT-IR spectra analysis

229 The UV spectra of crude r-EPS, r-EPS1, r-EPS2 and r-EPS3 were shown in Fig. 2d. Apart from

230 crude r-EPS and r-EPS3, no obvious absorption at 280 or 260 nm were observed for r-EPS1 and r-

231 EPS2 in the UV spectra, revealing the absence of nucleic acid and protein. The FT-IR spectroscopic

232 analysis was applied to research the polar bonds and vibrations of molecules between the different

233 atoms. The FT-IR spectra for three purified r-EPS fractions were very similar and in good agreement

234 with the typical peak of a polysaccharide (Fig. 2e). A obvious major broad absorption peak within the

235 range of 3411-3437 cm-1 corresponded to intensive hydroxyl group (O-H), and a weak band at between

236 2929 and 2940 cm−1 appeared, indicating the C-O and C-H stretching vibration, whereas the peaks at

237 around 1059 cm−1 and 1654cm−1 were corresponded to C-O groups (Wang et al., 2014a). The small

238 peak at 1233 cm−1 suggested the possible presences of sulfated groups in r-EPS2 and r-EPS3, which

239 was agreed with our previous result. An absorption peak at 1542 cm−1 was observed and represented

12
240 the symmetrical COO− stretching vibration in r-EPS2 and r-EPS3 (Wang et al., 2014b; Li et al., 2014a).

241 All of spectra appeared three weak peaks around 1000-1250 cm−1 on behalf of the existence of -

242 pyranose. What’s more, the relative stronger absorption peak at 1654 cm−1 for N–H bending vibration

243 might be related to relatively high content of protein in EPS-3 (Qiao et al., 2010). Considering that

244 lower heterogeneity of r-EPS3 and higher protein content in contract to lower sugar content, it was not

245 to be further structural studied by methylation and NMR analysis.

(a)

The concentration of NaCl (mol/L)

Absorbance at 490 nm

246 Tube number (10mL/tube）

13
 Retention
9.01time
min (min)
(b)

r-EPS1

9.15 min
Response (mV)

r-EPS2

9.10 min 9.72min

r-EPS3

247

(c)

r-EPS1
Detector Response (PA)

r-EPS2

r-EPS3

248

Retention
14 time (min)
249 Wavelength (nm)

250

251

252

Crude r-EPS (d)

r-EPS3

r-EPS2
Absorbance

r-EPS1

253

254 3.4. Linkage features of three r-EPS fractions

255 After methylation for five times, the hydroxyl group absorption at 3100-3700 cm-1 in FT-IR

256 disappeared (data not shown), indicating the accomplishment of methylation. The related linkage

257 patterns and identification are shown in Table 2. The molar ratio of monosaccharide residues was

258 calculated according to the peak areas and response factor of the total ion chromatogram (TIC) in

259 Agilent GC-MS system. Methylation analysis of r-EPS1 indicated 2,3,4,6-Me4Glcp, 2,3,6-Me3Galp,

260 2,3-Me2Glcp, 2,3-Me2Galp and 2,3,4-Me3Galp were the main methylated sugar derivatives with the

261 molar ratio of 2.53: 5.70: 1.65: 1.00: 2.12. Based on above available data, r-EPS1 was mainly

262 composed of (1→4)-linked-D-Galp, (1→6)-linked-D-Galp, (1→4,6)-linked-D-Glcp, (1→4,6)-linked-

15
263 D-Galp, and tail (1→)-linked-D-Glcp as the backbone. Methylation analysis of r-EPS2 indicated

264 presences of four components, namely 2,3,4,6-Me4Glcp, 2,3,6-Me3Galp, 2,3-Me2Glcp and 2,3,4-

265 Me3Galp with the molar percentages of 8.24: 1.01: 11.43: 23.43, respectively. In the same way, r-EPS2

266 was mainly consisted of (1→6)-linked-D-Galp, (1→4,6)-linked-D-Glcp, and tail (1→)-linked-D-Glcp

267 as the backbone with some (1→4)-linked-D-Galp which could exist in the backbone or side chains.

268 Additionally, the molar ratios of D-Glcp and D-Galp residues in r-EPS1 and r-EPS2 methylated

269 experiments were respectively about 1.00: 1.87 and 1.00: 1.19, which was in good match with the

270 monosaccharide composition of r-EPS1 and r-EPS2 obtained from GC analysis.

271 3.5. Determination of the sugar residues and their sequences by 1D and 2D NMR

272 In order to gain more insight into the structural characteristics of the r-EPS1 and r-EPS2 from L.

273 delbrueckii ssp. bulgaricus SRFM-1, both detailed 1D and 2D NMR analysis were performed. In the

274 1
H NMR spectrum of a polysaccharide, the anomeric region signals between 4.4 and 5.4 ppm were

275 usually used to differentiate the anomeric protons of the polysaccharide. As shown in the 1H NMR

276 spectrum of r-EPS1 (Fig. 3a), seven proton signals occurred at 5.25, 5.21, 5.20, 5.08, 4.66, 4.59 and

277 4.48 ppm. The 13C NMR spectrum of r-EPS1 showed anomeric C-1 signals were detectable at

278 109.15, 106.35, 107.13, 105.31, 104.23, 101.22 and 98.62 ppm (Fig. 3b). It also indicated seven

279 kinds of - or -linkages existed for the D-Galp and D-Glcp residues. The complete δ of 1H and 13C for

280 r-EPS1 were assigned by 2D COSY, TOCSY, HSQC, HMBC and NOESY experiments (Supporting

281 Information Fig. S2–S4). These results from COSY, based on gradually magnetization transfers from

282 the anomeric protons, might help to determine the chemical shifts of H-2. These signals cross link to

283 the proton signals at 5.25/4.22, 4.22/4.09, 4.09/4.08, 4.08/3.97 and 3.97/4.17. The 1H–1H TOCSY

284 spectrum can reveal the internal connection of all protons of the A-G, even difficult to discriminate in

16
285 COSY spectrum. The single-bond relevances between the protons and the corresponding carbons

286 procured from HSQC spectrum of r-EPS1 in D2O viable all the 13C NMR to be appointed. The C-1

287 signals at 109.15, 107.13, 106.35, 105.31, 104.23, 101.22 and 98.62 ppm might be assigned to the A,

288 D, B, G, E, C and F, respectively. The C2–C6 resonance signals were located at the region of 62.80–

289 83.25 ppm. It had no signal near 90 ppm indicated that there was no furan ring configuration in r-

290 EPS1. From the HMBC spectrum, the linkage of residue was procured. Inspection of the NOESY

291 spectrum indicated that the B and F H-1 track NOE concatenated with A and E H-6 in agreement with

292 the B-(1→6)-A and F-(1→6)-E linkages, respectively. In addition, inter-residue NOE crosspeaks

293 between D H-1 and A H-6 supported the D-(1→6)-A linkage, and between G H-1 and E H-4 supported

294 the G-(1→4)-E linkage. Therefore, a summarized result of the information from the 1D and 2D NMR

295 spectra provided a complete assignment of all the linkage patterns, which was shown in Table 3.

296 According to these results, it has been considered that residues C, D and G were substituted at C-4, C-6

297 and C-4 position. Based on the reported data, the anomeric proton signals at 5.25 and 4.66 ppm were

298 corresponded to →4,6--D-Glcp-(1→ and →4,6--D-Glcp-(1→ containing branching point,

299 respectively. the signals at 5.21, 5.20, 5.08, 4.59 and 4.48 ppm were assigned to the T--D-Glcp-

300 (1→, →4--D-Galp-(1→, →6--D-Galp-(1→, T--D-Glcp-(1→ and →4--D-Galp-(1→,

301 respectively. On the basis of above mentioned results, the suggested repeat unit of r-EPS1 was

302 concluded (Fig. 3c). According to the Mw of r-EPS1, we could calculate that the value of repeat

303 saccharide units was 204.

304 (a)

305

17
306

307 (b)

308

309

310 (c)

311

18
312

313 Furthermore, the general representative peaks of r-EPS2 sugar residue showed four obvious  signals

314 of anomeric protons were found at 5.25, 5.19, 5.12 and 5.06 ppm in 1H NMR spectrum, designated as

315 A, B, C’ and D (Fig. 4a) . The 13C NMR chemical shifts in the area of anomeric carbon atoms also

316 suggested four kinds of - or -linkages existed for the D-Glcp or D-Galp residues (Fig. 4b). The C-1

317 signals of the residues were detectable at 109.10, 107.13, 106.30 and 98.35 ppm, respectively. In

318 addition, the cross-peaks of 5.25/109.10, 5.19/106.30, 5.12/98.35 and 5.06/107.13 ppm were

319 observed in 1H-13C HSQC spectrum (Supporting Information Fig. S5), indicating that the C-1 signals

320 corresponded to the H-1 signals respectively. Combined with the result of monosaccharide analysis, the

321 A, B and D residues might reflect three different -type glycosidic bonds of r-EPS residues, and C’

322 residues might indicate a different -type glycosidic bond of r-EPS residue. The cross-peaks

323 5.25/4.22 and 4.22/4.09 ppm were detected in COSY spectrum (Supporting Information Fig. S6).

324 Since 5.25 was H-1 of residue A,  4.22 and 4.09 ppm were assigned to H-2 and H-3 of residue A.

325 Similarly, the resonances at 4.11, 3.97, 4.21 ppm were assigned to H-4, H-5, H-6 of residue A. Based

19
326 on these data from COSY and TOCSY spectra (Supporting Information Fig. S7), the carbon signals

327 in residue A were easily assigned from HSQC spectrum as C-1 of 109.10, C-2 of 71.38, C-3 of

328 68.58, C-4 of 83.18, C-5 of 66.52, C-6 of 62.91. Similarly, the complete assignments of the 1H

329 and 13C chemical shifts of the r-EPS2 were summarized in Table 3. The sequence of each residue in the

330 repeating unit was determined based on the cross-peaks of protons observed in NOESY (Supporting

331 Information Fig. S8). The cross peak  5.19/4.21 concluded that the B H-1 track NOE concatenated

332 with A H-6 with the B-(1→6)-A linkage. Similarly, inter-residue NOE crosspeaks between D H-1 and

333 A H-4, A H-1 and C’ H-6, C’ H-1 and D H-6 supported the D-(1→4)-A, A-(1→6)-C’ and C’-(1→6)-D

334 linkages, respectively. Based on the data presented above, residues A, B, C’ and D belonged to →4,6-

335 -D-Glcp-(1→, T--D-Glcp-(1→, →6--D-Galp-(1→ and →6--D-Galp-(1→, respectively. An

336 overall, structural repeating unit of r-EPS2 was proposed as shown in Fig. 4c. In addition, according

337 to the Mw of r-EPS2, we could calculate that the value of repeat saccharide units was 596.

20
(a)

(b)

21
 In general, the r-EPS backbones of Lactobacillus spp. (such as L. delbrueckii ssp. bulgaricus, L.

rhamnosus or L. helveticus) have repeated units consist of five to twelve sugar residues, where glucose

and galactose are the main sugar residues (Welman, 2014). In addtion, EPS from LAB can be classified

into homopolysaccharides (HoPS) and heteropolysaccharides (HePS) based on their monosaccharide

composition (Patel, Majumder & Goyal, 2012). HoPS consist of identical monosaccharides, in contrast,

HePS have a great variability in structures especially branched with different types of linkages. The

denominations of HePS are complex depending on the principal monosaccharide for example,

glucogalactan or galactoglucan and further they also concern ratio of each sugar. Obviously, both of the

r-EPS1 and r-EPS2 should be named galactoglucan according to the definition.

3.6. Antioxidant activities

The antioxidant properties of r-EPS1、r-EPS2 and r-EPS3 were assessed with hydroxyl, superoxide

and DPPH radicals scavenging activities and chelating activity on ferrous ion, which compared with

those of Vc or/and EDTA-2Na (Fig. 5a–d). As shown in Fig. 5, the scavenging abilities of r-EPS and

Vc on all free radicals were in a concentration-dependent way and were decreased in descending order

of r-EPS1 > r-EPS2 > r-EPS3. At 4.0 mg/mL, the scavenging ability of r-EPS1 on hydroxyl radicals

was up to 61.12% (Fig. 5a). The scavenging activities of r-EPS1 were significantly (P < 0.05) higher

than that of r-EPS2 and r-EPS3 between 0.25 mg/mL and 4.0 mg/mL. The results suggested that the r-

EPS was effective scavenger for hydroxyl radicals, and might be better advantageous for inducing

oxidative damage to adjacent biomacromolecules, which results in aging, as well as cancer and other

diseases. The r-EPS1 and r-EPS2 (4.0 mg/mL) also showed good scavenging activities on superoxide

radicals (Fig. 5b). At a concentration of 4.0 mg/mL, the scavenging activity on superoxide radicals for

r-EPS1 were 63.75%, and EC50 value of r-EPS1 was 1.36 mg/mL, indicating that r-EPS was also

22
effective in this antioxidant attribute. Similar results were obtained at the investigation of superoxide

radicals scavenging activity of LAB Streptococcus phocae PI80 (Kanmani, Yuvaraj, Paari, Pattukumar

& Arul, 2011). For DPPH free radical, both r-EPS1 and r-EPS2 (4.0 mg/mL) showed good DPPH

radical scavenging activities (59.94% and 53.31%, respectively, Fig. 5c). It has been reported that

DPPH free radical could accept electron or hydrogen radical to become a stable molecule (Qiao et al.,

2009). These results implied the r-EPS from L. delbrueckii ssp. bulgaricus SRFM-1 might be good

hydrogen or electron donors. In addition, less chelating ability on metal ion was observed with r-EPS

than with EDTA-2Na, but the chelating ability of EPS on Fe 2+ at 4.0 mg/mL was up to 62.33% (Fig.

5d). In general, the chelating ability of all samples was in the order of r-EPS1 > r-EPS2 > r-EPS3. The

r-EPSs from L. delbrueckii ssp. bulgaricus SRFM-1 showed good Fe2+ chelating activity as evidenced

by its EC50 value (1.25 mg/mL). These results suggest that three r-EPS fractions have the strong

antioxidant activities, but the antioxidant activities and chelating ability of r-EPSs are weaker than that

of Vc and EDTA-2Na.

(a)

Scavenging activity (%)

Concentration (mg/mL)

23
(b)

Scavenging activity (%)

Concentration (mg/mL)

(c)

Scavenging activity (%)

Concentration (mg/mL)

24
Concentration (mg/mL)

vChelating activity (%)

Chelating activity (%)

Chelating activity (%)

Chelating activity (%)

Recent studies have suggested that the moderate Mw are beneficial to r-EPS for exerting their

antioxidant activities (Sun, Wang, Shi, & Ma, 2009). Our results were in a good agreement with the

existing literature findings. Also, various monosaccharide compositions might result in different carbon

backbones and branch structure of the EPS, which may be in connection with their different antioxidant

activities (Lo, Kang, Wang, & Chang, 2007). Some literatures had been reported that the antioxidant

activities of the EPS associated with the ratio of different monosaccharides components, among which

rhamnose and galactose were significant factor related to antioxidant activities (Lo, Chang, Chiu, Tsay,

& Jen, 2011). According to the results of monosaccharide composition, r-EPS1, r-EPS2 and r-EPS3

obtained similar monosaccharide composition, but with different molar ratios, specifically, the higher

25
content of galactose in r-EPS1 and r-EPS2 leading to outstanding antioxidant activities compared to r-

EPS3. On the other hand, a variety of glycosidic bonds in polysaccharides usually have a key impact on

their antioxidant properties and higher complexity is associated with stronger radical scavenging ability

(Li, Liu, Fan, Ai, & Shan, 2011). In our research, a combination of  or -type glycosidic bond content

in r-EPS-1 with respect to the -type as major glycosidic bond observed in r-EPS2 could be of

relevance to account for the essentially different antioxidant activities. Furthermore, the difference in

antioxidant activities of polysaccharides is thus was not a function of a single factor but a combination

of several factors (Li et al., 2014a; Li et al., 2015; Pan & Mei, 2010; Qiao et al., 2010). The higher

antioxidant activities of r-EPS1 as compared to the other two fractions may be attributed to the Mw,

monosaccharide composition, repeated unit and the type and proportion of glycosidic bond.

4. Conclusion

In the present work, three r-EPS fractions from L. delbrueckii ssp. bulgaricus SRFM-1 were isolated,

purified and characterized by UV, FT-IR, GC, methylation, GC-MS, 1D and 2D NMR. We also

evaluated their antioxidant activities in vitro. Results indicated that r-EPS1 exhibited higher antioxidant

among the three r-EPS fractions. These results suggested the three r-EPS fractions from L. delbrueckii

ssp. bulgaricus SRFM-1 had a potentiality for used as antioxidant foods and drugs. In order to better

understand structure-bioactivity correlations in these r-EPS fractions, further researches on their

relationship are currently preceded in our laboratory and will be reported in future.

Acknowledgements

This work was co-financed by National Natural Science Foundation of China (No. 31571818;

31371807), Natural Science Foundation of Jiangsu Province (No. BK20161448) and was also

26
supported by the Project Funded by the Priority Academic Program Development of Jiangsu Higher

Education Institutions (PAPD).

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Fig. 1. Time course of cell growth and EPS production by L. delbrueckii ssp. bulgaricus SRFM-1 at 40

°C over time from 0 to 48 h.

Wavelength (cm-1)

Transmittance (%)

(e)

32
Fig. 2. DEAE-cellulose-52 anion exchange chromatograms (a), HPLC chromatograms of relative Mw

distribution of r-EPS1, r-EPS2 and r-EPS3 (b), GC chromatograms of r-EPS1, r-EPS2 and r-EPS3 (c),

UV spectra of crude r-EPS, r-EPS1, r-EPS2 and r-EPS3 (d) and FT-IR spectra of r-EPS1, r-EPS2 and r-

EPS3 in the range of 400–4000 cm−1 (e).

Fig. 3. The 500-MHz 1H (a) and 13C (b) NMR spectrograms of r-EPS1 from L. delbrueckii ssp.

bulgaricus SRFM-1 recorded on Bruker Avance DRX-500 spectrometer in D2O; Proposed structure of

r-EPS1 from L. delbrueckii ssp. bulgaricus SRFM-1 (c).

33
(c)

Fig. 4. The 500-MHz 1H (a) and 13C (b) NMR spectrograms of r-EPS2 from L. delbrueckii ssp.

bulgaricus SRFM-1 recorded on Bruker Avance DRX-500 spectrometer in D2O; Proposed structure of

r-EPS2 from L. delbrueckii ssp. bulgaricus SRFM-1 (c).

Chelating activity (%)

(d)

34
 Fig. 5. Scavenging activities on hydroxyl radical (a), superoxide radical (b) and DPPH radical (c) and

metal ion chelating activity (d) of three r-EPS fractions and positive control. Data are presented as

means ± SD of triplicates.

Table 1. Components analysis of crude r-EPS, r-EPS1, r-EPS2 and r-EPS3.

Samples Protein (%) Neutral sugar (%) Uronic acid (%) Sulfate group (%)

Crude r-EPS 6.98 72.45 2.65 1.84

r-EPS1 nd* 96.75 0.49 0.39

r-EPS2 nd* 93.54 1.47 0.51

r-EPS3 0.87 70.92 2.48 0.88

Table 2. Methylation analysis data of r-EPS1 and r-EPS2 from L. delbrueckii ssp. bulgaricus SRFM-1.

Retention
Fractions Methylated sugar Molar ratio Deduced linkage
time (min)

15.482 2,3,4,6-Me4Glcp 2.53 T-Glcp-(1→

15.905 2,3,6-Me3Galp 5.70 →4-D-Galp-(1→

r-EPS-1
16.548 2,3-Me2Glcp 1.65 →4,6-D-Glcp-(1→

16.655 2,3-Me2Galp 1.00 →4,6-D-Galp-(1→

17.171 2,3,4-Me3Galp 2.12 →6-D-Galp-(1→

15.485 2,3,4,6-Me4Glcp 8.24 T-Glcp-(1→

15.982 2,3,6-Me3Galp 1.01 →4-D-Galp-(1→

r-EPS-2
16.552 2,3-Me2Glcp 11.43 →4,6-D-Glcp-(1→

17.184 2,3,4-Me3Galp 23.43 →6-D-Galp-(1→

Table 3. Chemical shifts of 1H and 13C NMR signals for the r-EPS1 and r-EPS2, respectively.

Fractions Residues H-1 H-2 H-3 H-4 H-5 H-6

A →4,6--D-Glcp-(1→ 5.25 4.22 4.09 4.08 3.97 4.17

B T--D-Glcp-(1→ 5.21 4.20 3.96 3.17 3.73 3.71

r-EPS1
C →4--D-Galp-(1→ 5.20 3.98 3.72 4.24 3.59 3.68

D →6--D-Galp-(1→ 5.08 4.12 3.85 3.69 3.88 3.77

35
 E →4,6--D-Glcp-(1→ 4.66 3.75 3.73 3.62 3.69 3.41

F T--D-Glcp-(1→ 4.59 3.38 3.39 3.38 3.37 3.27

G →4--D-Galp-(1→ 4.48 3.59 3.56 3.96 3.50 3.43

C-1 C-2 C-3 C-4 C-5 C-6

A →4,6--D-Glcp-(1→ 109.15 71.40 68.63 83.25 66.42 62.85

B T--D-Glcp-(1→ 106.35 70.13 67.08 75.88 69.28 62.80

C →4--D-Galp-(1→ 101.22 71.53 70.58 73.72 73.71 65.70

D →6--D-Galp-(1→ 107.13 74.88 70.95 75.25 73.72 79.52

E →4,6--D-Glcp-(1→ 104.23 73.25 75.56 72.65 73.13 64.75

F T--D-Glcp-(1→ 98.62 75.51 75.52 80.05 73.66 81.25

G →4--D-Galp-(1→ 105.31 73.75 73.70 79.81 73.37 63.55

H-1 H-2 H-3 H-4 H-5 H-6

A →4,6--D-Glcp-(1→ 5.25 4.22 4.09 4.11 3.97 4.21

B T--D-Glcp-(1→ 5.19 4.20 3.94 3.16 3.66 3.67

C’ →6--D-Galp-(1→ 5.12 3.87 3.68 6.62 4.15 4.18

D →6--D-Galp-(1→ 5.06 4.12 3.83 3.75 3.90 3.89

r-EPS2
C-1 C-2 C-3 C-4 C-5 C-6

A →4,6--D-Glcp-(1→ 109.10 71.38 68.58 83.18 66.52 62.91

B T--D-Glcp-(1→ 106.30 70.09 67.00 75.88 69.28 62.99

C’ →6--D-Galp-(1→ 98.35 69.12 69.55 74.42 71.78 69.98

D →6--D-Galp-(1→ 107.13 74.88 70.95 75.12 70.52 67.02

36