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Accepted Manuscript

Isolation, purification and characterization of exopolysaccharide


produced by Leuconostoc pseudomesenteroides YF32 from
soybean paste

Yanfang Yang, Fang Feng, Qingqing Zhou, Fangkun Zhao,


Renpeng Du, Zhijiang Zhou, Ye Han

PII: S0141-8130(18)30082-5
DOI: doi:10.1016/j.ijbiomac.2018.03.162
Reference: BIOMAC 9384
To appear in:
Received date: 7 January 2018
Revised date: 9 March 2018
Accepted date: 26 March 2018

Please cite this article as: Yanfang Yang, Fang Feng, Qingqing Zhou, Fangkun Zhao,
Renpeng Du, Zhijiang Zhou, Ye Han , Isolation, purification and characterization of
exopolysaccharide produced by Leuconostoc pseudomesenteroides YF32 from soybean
paste. The address for the corresponding author was captured as affiliation for all authors.
Please check if appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.03.162

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ACCEPTED MANUSCRIPT

Isolation, purification and characterizati on of exopolysacchari de produced by Leuconostoc

pseudomesenteroides YF32 from soybean paste

Yanfang Yang, Fang Feng, Qingqing Zhou, Fangkun Zhao, Renpeng Du, Zhijiang Zhou, Ye Han*

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China

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*Author for correspondence: Phone number: +86-022-13920209057; Email address: hanye@tju.edu.cn

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Abstract: A water-soluble exopolysaccharide (EPS)-producing strain YF32 was isolated fro m soybean

paste, which was then identified as Leuconostoc pseudomesenteroides. After culturing the strain in

Man-Rogosa-Sharpe (MRS) mediu m containing 5% sucrose at 30°C for 48 h, the EPS was purified, and a

yield of 12.5 g/L was achieved. The weight-average molecular weight (M w) was 5.54×106 Da by

high-performance size-exclusion chromatography (HPSEC). The structural characterization of the purified

EPS was determined by gas chromatography (GC), Fourier transform infrared (FT-IR), 1 H, 13
C nuclear

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magnetic resonance (NMR) spectroscopy. The results demonstrated that the exopolysaccharide was glucan

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with a peak, a linear backbone co mposed of consecutive α-(1→6)-linked D-glucopyranose units. No

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branching was observed in the dextran structure. The degradation temperature (Td) of EPS was 307.62°C,

which suggested that dextran exh ibited high thermal stability. YF32 dext ran also showed high water

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solubility and emulsibility. All results suggested that dextran has the potential to be applied in food fields as

a food additive.
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Keywords: Exopolysaccharide; Dextran; Characterization; Leuconostoc pseudomesenteroides; Structure
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1. Introduction

Exopo lysaccharide (EPS), as a major part of extracellu lar poly meric substances produced by
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microorganis ms, has recently attained sizeable interest because of its numerous health benefits and various

industrial applicat ions [1]. EPS can exist in two types: attached to the cell wall to form a capsule, and
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secreted into the mediu m to form mucus. Many microorganisms, including bacteria, algae, and fungi, have

the ability to synthesize extracellu lar polysaccharides [2]. Lact ic acid bacteria (LA B) have gained special
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attention because they are generally recognized as safe (GRAS) [3]. EPS excreted by LA B can be regarded

as a safe biology poly mer that does not carry health risk and offers an alternative source of microbial

polysaccharides for use in the food or other industries [4]. In recent years, increasing attention has been

paid to the exp loration and utilization of mic robial polysaccharides for their potential industrial applicat ions.

For example, some EPSs with h igh thermal stability and solubility can be used as food additives in the food

industry [2]. EPS also have physiological activit ies that can enhance immun ity, antitumor activ ity, etc. [5].

To make better use of LA B EPS, many scholars have devoted themselves to studying LAB EPS. The usage

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of EPS-producing LAB could result in a safe, natural and healthy end product with improving texture,

feeling and stability [3]. These studies may have a significant impact on the development of novel products.

Microbial EPSs are added to or are present in a wide variety o f food products, where they serve as

viscosifying or gelling agents. EPSs with different compositions, sizes and structures are synthesized by

several strains of lactic acid bacteria, wh ich are used in different fields [6]. For examp le, the dextran

produced from Leuconostoc. mesenteroides NRRL B-640 contains D-glucose residues in a linear chain with

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consecutive α-(1→6) linkages and exhibits a typical non-Newtonian pseudoplastic behavior, which can be

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used as a thickening or gelling agent in food [7]. A novel water-soluble dext ran synthesized by Leuconostoc.

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citreum SK24.002 is main ly co mposed of α-1, 3 and α-1, 6 linked D-glucopyranose units with a rat io of 4:5,

which can be used in pharmaceutical factories [8]. Later, dextrans has been applied to many fields, such as

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food and cosmetic manufacture [9]. Therefo re, the current research focuses on screening more strains

producing extracellular polysaccharides.


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More and more LA B-producing EPS were recently isolated fro m traditional fermented food. There are

kinds of food that are fermented in Ch ina, such as Sauerkraut, wine and pickle [10]. Soybean, which
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originates fro m East Asia, is regarded as the most important legume for hu man consumption and animal
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feed in oriental countries [11]. Soybean paste is a co mmon dairy product in Northern China that is made of

soybean after fermentation. Many microorganisms are involved in this process, which can affect the taste
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and color of final products [9,12]. The EPS production of LA B already reported fro m soybean paste was

generally low and no special feature[11]. Thus, this study aimed to isolate LA B strains with h igher EPS
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production from soybean paste. The study of the structure and properties of EPS could p rovide a theoretical

basis for commercial applications of EPS.


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2. Materials and methods

2.1. Isolation and identification of LAB producing EPS

The sample named soybean paste was collected in Handan City of Hebei Province, which was made of

soybean paste after fermentation. It was transported to the laboratory at ambient temperature and stored at

4°C until used in experiments. Decimal dilution of the sample was plated in M RS agar p lates containing 50

g/L sucrose and incubated at 30°C for 24 h under static conditions. Later, the strain with mucoid and slimy

colony was used as the source of EPS in this work.

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The strain was identified through morphological, physiological, b iochemical tests and 16S ribosomal

DNA sequence analysis. Geno mic DNA was extracted fro m the fresh culture solution. The DNA was

amp lified using the primers 8F-A GA GTTTGATCATGGCTCA G and 1492R-ACGGTTACCTTGTTA

CGA CTT. PCR was performed in a 50-µL reaction mixture containing 1 µL Temp late DNA, 25 µL 2×Taq

Mixture, and 1 µL of each primer (10 mmo l/ L). The amp lification was init iated at 9 5°C for 3 min, fo llo wed

by 30 cycles of 95°C for 60 s, annealing at 55°C for 60 s, and extension at 72°C for 60 s. The final

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extension step was performed at 72°C for 7 min. The resulting sequence was compared to the 16S

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ribosomal DNA sequences of GenBank using BLAST [13].

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2.2. Extraction, purification and quantification of EPS

The YF32 strain was cultivated in MRS mediu m containing 50 g/L s ucrose for 48 h at 30°C in the Shaker

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Incubator (80 rp m), and the inoculation quantity was 2% (v/v) of the seed medium cu lture. EPS was

isolated and purified by Du’s method with modification [14]. At first, the strain cells were removed by
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centrifugation at 4,000 g for 40 min at 4°C. Then, the EPS was precipitated by adding a three-fold volume

of 95% (v/v) cold ethanol to the cell-free cu lture and kept at 4°C overnight. After centrifugation at 11,200 g
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at 4°C for 60 min, the crude EPS was collected and then suspended in pure water and stirred with a
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magnetic stirrer until co mplete dissolution. Then, 10% (v/v) trichlo roacetic acid (TCA) was added to the

EPS solution, making the final concentration 5%, and the mixture was stored overnight at 4°C. The proteins
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were eliminated by centrifugating at 4°C, 11,200 g for 60 min, and then, three-fold volu mes of 95% (v/v)

cold ethanol were mixed with the supernatant and kept at 4°C overnight. The EPS was obtained by
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centrifugation at 11,200 g at 4°C for 60 min and then dissolved in ultrapure water. The EPS solution was

dialy zed with a d ialysis bag (MWcut-off 14,000) to eliminate the small mo lecules or ions at 4°C for 2 days.
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Further purification of the EPS solution was conducted by gel chromatog raphy with a Sephadex G-100

column (1.6 cm×50 cm). The elution buffer was deionized water, and the flow rate was 0.2 mL/ min. The

collections were performed using an ultravio let spectrophotometer (Shanghai, China) to monitored A220nm .

Finally, the purified EPS was concentrated and lyophilized. The sugar content in the EPS was analyzed

using the phenol sulfuric acid method [13]

2.3. Monosacchari de composition analysis

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The monosaccharide composition of the EPS was analyzed through gas chromatography (GC). Briefly,

hydrolyzation of the purified EPS (10 mg ) was performed at 120°C for 6 h with 2 mL o f 2 mo l/L

trifluoroacetic acid (TFA), and the remanant TFA in the hydrolysate was eliminated by evaporation.

According to the methods of Yang [3], the dried hydrolysate was transformed to acetylated derivatives. GC

analysis was performed on an instrument equipped with a flame ionization detector (FID) using an HP -5

capillary colu mn (30 m×0.32 mm; I.d 0.25 m) (Agilent Technologies Co., Ltd., USA). The fo llowing were

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the operation conditions: injection temperature 250°C; in jection volu me 3 µL; detector temperature 250°C;

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split ratio 3:1. Co mpared with the standard sugars (glucose, fructose, arabinose, galactose, rhamnose and

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mannose), the monosaccharide composition of the EPS was identified.

2.4. Estimation of EPS molecular weight

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The molecu lar weight of the EPS produced from L. pseudomesenteroides YF32 was analyzed by

high-performance size-exclusion chromatography (HPSEC) with a refractive index detector. The samp le (2
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mg/ mL) was filtered through a 0.22-µm filter (Sartorius, USA) before inject ion. A Shodex OH-pak SB-805

column fo llo wing an OH-pak SB-G guard colu mn (8.0 mm×300 mm, Japan) was used at 25°C and the
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column was eluted with PBS at a flow rate o f 0.8 mL/ min. The volu me of injection was 20 µL . T-series
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dextran standards were used to obtain the standard curve. The molecular mass of the sample was

extrapolated according to the calibration curve of standard dextrans.


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2.5. Ultraviolet (UV) and Fourier transform infrared (FT-IR) spectrum analysis

The purified EPS was dissolved in ultrapure water (1 mg/ mL), and the UV spectrum o f the EPS solution
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was obtained using a UV-v isible spectrophotometer (UV-1603, Kyoto, Japan) scanning in a wavelength

range of 190-350 nm.


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FT-IR spectra of the purified EPS were measured by a BIO-RAD FTS3000 IR spectra scanner at roo m

temperature. The spectroscopy was recorded between 400 cm-1 and 4000 cm-1 at a resolution ratio of 1 cm-1 .

A metal mold was obtained by mixing the powder of the dried EPS (1 mg) with KBr (100 mg) and then

pressing the mixture. Finally, the FT-IR spectrum was obtained by scanning the metal mold.

2.6. Nuclear magnetic resonance (NMR) spectroscopy analysis

According to the procedure described by Maina [15], the purified EPS (20 -40 mg/ mL) was vacuum dried

and exchanged three times with D2 O (99.9%), finally placed in NM R tubes. NM R spectra of the

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polysaccharide were recorded using a Bruker 400 M Hz liquid NMR s pectrometer (Bruker, Avance Ш,

Switzerland). All experiments were conducted at 25°C.

2.7. Scanning electron microscopy (SEM) analysis

The microstructure and surface morphology of the EPS was observed using scanning electron microscopy

at an accelerat ion voltage of 10.0 KV and under 200×, 400×, 1000× magnification. The lyophilized EPS

sample was fixed to the SEM stubs with conductive tape and coated with a layer of 10 n m Au before SEM

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observation.

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2.8. Water solubility index (WSI)

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The WSI of the samp le (EPS) was measured using the modified method of Yang [3]. Fifty milligrams of the

EPS was placed in a centrifugal tube, and 0.5 mL pure water was added. A vortex mixer was used to agitate

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the liquid in tube vigorously to fo rm a un iform suspension for 2 h at room temperature, and the precipitant

was obtained through centrifugation at 12,000 g for 25 min and then freeze dried. The WSI was calculated
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according to Eq. (1)

WSI=(m0 -m1 )/m0 ×100%. (1)


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where m1 is the weight of precipitant lyophilized and m0 is the weight of the initial EPS sample.
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2.9. Determination of emulsifying activity (EA)

According to the method described by Dev i [16], three milliliters of sunflower o il was mixed with 2 mL of
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sample solution (1%, w/v) in a glass tube with screw lid (100 mm×13 mm), vortexed for 3 min at 40 Hz.

The mixtu re was preserved at 25°C for 1, 24, 168 and 360 h, and the emulsify ing activity (EA 1 , EA 24 ,
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EA 168 and EA 360 ) was calculated according to Eq. (2):

Emulsifying activity(EA)=(He/Ht )×100% (2)


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where He is the height of the emulsifying layer, Ht is the full height of the mixture.

2.10. Analysis of thermal properties

To study the thermal properties of the obtained EPS, we used a thermal analysis instrument with Ar 2 as

protective gas. The system was controlled by a compatible PC, which reg istered the temperature measured

by a thermocouple placed in the crucible. The crucible was made of Al2 O3. The EPS (3-5 mg ) was placed in

a platinum crucible and heated at a linear heating rate of 10°C/min over a temperature range of 40-800°C.

3. Results and discussion

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3.1. Isolation and purification of the EPS

A bacterial strain cultured on MRS p lates containing sucrose was able to form a slimy and liquid colony,

revealing the presence of EPS (Fig. 1a). Th rough morphological, physico-chemical tests and 16S rDNA

sequence analysis (accession number: LC274615.1), the bacteriu m was identified as L.

pseudomesenteroides and named YF32. After incubating the strain in MRS mediu m containing 5% sucrose

at 30℃ for 48 h, the crude EPS was harvested by the method of ethanol precip itation, then further purified

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by Sephadex G-100 gel-filt ration chro matography and used pure water as eluent. The elution profile

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presented a single peak, which indicated that the corresponding fraction was homogeno us. The sugar

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content of the collected fraction was analyzed using the phenol sulfuric acid method and then frozen,

lyophilized, and weighed. The yield of the purified EPS (Fig. 1b) was 12.5 g/ L. In general, the yield of EPS

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varies with changes in the type of bacteria, culture conditions and processing methods [17]. For instance,

Leuconostoc. mesenteroides BD170 was cultured in to mato ju ice containing 15% sucrose, and the
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production of EPS achieved 32 g/L [18], but Weissella. cibaria CMGDEX3 was cultured in MRS mediu m

containing 15% sucrose, and the yield was only 2.4 g/L [19]. Therefore, L. pseudomesenteroides might
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have a potential capacity for the high production of EPS co mpared with the other strains, which can
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produce EPS.

3.2. The monosaccharide composition and molecular weight of the EPS


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The monosaccharide composition of the EPS fro m strain YF32 was analyzed using GC, and the results

revealed that the EPS was homopolysaccharide with a single peak. According to the retention time of
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different standard monosaccharides, monosaccharide of the purified EPS was glucose; therefore, the EPS

was composed of glucose units.


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The weight of the EPS produced fro m L. pseudomesenteroides YF32 was determined by HPSEC. The

HPSEC elut ion profile (Fig. 2) showed a single symmetrical narro w peak, verifying that the purified EPS

was homogeneous. Based on the calibrat ion curve of the elution retention time of various standard dextrans,

the weight-average mo lecular weight (Mw) of the EPS was estimated to be 5.54×10 6 Da. According to

Freitas [20], the molecu lar weight of dextran fro m bacteria was in the range of 10 6 -109 Da. Yang [3]

reported the molecu lar weight of the EPS fro m Leuconostoc. citreum NM105 was 1.01×108 Da, which is

higher than that of dextran in this study. In general, the diversity in the mo lecular weight of EPS could be

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explained by the differences in strains, fermentation factors and EPS structures. According to Wang et al.

[21], the polydispersity index is a measure of the width of molecular mass distribution, wh ich is an

important element influencing the functional properties of EPS.

3.3. UV and FT-IR spectrum analysis

UV spectra of the EPS showed only a single peak in the range of 190-210 n m, which is a co mmon

characteristic of carbohydrates [22]. The UV spectrum had no other peak at 260 or 280 n m, indicating that

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the purified EPS did not have any protein or nucleic acid.

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FT-IR has been a very useful and potent tool for analyzing and speculating complex carbohydrate

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compositions. The FT-IR spectrum (Fig. 3) of the sample EPS showed different peaks between 3910 and

526 cm-1 . The broad absorption peak at 3417 cm-1 suggested that the sample contained massive hydroxyl

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groups (O-H) stretching vibration, which was a characteristic of polysaccharide and further confirmed that

the substance was polysaccharide [4]. The absorption peaks at 2926 and 1639 cm-1 were due to C-H
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stretching vibration [23]. The existence of associated water led to the absorption peak at 1639 cm-1 [24,25].

The narrow peak at 1154 cm-1 was related to the glycosidic bridge bond and the valent vibrations of C-O-C
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[26]. The band at 850 cm-1 suggested that the EPS contained pyranose residues of the α-configuration. No

absorption peak at 890 cm-1 indicated no ß-configuration in the EPS fro m strain N21. The appearance of an
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absorption peak at 1015 cm-1 was attributed to the dextran possessing a highly flexib le chain around the
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α-(1→6) linkages [5], which was also thought to be a feature of the link pattern of the interunit [27].

According to the FT-IR spectra, L. pseudomesenteroides YF32 dext ran contained pyranose glycosidic
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bonds of the α-configuration, and glucan units were connected with α-(1→6) linkages. This was further

confirmed by subsequent NMR analysis .


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3.4. NMR spectroscopy analysis

NMR spectra are a powerful tool fo r analyzing the structure of complex substances. The 1 H NMR and 13
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NMR spectroscopy of the EPS fro m strain YF32 are shown in Fig. 4. The typical signal of polysaccharides

was that the peaks were narrow in a range of 3-5 pp m in the 1 H NMR spectrum [28]. The signals in δ

95-101 pp m were related to ɑ -ano meric carbons, and the signals in δ 101-105 pp m were due to

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ß-anomeric carbons in C NMR spectra, which corresponded with the 1 H NMR spectra [28]. Fro m the 1 H

NMR spectra of the EPS, only one obvious chemical shift signal in 4.8-5.5 pp m (ano meric reg ion) was

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found at 4.89 ppm. This was due to H1 resonance of α-glucose residues, and the signal at δ97.62 pp m in

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C NMR spectra supported this deduction. There was no peak between δ101 and 105 ppm in C NMR

spectra, which indicated that the type of glycosidic bond composed of the polysaccharide was only

α-configuration [29]. The peaks at 69.67, 70.31, 71.53 and 73.52 ppm corresponded to C-5, C-4, C-3 and

C-2, respectively [30]. No addit ional peak appeared in the range of 75-85 ppm, wh ich indicated that there

was no branched chain in the EPS. The two g lucose units in the backbone chain of the polymer were

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connected with α-(1→6) linkages, which could be confirmed by the C-6 carbon signal of the dextran un it at

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65.69 pp m in the down field shift. This study confirmed that the EPS produced fro m L.

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pseudomesenteroides YF32 was a highly linear glucan and lin ked with the α-(1→6) glycosidic bond, with

no branched chain. These results were also confirmed by FT-IR analysis. It was different fro m the dextran

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synthesized by L. citreum NM105 [3].

It is possible to correlate each 1 H with its directly attached 13


C using the Heteronuclear Multiple
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Quantum Co rrelation (HM QC) technique [7]. HM QC correlations between 1 H and their corresponding 13 C
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of dextran fro m L. pseudomesenteroides YF32 showed six correlations (Fig. 5). This fu rther confirmed that

the dextransucrase elaborated by this microorganism synthesizes only a linear dextran with α-(1→6)
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glycosidic bonds.

3.5. SEM analysis of the EPS


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SEM is a very powerful tool to exh ibit the surface morphology of bio -macro molecules and indicate the
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characteristics of poly mers, wh ich helps in understanding their physical propert ies. The microstructure and

surface morphology micrography at 200×, 400×, 1000× of the EPS are shown in Fig. 6. As observed by
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SEM, the EPS appeared spherical, smooth glittering surface and mu ltip le branches, wh ich were most likely

to be utilized in food industries as texturing, thickening, stabilizing and emulsifying agents and could also

be a good choice for preparing edible films and coatings for food application. Part of the surface

morphology of the EPS was similar to that of polysaccharide produced by Leuconostoc. lactis KC117496,

which revealed the compact and porous structure and Leuconostoc. plantarum YW11, wh ich appeared

porous and glittering surface [3,21], but there are many differences between the dextran synthesized by L.

mensenteroides NRRL B-640, which appeared as irregular blocks and cellulosic structures, and the EPS in

this study [5]. The special micromorphology gave soybean paste better water absorption during production .

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3.6. WSI and EA of the EPS

The EPS had strong solubility, and the WSI of the dextran was 90.5%(81.15 g/ L), wh ich was related

to the stronger hydrogen bonds with water in the EPS. Four guar gu m and xanthan as thickener had been

shown that their WSI was found to be 100% (83.3 g/ L) before [31]. Whereas the WSI of the EPS fro m

Leuconostoc. kefiranofaciens ZW3 and Leuconostoc. lactis were 14.2% (11.83 g/L), wh ich was reported by

Ahmed and Saravanan [32,13]. Fro m the literature that has been reported, the higher the solubility, the

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more beneficial as a biosurfactant and stabilizer [2,7,8]. Therefore, the EPS in th is study could b e applied to

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the food fields and industrial production as hydrocolloids, stabilizers and biothickeners.

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EA of the EPS is measured by its capacity to retain the hydrocarbon emulsion in water. An effective

emu lsifier should have the ability to keep at least 50% of the primal volu me of emu lsion until 24 h because

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it has formed [33]. After 1, 7 and 15 days, emulsions mixed with the dextran solution (1 mg/ mL) and

sunflower oil were compared for their stability; the results are presented in Table 1. The dextran p roduced
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by strain YF32 possessed an emu lsify ing activity of 67.25% after 1 h, and a slight decrease of 64.36% was

recorded at 24 h. The same trend could be observed with locust gum, guar gum and purified EPS fro m L.
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kefiranofaciens, revealing EA of 65.46, 37.47 and 84.12%, respectively, after 1 h [34]. A lthough the
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activity decreased slightly after 7 and 15 days of storage and was 63.43% and 63.39%, respectively, the

decrease in emulsion stability was not significant after 24 h. The EPS fro m Bifidobacterium longum CCUG
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52486 and NCIM B 702205 could maintain emu lsifying stability for 7 days at the temperature o f 25℃ with

oil fro m sunflower seed [35]. Co mpared to these EPSs, the EPS in this study had a higher Emulsifying
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property. In summary, the high water solubility and emulsibility could make soybean paste good

rheological propert ies and taste. Therefore, EPS could be used as food additives in the food field to
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improve viscosity and taste.

3.7. Analysis of thermal properties

The literature has reported that the thermal degradation of different typical carbohydrates can generate

certain substances, including volat ile and non-volatile. With the in itial increase in temperature, the primary

two events are gelatinization and swelling in the thermogravimetric curve. As the temperature goes further

up, the following three events begin to occur: first dehydration, fo llo wed by pyrolysis and the

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recomb ination of chemical bonds [36]. These processes include breakdown of the old bonds in the EPSs

and new bonds forming many products, which leads to the generation of volatile or non -volatile products.

In the TGA curve of the EPS (Fig. 7), three events could be found with the increase of the temperature.

The first event appeared at approximately 100°C, which occurred as a sma ll weight loss because of the loss

of mo isture [4,37,38]. As the temperature further increased fro m 130.26°C, the energy was released, and

the second event occurred at a temperature of appro ximately 300°C. An exothermic peak with a maximu m

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at 307.62°C represented the energy released in the second event. A large weight loss could be observed at

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this point (307.62°C). Co mparing these results with those observed by the EPSs obtained from the

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commercial kefir product, the major mass loss occurred at the tempera ture of 250-350°C fro m the

degradation temperature (Td) curve [39]. The TGA curve of the EPS revealed water evaporation at

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approximately 100°C, and the degradation of the EPS occurred at appro ximately 300 ℃; this finding is also

in line with the TG results [40]. Fro m the co mprehensive analysis of thermal characterization of the EPS,
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we found that the degradation temperature of the dextran was relatively high – that is, the obtained EPS

possessed high thermal stability, wh ich indicates that the soybean paste could not lose nutrients during the
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heating process. This process could be applied in the food industry to improve thermotolerance and
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applicability.

4. Conclusions
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Currently, it is critical to reduce costs for producing the same good. In this paper, an LAB s train with high

relative production of EPS was isolated fro m soybean paste, which was identified as L.
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pseudomesenteroides. Based on GC, FT-IR and NMR experiments, the EPS was determined to be a dext ran

with a linear backbone co mposed of consecutive α-(1 →6)-lin ked D-g lucopyranose units. The EPS
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possesses largely valuable characteristics for various commercial applications. For example, the unique

spherical porous microstructure and high solubility of dextran may suggest potential applicat ions in the

food industries, such as texturing, bioflocculants and drug delivery agents. The high thermal and

emu lsifying activ ity indicated that it could be used as a stabilizing and emulsify ing agent. In summary, L.

pseudomesenteroides YF32 could be widely used to produce linear EPS, and EPS could be applied in food

fields as a food additive. To understand the more practical value of dext ran in food and other fields, fu rther

studies are necessary.

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Acknowledgements

This work was financially supported by National science and technology support plan project (No.

2015BAD16B01).

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Table 1 EA of EPS at different intervals

Time(h) EA (%)

1 67.25±0.69a

24 64.36±0.87b

168 63.43±1.08b

360 63.39±0.83b

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EA having superscript a, b letters denotes the statistically significant difference at P < 0.05. Same letters

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represent no significant differences (p<0.05).

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Figures

Fig. 1. The viscous colony morphology (a) and pure EPS (b) from L. pseudomesenteroides YF32.

Fig. 2. Hig h-performance size-exclusion chromatograms of the EPS produced by L.

pseudomesenteroides YF32.

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Fig. 3. FT-IR spectrum of EPS produced from L. pseudomesenteroides YF32.

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Fig. 4. 1 H (a) and 13
C (b) NMR spectra of the purified EPS from L. pseudomesenteroides YF32.

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Fig. 5. 13 C/ 1 H 2D HMQC NMR spectrum of the purified EPS from L. pseudomesenteroides YF32.
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Fig. 6. S EM showi ng the surface morphology of EPS from L. pseudomesenteroides YF32 at vari ous
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magnifications at 200×(a), 400×(b), 1000×(c).


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Fig. 7. TG, DSC and DTG of EPS from L. pseudomesenteroides YF32.


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Highlights

A Leuconostoc pseudomesenteroides strain with high production (12.5g/L) of dextran was isolated

from soybean paste.

The exopolysaccharide produced from Leuconostoc pseudomesenteroides YF32 was a highly linear

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dextran.

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The exopolysaccharide produced from Leuconostoc pseudomesenteroides YF32 had high solubility

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(81.15 g/L), emulsifying properties and thermal stability.

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The degradation temperature (Td) of the EPS was 307.62℃, which indicated the EPS could be applied

in food and pharmaceutical fields as stabilizers.


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Graphics Abstract
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

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