Preparation screening, production optimization and characterization of exopolysaccharides produced by Lactobacillus sanfranciscensis Ls-1001 isolated from Chinese traditional sourdough

International Journal of Biological Macromolecules 139 (2019) 1295–1303

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Preparation screening, production optimization and characterization of exopolysaccharides produced by Lactobacillus sanfranciscensis Ls-1001 isolated from Chinese traditional sourdough Guohua Zhang a,⁎, Weizhen Zhang a, Lijun Sun b, Faizan A. Sadiq c, Yukun Yang a, Jie Gao d, Yaxin Sang d a

School of Life Science, Shanxi University, Taiyuan 030006, China College of Food Science and Engineering, Northwest A & F University, Yangling 712100, China c School of Food Science and Technology, Jiangnan University, Wuxi 214122, China d College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China b

a r t i c l e

i n f o

Article history: Received 22 July 2019 Received in revised form 2 August 2019 Accepted 8 August 2019 Available online 08 August 2019 Keywords: Lactobacillus sanfranciscensis Exopolysaccharides Chinese traditional sourdough Optimization Characterization

a b s t r a c t The present study focused on preparation screening, production optimization and characterization of exopolysaccharides (EPS) produced by Lactobacillus sanfranciscensis isolated from a Chinese traditional sourdough sample. In addition, in vitro antioxidant activity of pure EPS was determined. A total of 51 strains of L. sanfranciscensis were screened, and L. sanfranciscensis Ls-1001 was proved as the most abundant crude EPS producing strain, which was selected for further EPS optimization and characterization. The optimized conditions resulted in around 31% increase in EPS production (final yield: 249.30 mg/L) and included: maltose 25 g/L, yeast peptone 10.24 g/L and fresh yeast extractives (FYE) 12.92 mL/L. HPLC analysis showed that pure EPS contained only glucose monomers, indicating that it was a kind of homopolysaccharides. FTIR spectroscopy revealed the presence of hydroxyl and carbonyl groups. NMR analysis confirmed that EPS contained α-glycosidic bond and pyranose residue. The EPS showed strong in vitro antioxidant activity. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Sourdough is a mixture of water and flour (mainly wheat flour), fermented by lactic acid bacteria (LAB) and yeasts [1,2]. The use of sourdough as a leavening agent for production of baked artisan and steamed breads is well acknowledged throughout the world. Chinese wheatbased steamed bread or Chinese Mantou, is a traditional and favorite food in China that is made by wheat flour dough and cooked by steaming after fermentation using a handful of sourdough [3]. It was originated from northern China N1700 years ago, and spread to other Asian countries including Japan, Korea and Southeast Asia, where it has now become popular as instant foods or snacks [4]. Sourdough, as a result of the metabolic activities of included complex microbiota, mainly comprises yeasts and lactic acid bacteria. The complex interplay between the flour ingredients and indigenous enzymes of microbiota, plays a key role in increasing the shelf-life and improving the quality characteristics of breads. Breads fermented by sourdoughs have been reported to possess better crumb structure and texture, volume, nutritional, and sensory properties as compared to non-sourdough-based breads [5]. Until now, N80 bacterial species have been identified from sourdough ecosystem, which mainly belong to the genera Lactobacilli, ⁎ Corresponding author at: Shanxi University, Wucheng Road 92, Taiyuan, China. E-mail address: [email protected] (G. Zhang).

https://doi.org/10.1016/j.ijbiomac.2019.08.077 0141-8130/© 2019 Elsevier B.V. All rights reserved.

Leuconostocs, Lactococci, Enterococci and Streptococci [6]. However, Lactobacillus sanfranciscensis is considered as the most predominant and key lactic acid bacterium in traditionally fermented sourdoughs [7–10]. L. sanfranciscensis, is firstly discovered and isolated from San Francisco sourdoughs by Kline and Sugihara [11]. It is an obligate heterofermentative species with phylogenetic relationship to the Lactobacillus casei–Pediococcus group. This species uses maltose as the main carbon source [12], and its growth has been reported to be affected by the carbon dioxide produced by Saccharomyces cerevisiae during coculture fermentation [13]. During sourdough fermentation, L. sanfranciscensis converts glutamine to glutamate through chemical deamidation, which improves bread flavor [14]. In addition to its positive contribution towards aroma, L. sanfranciscensis has also been reported to play an important role in extending the shelf-life and the overall textural properties of sourdough-based breads [5,15]. L. sanfranciscensis strains confer various value-added traits to breads. For instance, it increases the bioavailability of minerals and bioactive compounds in breads, by producing higher levels of phytase [16]. Exopolysaccharides produced by LAB are a kind of typically high molecular weight polymers [17]. The classification of EPS is based on the composition of monosaccharides or its chain units. A homopolysaccharides (HoPSs) contains only one type of monosaccharide that serves as a repeating unit, while a hetero-polysaccharides (HePSs) contains two or more monosaccharides [18]. It is well known

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that the EPS produced by lactic acid bacteria shows physiochemical properties like commercial hydrocolloids or gums, in terms of water binding capacity and resultant hydrocolloid formation. Therefore, EPS produced by L. sanfranciscensis is an ideal alternative to xanthan and guar gum, both of which are generally used to improve the rheology of dough and bread texture [19]. In addition, EPS have exceptional some other bioactivities, such as cholesterol-lowering potential and immunomodulating, antioxidant, antiviral and anticoagulant effects, making the biopolymers industrially and functionally more important [20]. According to the report, EPS produced by L. sanfranciscensis are known to have positive influence on bread rheology, volume, texture and shelf-life [21]. Therefore, this study aimed to screen high yield EPS-producing L. sanfranciscensis strains from Chinese traditional sourdough followed by the optimization of their production conditions and EPS characterization including component analysis using FTIR spectrum, analysis of the monosaccharide composition, glycosidic bond type, AFM and the in vitro antioxidant activity. The results obtained in this study may enrich the information on metabolites of L. sanfranciscensis in traditional sourdough and its application potentials. 2. Materials and methods

medium constant. For screening the nitrogen source, peptone, yeast peptone, soya peptone, or tryptone were used while keeping other components constant. The above carbon and nitrogen sources have concentrations of 20 g/L and 10 g/L, respectively. The mMRS medium including two growth promoting factors, which are B vitamin mix (1 g/L) [25] and fresh yeast extractives (FYE, 15 mL/L) [11]. The medium containing the carbon source was sterilized at 115 °C for 20 min.

2.4.2. Response surface optimization experiment The results of single factor screening showed that maltose, yeast peptone, and FYE had a significant effect on the crude EPS production by L. sanfranciscensis. Therefore, these three factors were selected for response surface design. These factors are denoted as A, B and C, respectively. Then, the response surface methodology was used to optimize the screening variables for the EPS production based on the central composite design. The test was carried out in accordance with Table 1 (A and B). The experimental matrix was designed by design using DesignExpert 8.0.6 (Stat-Ease Inc., East Hennepin Ave., Minneapolis, MN, USA). The experiment was carried out 20 times, and the software was used to perform regression analysis on the data obtained from the design experiment.

2.1. Materials 2.5. EPS characterization 51 L. sanfranciscensis isolated from Chinese steamed bread were provided by the college of Life Science, Shanxi University, China. All strains are stored at −80 °C. 2.2. Composition of the culture medium The strains of L. sanfranciscensis was cultured at 30 °C in mMRS medium containing the following components per liter: peptone, 10 g; yeast extract, 5 g; beef extract, 10 g; maltose, 20 g; sodium acetate, 5 g; K2HPO4, 2 g; ammonium citrate, 2 g; MgSO4, 0.2 g; MnSO4, 0.5 g; Tween 80, 1 mL; pH 5.4. The medium was sterilized by heating to 121 °C for 20 min.

2.5.1. Fourier-transform infrared (FTIR) spectroscopy and ultraviolet spectral (UV) analysis The pure EPS were analyzed by FTIR spectroscopy. The spectra were recorded with a light source in the middle infrared range in a Thermo Scientific Nicolet IS50. Chemical characterization included UV was carried out using UV spectroscopy.

Table 1 Levels of process variables (A), central composite design and the crude EPS production by L. sanfranciscensis Ls-1001 (B).

2.3. Isolation and purification of EPS A: Levels of process variables used in central composite design.

For the isolation of EPS, all strains were grown in mMRS culture, inoculated at 1% (v/v) with an overnight culture then incubated at 30 °C for 24 h anaerobically [22]. Protein was removed by adding trichloroacetic acid to the fermentation solution. The cells were separated from the culture medium by centrifugation at 10,000 r/min for 20 min at 4 °C. The supernatant was collected and 4 volumes of chilled ethanol was added. The mixture was stored at 4 °C overnight to precipitate the carbohydrate and centrifuged again [23]. The separated precipitate was dissolved in distilled water and dialyzed for 8–12 h to obtain the crude EPS. The crude EPS was freeze dried and then fractionated through anionexchange chromatography on a DEAE-Berpharose FF column (1.6 cm × 30 cm). The crude sample (10 mg/mL) was dissolved in Tris-HCl buffer (0.05 mol/L, pH 7.60) injected into the chromatographic column at a flow rate of 1 mL/min and eluted by applying a linear gradient of sodium chloride (NaCl) concentration (0.2–1.0 M). The phenol sulfuric acid method was used for the determination of EPS content, with glucose as standard. Each single peak was collected separately and freeze dried to obtain the pure EPS fractions [23,24]. 2.4. Response surface optimization experiment 2.4.1. Selection of carbon source, nitrogen source and growth promoting factor For screening carbon source, glucose was replaced by maltose, sucrose, fructose, galactose while keeping other components in the

Factor

Levels

A Maltose (g/L) B Yeast peptone (g/L) C FYE (mL/L)

1.682

1

0

−1

−1.682

11.59 6.64 8.29

15 8 10

20 10 12.5

25 12 15

28.41 13.36 16.7

B: Central composite design and the crude EPS production by L. sanfranciscensis Ls-1001. Run order

A

B

C

Crude EPS (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

11.59 25 20 20 20 20 20 25 15 20 25 15 20 15 15 20 20 20 25 28.41

10 12 10 10 13.36 10 10 12 8 10 8 12 10 12 8 6.64 10 10 8 10

12.5 15 12.5 8.29 12.5 12.5 16.7 10 15 12.5 10 15 12.5 10 10 12.5 12.5 12.5 15 12.5

177.18 ± 2.54 236.70 ± 4.95 232.53 ± 5.80 224.40 ± 3.86 224.58 ± 4.23 240.99 ± 3.14 219.79 ± 0.60 236.70 ± 3.14 200.75 ± 3.02 238.67 ± 1.69 230.55 ± 1.33 199.39 ± 0.85 233.55 ± 4.59 202.03 ± 3.86 199.56 ± 0.36 206.72 ± 4.11 237.99 ± 5.92 245.94 ± 5.55 242.85 ± 7.61 245.75 ± 0.24

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solution until the absorbance was 0.70 ± 0.02. Different concentrations of sample and ABTS•+ working solution were mixed and protected from light, and blanks and sample controls were set. These mixtures could stand at 37 °C for 8 min, the absorbance was measured at 734 nm. Ascorbic acid was used as the positive control. The formula for calculating the free radical scavenging activity was as follows:

2.5.2. Determination of monosaccharide composition The monosaccharide composition of pure EPS was determined by high performance liquid chromatography (HPLC), adjust slightly according to Zhu et al. [26]. The pure polysaccharide solution (1 mg/mL) was placed in a glass stopper tube, added to 0.5 mL of 2 M trifluoroacetic acid (TFA), and heated at 120 °C for 2 h to hydrolyze the polysaccharide into component monosaccharides. After being cooled to room temperature, the supernatant was collected by centrifugation (8000 r/min, 5 min), dried by adding 500 μL of methanol and purged with stream of nitrogen. This process was repeated three times to completely remove TFA. The dried sample is re-dissolved in distilled water and filtered with 0.45 mm filter membrane, resulting in 1-phenyl-3 methyl5-pyrzolone (PMP) derivatization procedure. According to the previously described, the PMP derivative program was properly modified [26–28]. The hydrolyzed sample or the monosaccharide standard (100 μL) was mixed with 0.3 M aqueous NaOH (120 μL) and 0.5 M methanol solution (120 μL) of PMP. The mixture was heated in a water bath at 70 °C for 30 min, then cooled to room temperature and neutralized with a 0.3 M HCl solution (200 μL). The obtained solution was extracted with 1 mL of chloroform, and the process was repeated three times and the upper aqueous phase was passed through 0.45 μm membrane to be analyzed. Monosaccharides such as D(+)-Glucose (Glu), DL-Arabinose (Ara), D-Galacturonic acid (GalA), D-N-acetylglucosamine (GlcNAc), L(−)Fucose (Fuc) were used as standard. HPLC analysis was carried out on a Waters 1525 Binary HPLC Pump equipped with an UV/Visible Detector (Waters 2489). The analytical column used was Thermo C18 (250 × 4.6 mm, 5 μm). Use the following conditions for analysis: mobile phase A:B = 73:27 (A: deionized water, B: acetonitrile); flow rate: 1.0 mL/min; injection volume: 10 μL; UV detector: 250 nm; column temperature: 35 °C.

Inhibition=% ¼ ð1−ðA1 −A2 Þ=A0 Þ  100 In the formula: A0: black control group; A1: sample group; A2: sample control group. 2.7. Statistical analysis Statistical comparisons were performed using one-way analysis of variance (ANOVA). All data were presented as the mean ± standard deviation (SD). P values of b0.05 were considered statistically significant. 3. Results and discussion 3.1. Screening for high-yield EPS strains The results regarding the extraction and production of crude EPS by 51 strains of L. sanfranciscensis (Fig. 1), showed that L. sanfranciscensis Ls-1001 yielded the highest EPS production with the EPS concentration of 190.32 ± 2.36 mg/L in fermentation broth. 3.2. Response surface optimization experiment 3.2.1. Selection of carbon source, nitrogen source and growth promoting factors The effects of different carbon and nitrogen sources and growth promoting factors on crude EPS production by L. sanfranciscensis Ls-1001 are suggested in Table 2. There is a statistically significant difference (p b 0.05) between the two carbon sources, with maltose (162.96 mg/L) as more favorable for EPS production by L. sanfranciscensis Ls-1001. These findings are consistent with the results obtained by Gobbetti and Corsetti [12], in which L. sanfranciscensis preferentially fermented maltose rather than glucose for growth. Surprisingly, L. sanfranciscensis Ls-1001 did not grow in the medium containing sucrose, fructose, and galactose (data not shown). Therefore, maltose was chosen as the sources of carbon for further optimization of production. The results for the screening of nitrogen source show that yeast peptone was more beneficial to the production of EPS (182.60 mg/L) by L. sanfranciscensis Ls-1001. The study confirmed that there is a mechanism of metabolite complementation between yeasts and lactic acid bacteria, i.e., yeasts provide nutrients for the growth of lactic acid

2.5.3. Nuclear magnetic resonance (NMR) spectroscopy analysis of EPS Pure EPS (18 mg) was dissolved in 1.0 mL of D2O and was analyzed. 1 H and 13C NMR spectra were recorded on a BRUKER AVANCE III 600 MHz spectrometer. 2.5.4. Morphological characterization of EPS Morphology of pure EPS was determined by atomic force micrograph (AFM). The exopolysaccharides concentration was 10 μg/L. 2.6. Antioxidant activity of EPS The antioxidant activity of pure EPS was determined using an ABTS radical cation scavenging assay as slightly modified as previously described [29]. 7 mM ABTS solution was mixed with 2.45 mM K2S2O8 solution in an equal volume, and reacted in the dark at room temperature for 16 h to obtain ABTS•+ working solution. Before the measurement, the working solution was diluted with PBS (pH = 7.4)

250

200 ** **

** **

**

** ** ** ** **

** ** ** ** ** **

**

**

** ** *

**

150

** **

**

**

100 **

** ** **

**

50

**

**

**

** **

**

** **

** **

**

**

Fig. 1. EPS content of different L. sanfranciscensis strains. *: significant (p b 0.05); **: very significant (p b 0.01).

Zj3

Gs5

Gs1

Gm5

Zj2

Xj3

Sx13

Zj6

Hr8

Sx4

Gs9

Sx2

Ts11

Ah4

Zj15

Sx5

Ah10

Ah9

Wf14

Wf13

Gs2

Xj15

Sd1

Ah7

Gs11

Sd1-3

Ts4

Sd3

Gm2

Zj9

Ls-1001

Zj7

Ah8

Wf12

Gs14

Sd4

Gs7

Sd2

Ah6

Gs3

Ah5

Bj2

Ts14

Bj1

Ts8

Hb1

Ts3

Zj4

Sx10

Ts9

0 Sx1

EPS (mg/L)

**

Strain

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G. Zhang et al. / International Journal of Biological Macromolecules 139 (2019) 1295–1303 Table 2 Effects of different carbon source, nitrogen source and growth promoting factors on crude EPS production of L. sanfranciscensis Ls1001. Factor

Crude EPS (mg/L)

Carbon source Maltose Glucose

162.96 ± 1.33a 130.86 ± 3.40b

Nitrogen source Peptone Tryptone Soya peptone Yeast peptone

161.79 ± 1.46c 163.46 ± 3.13c 169.72 ± 1.76b 182.60 ± 0.33a

Growth promoting factors mMRS mMRS+VB mMRS+FYE

177.75 ± 1.42b 181.49 ± 2.04a 184.46 ± 1.62a

Data based on three trials. Values are the mean ± SD. Different letters represent significant differences between the data (p b 0.05).

bacteria in the fermentation process, and the metabolites of lactic acid bacteria serve as energy sources for yeasts [30]. This also confirms that yeast-derived peptone promotes the growth of L. sanfranciscensis. Thus, yeast peptone was selected as a nitrogen source for further optimization. Among growth promoting factors, FYE was proved to be more beneficial for EPS production, the quantity of EPS was 184.46 mg/L. Kline and Sugihara [11] mentioned that the FYE stimulated the growth of L. sanfranciscensis. Therefore, FYE was chosen as the growth promoting factor for further optimization. 3.2.2. Response surface optimization experiment The three factors (maltose, yeast peptone and FYE) that significantly influenced EPS production were taken into account for the central composite design under response surface optimization to optimize crude EPS production by L. sanfranciscensis Ls-1001. The response values of 20 experiments are given in Table 1B. By fitting the experimental results into the central composite design under response surface optimization experiment, the equation (in terms of actual factors) used to predict the maximum EPS production is given below:

EPS ¼ −315:56 þ 16:22A þ 42:78B þ 21:31C−0:014AB þ 0:14 AC−0:40BC−0:35A2 −1:82B2 −0:80C2 where A is maltose, B is yeast peptone, C is FYE, respectively. Table 3 Optimization of variance analysis of EPS production. Source

Sum of squares

Df

Mean square

F value

P-value (Prob N F)

Model A-Maltose B-Yeast peptone C-FYE AB AC BC A2 B2 C2 Residual Lack of fit Pure error Cor total

6973.91 4964.83 71.01 0.7 0.15 23.63 32.56 1101.82 760.81 358.13 299.33 177.61 121.72 7273.23

9 1 1 1 1 1 1 1 1 1 10 5 5 19

774.88 4964.83 71.01 0.7 0.15 23.63 32.56 1101.82 760.81 358.13 29.93 35.52 24.34

25.89 165.87 2.37 0.023 5.15E-03 0.79 1.09 36.81 25.42 11.96

b0.0001⁎⁎ b0.0001⁎⁎ 0.1545 0.8816 0.9442 0.3952 0.3215 0.0001⁎⁎ 0.0005⁎⁎ 0.0061⁎⁎

1.46

0.3442

R2 = 0.9588; R2Adj = 0.9218; Adeq precision = 16.579. ⁎⁎ Very significant (p b 0.01).

The analysis of variance on the results of response surface method to optimize EPS production by L. sanfranciscensis Ls-1001 is shown in Table 3. The model F value = 25.89 (p b 0.01), indicated that the fitted model was significant. The correlation coefficient (R2) was calculated as 95.88%, indicating that the regression, of the model was good. Similarly, the correction decision coefficient (R2Adj) was 92.18%, indicated that the variability of most test data can be explained by the model. Adeq precision measures the signal to noise ratio. A ratio N4 is desirable. The precision of this test reached at 16.6, which was considered reasonable. This model is reliable and can predict the quantity of the EPS satisfactorily. Three-dimensional response surface graphs and contour plots were produced using two independent variables to study the optimal level of each variable and its effect on EPS production, while the other variables remained constant at the central level (Fig. 2). Fig. 2 shows the interaction between each two factors. As the concentration of maltose increased, the yield of EPS increased gradually (Fig. 2A and B). There was a noticeable interaction between yeast peptone and FYE (Fig. 2C). Through analysis of the study, the model predicted that when maltose, yeast peptone and FYE were set to 25 g/L, 10.24 g/L and 12.92 mL/L, respectively, maximum EPS yield can be achieved. The maximum predicted value of EPS production was 248.79 mg/L. The experimental EPS yield was 249.30 mg/L under the selected optimal conditions, which was comparable to the predicted one. Besides, the yield was increased by 30.99% when compared with that obtained under the original medium and fermentation conditions (190.32 mg/L). Therefore, the model developed in this study accurately and reliably predicts the crude EPS produced by L. sanfranciscensis Ls-1001. 3.3. EPS characterization 3.3.1. Fourier-transform infrared (FTIR) spectroscopy and ultraviolet spectral (UV) analysis The FTIR spectra of the pure EPS (Fig. 3-A) exhibited bands at various levels. The intense peak at 3295.86 cm−1 generated from the hydroxyl group stretching region. The peak at 2925.89 cm−1 was due to C\\H stretching vibration. The band at 1651.74 cm−1 was associated with the stretching vibration of C_O. The peak at 1517.21 cm−1 and 1375.78 cm−1 possibly represented the vibration stretching of alkyl hydrogen (CH2-CH3) in aliphatic alkyl group (R-CH2-CH3) [31]. The absorption peak at 1026.23 cm−1 was due to the α-(1 → 6) glycosidic bond in the polysaccharide [32]. The absorption peak at 914.36 cm−1 was attributed to the asymmetric stretching vibration of the pyran ring. The absorption peak at 813.87 cm−1 was the result of the stretching vibration of α-anomeric carbon. The comprehensive analysis indicated that the polysaccharides contained a pyranose residue and an α-glycosidic bond, whereas, it did not contain a β-glycosidic bond. UV analysis showed (Fig. 3-B) that the maximum absorption peak of EPS was at 190 nm, containing no protein. 3.3.2. NMR spectroscopy analysis In the 1H NMR spectrum, the signal of the terminal proton located at around the chemical shift value of 4.5–5.5 ppm, which was in a relatively low field and could be used to analyze the configuration and composition of sugar residues. The signals of other hydrogen protons on the sugar ring mostly focused between 3.0 and 4.0 ppm, which may overlap. As shown in Fig. 3C, three signals appear in the anomeric proton region with chemical shifts values of 4.91, 5.02, and 5.27 ppm, respectively. In the 13C NMR spectrum, the signal of the terminal carbon is mostly concentrated between 90 and 110 ppm, and the signal of the non-endgroup carbon is concentrated between 60 and 90 ppm. Since the range of the displacement value of the carbon spectrum signal was relatively large, the signal overlap is hardly observed, which is important for inferring the configuration and proportion of the sugar residue. The results in Fig. 3D show that three anomeric carbon signals are located at 98.08, 100.45, and 102.13 ppm. Since the alpha carbon signal of α-pyranose

G. Zhang et al. / International Journal of Biological Macromolecules 139 (2019) 1295–1303

A-1

B-1

C-1

1299

A-2

B-2

C-2

Fig. 2. Three-dimensional grid map and two-dimensional contour plot of the production of crude EPS from L. sanfranciscensis Ls-1001. A-1, A-2: The effect of maltose and yeast peptone on EPS yield with other components set at center level. B-1, B-2: The effect of maltose and FYE on EPS yield with other components set at center level. C-1, C-2: The effect of yeast peptone and FYE on EPS yield with other components set at center level.

is generally between 97 and 101 ppm, and β-pyranose residue is generally between 103 and 105 ppm [33]. Therefore, it can be judged that the three sugar residues of the polysaccharides are all pyranose residues in the α configuration, which confirms the results obtained by FT-IR. 3.3.3. Monosaccharide composition of EPS The polysaccharides were hydrolyzed by trifluoroacetic acid and analyzed by high performance liquid chromatography (HPLC). The results are shown in Fig. 4. By comparing the peak values obtained for the polysaccharide with the peak time values obtained from standards of various monosaccharides of control, it was confirmed that the polysaccharide contained only one monosaccharide that was glucose, indicating that the pure polysaccharide was a glucan. The structure and composition of the microbial polysaccharides depend on several factors, such as the composition of the culture medium, carbon and nitrogen sources, mineral salts, trace elements, type of

strain, and fermentation conditions (pH, temperature, oxygen concentration, agitation, etc.) [20,34,35]. Kaditzky, Seitter, Hertel, and Vogel [36] showed that the EPS produced by L. sanfranciscensis TMW 1.392 is a homopolysaccharide (HoPS) which is composed of fructose. Besides, some studies have shown the production of heteropolysaccharides by L. sanfranciscensis during growth in wheat sourdough-heterooligosaccharides (HeOS), comprising fructan and 1-kestose [21,25,37]. These studies have also shown that a single bacterial species can produce even different EPS. 3.3.4. Morphological characterization of EPS AFM can be used to observe the three-dimensional structure and microscopic surface morphology of polysaccharides [38]. As shown in Fig. 5, the shape of the pure polysaccharide appeared as regular droplets which were evenly distributed in liquid (distilled water). The minimum peak depth and maximum peak depth were 3.19 nm and 6.58 nm,

1300

G. Zhang et al. / International Journal of Biological Macromolecules 139 (2019) 1295–1303 104

1

102 100

0.8 Absorbance

914.36 813.87 668.77

1517.21

1375.78

92

1651.74

94

2925.89

96

3295.86

Transmittance (%)

98

90 88

0.6

0.4

86

82 80 4000

0.2

1026.23

84

3500

3000

2500

2000 -1

Wavenumber (cm )

1500

1000

500

A

0 190.0

240.0 290.0 Wave Number (nm)

340.0

B

C

D Fig. 3. FTIR spectra (A), UV analysis (B), 1H (C) and 13C NMR (D) analysis of pure EPS produced by L. sanfranciscensis Ls-1001.

respectively. It has been reported that the EPS produced by L. plantarum RS20D was spherical in water and widely distributed [26], indicating that the molecular aggregation may possibly occurs in polysaccharides. Therefore, EPS produced from different lactic acid bacteria strains has morphological features that are not identical.

3.4. Antioxidant activity of EPS ABTS is commonly used to determine the antioxidant capacity of natural products. During the measurement, ABTS directly reacts with K2S2O8, forming a stable free radical ABTS•+ to promote the

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1301

Fig. 4. HPLC analyses of (A) monosaccharide standard samples and (B) hydrolysate of pure EPS produced by L. sanfranciscensis Ls-1001 (1: D-N-acetylglucosamine (GlcNAc); 2: D(+)Glucose (Glu); 3: DL-Arabinose (Ara); 4: L(−)Fucose (Fuc); 5: D-Galacturonic acid (GalA)).

decolorization reaction [29,38]. Evaluation of free radical (ABTS•+) scavenging capacity is used to determine the antioxidant activity of the obtained polysaccharides. As shown in Fig. 6, an increase in the

concentration led to a gradual increase in the scavenging ability of ABTS•+ by pure EPS, which proved its antioxidant properties. When the concentration of pure polysaccharide reached to 1 mg/mL, the

Fig. 5. Molecular morphology of pure EPS as observed by atomic force microscopy (AFM).

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Author contributions Guohua Zhang and Weizhen Zhang mainly designed and performed experiments and drafted the manuscript. Lijun Sun, Faizan A Sadiq, Yukun Yang, Jie Gao and Yaxin Sang mainly participated in the writing of experimental methods and papers.

Declaration of competing interest The authors declare no conflict of interest. References

Fig. 6. The ABTS•+ method detects the antioxidant effect of pure EPS of L. sanfranciscensis Ls-1001.

clearance rate was 93.43%, close to ascorbic acid (99.94%). In addition, EPS exhibited a certain ABTS•+ scavenging capacity even at a very low concentration. For example, when the concentration of the pure EPS was 0.0625 mg/mL, it still had a scavenging effect and the clearance rate was 10.42%. According to one previous study [39], EPS produced by L. plantarum KX041 also had high antioxidant activity. These results indicate that the EPS produced by lactic acid bacteria has a significant effect on the removal of ABTS free radicals, and may be used as a natural substitute for commercial antioxidants, and has certain potentials of commercial application. EPS are produced by lactic acid bacteria in the fermentation of sourdough, which not only has various functional activities, but also plays a positive role in dough's rheological properties and products' texture characteristics. EPS is used in sourdough to have a positive effect on sensory quality of bread samples. Compared to the EPS-samples, the EPS+ samples showed a lighter brown crust, lighter crumb color, a more open crumb structure and better chewiness and hardness [40]. In addition, when EPS is dextran, it has a positive influence on dough rheology, such as softer dough, especially in sourdough containing buckwheat and teff [41]. The effects of EPS on the quality, such as textural and sensory properties of fermented paste, as well as bioactivities of the polysaccharides are being explored to provide more information for its application.

4. Conclusions L. sanfranciscensis Ls-1001 strain isolated from a sourdough sample was proved to be an abundant EPS producing strain, yielding 249.30 mg/L EPS under the optimized conditions. The EPS produced by this strain was a homopolysaccharide, containing only glucose, with α-(1 → 6) linked glucan. This study based on in vitro trials also suggested that EPS produced by L. sanfranciscensis has comparable antioxidant activity with ascorbic acid, effectively scavenging ABTS•+ free radicals. In the future, this EPS may be widely used in the food industry as well as an auxiliary culture in fermented foods.

Funding This work was supported by the National Natural Science Foundation of China [grant numbers 31601461]; and the Natural Science Foundation of Shanxi Province [grant numbers 201701D221151].

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