Accepted Manuscript Title: Characterization of an exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibet Kefir Author: Ji Wang Xiao Zhao Zheng Tian Yawei Yang Zhennai Yang PII: DOI: Reference:

S0144-8617(15)00186-1 http://dx.doi.org/doi:10.1016/j.carbpol.2015.03.003 CARP 9743

To appear in: Received date: Revised date: Accepted date:

12-9-2014 27-2-2015 2-3-2015

Please cite this article as: Wang, J., Zhao, X., Tian, Z., Yang, Y., and Yang, Z.,Characterization of an exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibet Kefir, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights (for review)

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1. Physicochemical, rheological properties, structural characterization and

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microstructure of the EPS from L. plantarum are reported.

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at acidic pHs.

4. Unlike reported before, the EPS from L. plantarum YW11 shows a

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3. The EPS has higher viscosity in skim milk, at lower temperature, or

highly branched and porous structure.

5. The EPS has high thermal stability.

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for exploitation in food industry.

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2. The EPS produced by L. plantarum YW11 is a promising candidate

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Characterization of an exopolysaccharide produced by

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Lactobacillus plantarum YW11 isolated from Tibet Kefir

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Ji Wanga,b, Xiao Zhaoa, Zheng Tiana, Yawei Yanga, Zhennai Yanga,b,* a. Beijing Laboratory of Food Quality and Safety, Beijing Technology and Business University, Beijing, China;1 b. School of Biological and Agricultural Engineering, Jilin University, Changchun, China

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Abstract: An exopolysaccharide (EPS)-producing strain YW11 isolated from Tibet

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Kefir was identified as Lactobacillus plantarum, and the strain was shown to produce

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90 mg L-1 of EPS when grown in a semi-defined medium. The molecular mass of the

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EPS was 1.1 × 105 Da. The EPS was composed of glucose and galactose in a molar

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ratio of 2.71:1, with possible presence of N-acetylated sugar residues in the

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polysaccharide as confirmed by NMR spectroscopy. Rheological studies showed that

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the EPS had higher viscosity in skim milk, at lower temperature, or at acidic pHs. The

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viscous nature of the EPS was confirmed by observation with scanning electron

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microscopy that demonstrated a highly branched and porous structure of the

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Keywords: Exopolysaccharide; Lactobacillus plantarum; Rheology; Structural

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polysaccharide. The atomic force microscopy of the EPS further revealed presence of many spherical lumps, facilitating binding with water in aqueous solution. The EPS had a higher degradation temperature (287.7 °C), suggesting high thermal stability of the EPS.

*Corresponding author. Beijing Laboratory of Food Quality and Safety, Beijing Technology and Business University, No. 11 Fu-Cheng Road, Hai-Dian District, Beijing, 100048 China. Tel.: +86 10 68984870; Fax: +86 10 68985456. Email: [email protected] 2

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characterization

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Chemical compounds studied in this article

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Glucose (PubChem CID: 79025); Fructose (PubChem CID: 5984); Rhamnose

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(PubChem CID: 25310); Glycerol (PubChem CID: 753); Glucuronic acid (PubChem

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CID: 444791); Sodium nitrate (PubChem CID: 24268); Sodium chloride (PubChem

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CID: 5234); Calcium chloride (PubChem CID: 21226093); Trifluoroacetic acid

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(PubChem CID: 6422); Phenol (PubChem CID: 996); Sulfuric acid (PubChem CID:

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1118).

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41 1. Introduction

Exopolysaccharides (EPSs) are extracellular biopolymers that are produced during

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the metabolic process of microorganisms such as bacteria, fungi, and blue-green algae

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(Amjres et al., 2014). The EPSs could be either covalently associated with the cell

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surface forming a capsule, or be loosely attached, or totally secreted into the surrounding environment during the cell growth (Yang et al., 2010). Among the wide variety of EPS-producing microorganisms, lactic acid bacteria (LAB) are generally regarded as safe due to their long history of safe use in human consumption (Nikolic

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et al., 2012). In recent years, owing to the unique physicochemical properties and

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biological activities, the EPSs produced by LAB have been used in a wide range of

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applications including food products, pharmaceuticals, bioflocculants, bioemulsifiers

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and chemical products (Liu et al., 2010; Ye, Liu, Wang, Wang, & Zhang, 2012; Ismail 3

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& Nampoothiri, 2010). In the fermented food industry, LAB EPSs are usually used as

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natural alternatives to commercial stabilizers because of their viscosifying, stabilizing,

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emulsifying or gelling properties for improving the rheology, texture and mouth-feel

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of the fermented products including yoghurt and cheese (Ahmed, Wang, Anjum,

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Ahmad, & Khan, 2013). In addition, extensive research has revealed that some EPSs

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produced by LAB may be correlated with promoting human health and preventing

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diseases due to their pharmacological activities, such as immunostimulatory,

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immunomodulatory, antitumor, antibiofilm, antioxidant and cholesterol lowering

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activities (Liu, Chu, Chou, & Yu, 2011; Kanmani et al., 2011; Li et al., 2014a;

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Lindström, Holst, Nilsson, Öste, & Andersson, 2012). Since bacterial EPS are generally

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of protective nature, production of EPS may increase the resistance of the bacteria

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against unfavorable environmental factors, e.g. resistance to high acidity and bile salts

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(de los Reyes-Gavilán et al., 2011).

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In the last decade, a large number of EPS-producing LAB have been isolated from

a variety of fermented foods such as yoghurt, cheese, sausages, kefir, wine and sauerkraut. These LAB strains belong to the species of Streptococcus, Lactobacillus, Pediococcus, Lactococcus and Bifidobacterium (Ramchandran & Shah, 2010; Zhang

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et al., 2013; Song, Jeong, Cha, & Baik, 2013; Costa et al., 2010; Prasanna, Bell,

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Grandison, & Charalampopoulos, 2012). Among EPS-producing LAB, Lactobacillus

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plantarum is famous for its potential probiotic properties and has received

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considerable attention in recent years (Zhang et al., 2013; Wang et al., 2014; Wang et 4

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al., 2010). It has been shown that the EPS yield, monosaccharide composition and

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structure are greatly dependent on the producing microorganisms, their culture

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conditions and media compositions (Salazar et al., 2009). L. plantarum C88, an

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isolate from fermented dairy tofu, is able to synthesize a high molecular mass capsular

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polysaccharide (1.15 × 106 Da) composed of galactose and glucose (1:2) when grown

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in a semi-defined medium (Zhang et al., 2013). L. plantarum 70810 isolated from

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Chinese Paocai could produce two released EPSs with average molecular weights of

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204.6 and 202.8 kDa, and composed of glucose, mannose and galactose with molar

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ratios of 18.21:78.76:3.03 and 12.92:30.89:56.19, respectively (Li et al., 2014b).

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Besides, L. plantarum KF5 isolated from Tibet Kefir was reported to compose of

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mannose, glucose and galactose in an approximate ratio of 1:4.99:6.90 (Wang et al.,

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2010).

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EPSs produced by L. plantarum will be increasingly applied in the fermented

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products due to their safe, healthy and desired physicochemical properties in the future, and thus more research is required on the physicochemical, structural and rheological parameters of the EPS. However, so far most of the reports about EPS are focused on the functional characteristics of the EPS-producing L. plantarum strains,

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Therefore, in the present study, the EPS from L. plantarum YW11 was prepared and

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characterized by gas chromatography–mass spectrometer (GC–MS), nuclear magnetic

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resonance (NMR), scanning electron microscopy (SEM), atomic force micrograph

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(AFM), thermogram analysis (TGA) and differential scanning calorimeter (DSC) in 5

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order to evaluate its potential application in food industry.

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2. Materials and methods

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2.1. Bacterial strain and media

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The EPS-producing strain YW11 used in this study was isolated from Kefir grains

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collected from Tibet. The strain was maintained as frozen (−80 °C) stocks in MRS

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broth (de Man et al. 1960) supplemented with 20% (v/v) glycerol. The sterile

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semi-defined medium (SDM) broth contained (per 1 L): 10 g of bactocasitone (Difco),

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5 g of yeast nitrogen base (Difco), 2 g of ammonium citrate, 5 g of sodium acetate,

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0.1 g of MgSO4·7H2O, 0.05 g of MnSO4, 2 g of K2HPO4, 20 g of glucose and 1.0 mL

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of Tween 80, adjusted to pH 6.6 with 1 M acetic acid (Kimmel & Roberts, 1998).

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2.2. Identification of strain YW11

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Strain YW11 was primarily identified based on Gram reaction, catalase tests and

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cell morphology. Then, the strain was further identified to the species level by API 50

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CHL test (bio-Mérieux, France) and 16S rDNA sequencing analysis. The primers with the

following

sequences:

(5’-AGCGGATCACTTCACACAGGACTACGGCTACCTTGTTACGA-3’)

A27F and

A1495R

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(5’-GCAGAGTTCTCGGAGTCACGAAGAGTTTGATCCTGGCTCAG-3’),

were

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provided by China Agricultural University (Beijing, China). The nucleotide sequences

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were compared with standard strains for the sequence similarity through BLAST

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(http://www.ncbi.nlm.nih.gov/blast). 6

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2.3. Analysis of bacterial growth and EPS production Strain YW11 was inoculated in 2000-mL Erlenmeyer flasks containing 1000 mL of

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SDM broth as mentioned above and incubated at 37 °C. Samples (50 mL) were

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withdrawn at different time intervals from 0 to 56 h. The pH was determined with a

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pH meter (FE20, Mettler Toledo, Switzerland). The cell viability was determined by

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dilution plating with MRS agar medium incubated at 37 °C for 48 h. EPS yield

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(expressed as mg L-1) was estimated by phenol-sulfuric acid method using glucose as

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a standard (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956).

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2.4. Isolation and purification of EPS

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The EPS was isolated by using the method of Zhang et al. (2013). The crude EPS

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solution (20 mg mL-1, 5mL) was fractionated with an anion exchange chromatography

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on a DEAE-cellulose column (26 mm × 40 mm), eluted with deionized water, 0.2 and

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0.5 M NaCl solution at a flow rate of 1 mL min-1. Every 5 mL of elution was collected

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automatically and the carbohydrate content was determined by phenol-sulfuric acid method. Peak fractions containing polysaccharides were pooled, dialyzed and lyophilized. Further purification of the EPS was performed by a Sepharose CL-6B column (25 mm × 50 mm) and eluted with 0.9% (w/v) NaCl at a flow rate of 0.5 mL min-1. The polysaccharide fractions were detected, pooled, dialyzed and lyophilized.

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The purity of the final purified EPS sample was checked. The total carbohydrate

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content of the sample was determined by the phenol-sulfuric acid method using

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glucose as standard. The total protein content of the sample was determined by the 7

Page 7 of 37

method of binding of Coomassie Brilliant Blue G-250 to protein, using bovine serum

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albumin as a standard (Bradford, 1976). The uronic acid content was determined by

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the Dische method, using glucuronic acid as standard (Dische, 1947).

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The moisture content of the sample was measured by the method of Vijayendra,

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Palanivel, Mahadevamma, and Tharanathan (2008). The EPS taken in a dish, which

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was previously dried and weighed, was placed along with its lid in an oven

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maintained at 105 °C for 5 h and cooled in a desiccator. Drying was repeated till a

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constant weight was obtained and the percentage of moisture content was calculated.

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2.5. Molecular mass determination of EPS

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The molecular mass of the purified EPS was measured by gel-permeation

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chromatography (GPC). The GPC system consisted of a Shodex SB-806m-HQ 13 μm,

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300 × 8.0 mm column, connected with a SB-G 10 μm, 50 × 6.0 mm guard column.

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The EPS were detected using a refractive index detector (RI) (Optilab Wyatt, USA)

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and a multi angle laser-light scattering detector (MALLS) (DAWN HELEOS-II Wyatt, USA), at an internal temperature of 40 °C. The column was eluted with 0.1 M NaNO3 solution at a flow rate of 0.5 mL min-1, and the injection volume of sample was 200

μL, and dn/dc of 0.146 as a refractive index increment was used for polysaccharides

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solution (Ai et al. 2008). Data processing was performed with Wyatt Astra software

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(Version 5.3.4.14, Wyatt Technology, USA).

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2.6. Determination of monosaccharide composition of EPS

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Five milligrams of the purified EPS were hydrolyzed with 2 mL (2 M) 8

Page 8 of 37

trifluoroacetic acid (TFA) at 120 °C for 2 h. The hydrolysates were then subjected to

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GC-MS analysis. GC-MS was performed on an Agilent 7890A GC fitted with a flame

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ionization detector (FID) and a DB-WAX column (30 m length × 0.25 mm inner

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diameter × 0.25 µm film thickness; J & W Scientific, Folsom, CA). The operating

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conditions were determined according to Li et al. (2014a). Sugar identification was

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done by comparison with standard rhamnose, arabinose, galactose, glucose, mannose

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and fructose.

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2.7. UV-vis and FTIR spectral analysis of EPS

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Ultraviolet-visible (UV-vis) spectroscopy analyses of the EPS were conducted on

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UV-vis spectrophotometer (U-3900, Hitachi Ltd., Japan). The EPS solution was

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prepared by suspending the sample in distilled water for UV-vis measurement in the

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wave-length range of 190-550 nm.

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The major structural groups of the purified EPS were detected using Fourier

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transform infrared (FTIR) spectroscopy, and the spectrum of the EPS was obtained using a KBr method. The polysaccharide samples were pressed into KBr pellets at sample: KBr ratio 1:100. The FTIR spectra were recorded on a Bruker Tensor 27 instrument (Germany) in the region of 4000-400 cm−1. FTIR spectrum was

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determined in transmission mode and the number of scans was 32. The infrared

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spectral resolution was 4 cm-1.

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2.8. Nuclear magnetic resonance (NMR) spectroscopy analysis of EPS

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NMR spectrum of the EPS from strain YW11 were obtained using a Bruker 9

Page 9 of 37

AVANCE 600 MHz spectrometer (Bruker Group, Fällanden, Switzerland) operated at

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27 °C with a 5 mm inverse probe. Prior to analysis, samples were exchanged twice in

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D2O (99.9 at % D, Cambridge Isotope Laboratories, Inc., Andover, MA, USA) with

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intermediate lyophilization, and then dissolved in D2O at concentrations of 5 mg/mL

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(for 1 H NMR) and 40 mg/mL (for 13 C NMR). Chemicals shifts (δ) were expressed in

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parts per million (ppm). The 2D

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decoupling during acquisition of the 1 H FID and used to assign signals.

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2.9. Rheological properties of EPS

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H–13 C HSQC experiment was recorded with

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The rheological behavior of the EPS solutions was carried out in a Brookfield

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DV-III ultra programmable rheometer (Brookfield Engineering Laboratories Inc.,

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Stoughton, Massachusetts, USA) with a SC4-18 spindle that rotated in chamber

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equipped with temperature control system. The lyophilized EPS sample was dissolved

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in 0.1 M CaCl2, 0.1 M NaCl solutions, 11% (w/v) skim milk and distilled water at a

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concentration of 2 mg mL-1, respectively. To investigate the effect of pH on the

viscosity of EPS, the pH of EPS solution was adjusted at levels of 4.0, 6.0 and 7.0 by 1 N HCl and NaOH solutions. To investigate the effect of temperature on the viscosity of EPS, the EPS solution was exposed to different temperatures (25 °C, 35 °C and 45

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°C). The rheological behavior of the EPS was studied by measuring viscosity as a

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function of shear rate from 10 to 300 s−1.

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2.10.

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Scanning electron microscopy (SEM) analysis of EPS

The lyophilized samples of the purified EPS (5 mg) were fixed to the SEM stubs 10

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with double sided tape, then coated with a layer of gold, ∼10 nm thick. The samples

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were observed in a scanning electron microscope (S-4800, Hitachi Ltd., Japan) at an

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accelerating voltage of 3.0 kV.

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2.11.

Atomic force micrograph (AFM) analysis of EPS

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A stock solution (1 mg mL-1) was prepared by adding the purified EPS into distilled

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H2O. The aqueous solution was stirred for about 1 h at 40 °C in a sealed bottle under

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N2 stream so that EPS dissolved completely. After cooling to room temperature, the

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solution was continuously diluted to the final concentration of 0.01 mg mL-1. About 5

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μL of diluted EPS solution was dropped on the surface of a mica sample carrier,

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allowed to dry at a room temperature. The AFM images were obtained using a

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Dimension® Icon instrument (Bruker Instruments Co., Germany) in tapping mode.

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2.12.

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Thermogram analysis (TGA)

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of 100 mL min-1.

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2.13.

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The pyrolysis and combustion were carried out in TA SDT-Q600 thermal analyzer

operating at atmospheric pressure. The purified sample of EPS (3 mg) was placed in an Al2O3 crucible and heated at a linear heating rate of 10 °C min-1 over a temperature

range of 25–800 °C. The experiments were performed in air atmosphere at a flow rate

Differential scanning calorimeter (DSC)

The thermal property of the EPS was analyzed using a DSC (DSC Model Q 100, 11

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TA instruments). The purified EPS sample (5 mg) was placed in an aluminum pan,

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which was sealed and analyzed using empty pan as a reference. The melting point and

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enthalpy changes were determined by increasing the heating rate at 10 °C min-1 from

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10 to 400 °C (Wang et al., 2010).

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3. Results and discussion

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3.1. Identification of strain YW11

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Strain YW11 was primarily identified as L. plantarum by API 50 CHL test. Further

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identification by 16S rRNA sequencing confirmed that the sequence of strain YW11

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(Acc. No. KM265361) was identical to those of L. plantarum lp-15 (Acc. No.

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FJ763580), L. plantarum LW4 (Acc. No. KJ779096) and L. plantarum TW57-4 (Acc.

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No. KJ026699). Therefore, the strain was designated as L. plantarum YW11.

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3.2. Bacterial growth and EPS production

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Change of pH of the medium and EPS production by L. plantarum YW11 during

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growth are shown in Fig. 1. L. plantarum YW11 exhibited a fast growth with a rapid decrease in pH of the medium during the first 8 h of incubation, and the viable cell count reached a maximum of 9.77 log cfu mL-1 at 16 h with pH about 4.0. In the late

stationary phase of growth (after 32 h), the viable count decreased gradually to about

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8.60 log cfu mL-1, with a slight decrease in pH to about 3.90 at 56 h. EPS production

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by strain YW11 increased rapidly during the initial phase of growth, and continued to

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increase to 90 mg L-1 at 56 h. It seemed that the EPS was not degraded during the late

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stationary phase of L. plantarum YW11 although the bacterial growth decreased, as 12

Page 12 of 37

previously reported with other EPS-producing L. plantarum strain (Yang et al., 2010).

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However, EPS production by L. plantarum C88, L. plantarum 70810, and L.

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helveticus MB2-1 decreased after prolonged incubation, probably due to presence of

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glycohydrolases in the culture that catalyzed the degradation of polysaccharides

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(Zhang et al., 2013; Wang et al., 2014; Li et al., 2014a).

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3.3. Isolation and purification of EPS

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The crude EPS obtained by ethanol precipitation of the culture supernatant of L.

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plantarum YW11 was first separated by anion-exchange chromatography of

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DEAE-52. Fractions corresponding to the major peak eluted with 0.2 M NaCl were

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found to contain polysaccharides. These fractions that contain the EPS, being an

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acidic polysaccharide as it was eluted with NaCl (Gan, Ma, Jiang, Xu, & Zeng, 2011),

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were further purified by Sepharose CL-6B gel permeation chromatography. A single

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elution peak was generated, and the corresponding fractions were collected and

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lyophilized to obtain the purified form of the EPS that was used for the following physicochemical characterization. Analysis of the purified EPS sample showed that it contained 92.35 ± 2.38% of carbohydrate, 2.52 ± 0.12% of moisture, 1.38 ± 0.25% of protein, and 1.56 ± 0.09% of uronic acids. 3.4. Molecular mass and monosaccharide composition of EPS

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The molecular mass of the EPS of L. plantarum YW11 was determined by

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GPC-MALLS-RI (Fig. 2). The chromatogram of the EPS appeared as a single

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symmetrical narrow peak, confirming the homogeneity of the purified EPS sample. 13

Page 13 of 37

The molecular mass of the EPS was determined to be 1.1 × 105 Da (error 3.2%),

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which was similar to that (169.6 kDa) of the acidic EPS from L. plantarum 70810

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(Wang et al., 2014), lower than that (1.15 × 106 Da) of the neutral EPS of L.

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plantarum C88 (Zhang et al., 2013), but higher than that (4.4 × 104 Da) of the EPS of

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L. plantarum EP56 (Tallon, Bressollier, & Urdaci, 2003). The polydispersity index of

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the EPS of L. plantarum YW11 was determined to be 1.2 (error 2.5%), which was

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generally low, indicating no large aggregates formed upon dispersion of this EPS in

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aqueous solution. The polydispersity index, as a measure of the width of molecular

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mass distribution, was thought to be important due to the relevance and significant

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influence of molecular mass distribution on the functional properties of EPS (Zheng et

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al., 2014).

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GC-MS analysis of the monosaccharide composition of the EPS of L. plantarum

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YW11 showed that the EPS was composed of glucose and galactose in a molar ratio

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of 2.71:1. Previously, the EPS from L. plantarum C88 was shown to contain glucose

and galactose, but in different molar ratio (Zhang et al., 2013). Other L. plantarum strains, e.g. both EP56 and KF5 produced EPSs consisted of glucose and galactose, and additionally N-acetylgalactosamine (Tallon et al., 2003) and mannose (Wang et

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al., 2010), respectively. The monosaccharide composition of EPS produced by LAB

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can be affected by the type of strains, culture conditions and medium compositions

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(Wang et al., 2014).

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3.5. Spectra analysis of the EPS functional groups 14

Page 14 of 37

The UV-vis spectrum of the purified EPS from strain YW11 showed no absorption

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at 260 nm or 280 nm, indicating no nucleic acid present in the EPS sample. The FTIR

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spectrum (Fig. 3) of the EPS indicated that the polysaccharide contained a significant

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number of hydroxyl groups as it displayed a broad and intense stretching peak around

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3408 cm-1 (Wang et al., 2010). The stretching band around 2933 cm-1 was due to C–H

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stretching vibration (Melo, Feitosa, Freitas, & de Paula, 2002). The absorptions at

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1726 cm-1 and 1646 cm-1 were due to the stretch vibration of C=O bond (Ye et al.,

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2014), and a peak at 1550 cm-1 could be assigned to N–H bending of amides II of

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protein (Lin et al., 2005). The absorption at 1384 cm-1 was possibly due to symmetric

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CH3 bending (Pan & Mei, 2010). The bands within the 900–1150 cm-1 region were

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attributed to the vibration of C-O-C bond (Ye et al., 2014).

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3.6. NMR analysis of EPS

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The 1 H NMR and 13 C NMR spectroscopy of the EPS from strain YW11 are shown

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in Fig. 4. Three major chemical shift signals in the anomeric region (δ 4.8–5.5 ppm) were found at δ 5.22, 5.07 and 4.97 ppm in 1 H NMR (Fig. 4A). This indicates that the

EPS from strain YW11 mainly consists of three monosaccharide residues. Based on their chemical shift and the value of the coupling constant for anomeric signals in the

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The

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carbons regions (95–110 ppm) and ring carbons (50–85 ppm) regions. Based on the

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data reported previously (Li et al., 2014; Wang at al., 2014), the three main signals in

H NMR spectrum, three signals were corresponds to C-1 of α-type configurations. 13

C NMR spectrum (Fig. 4B) of EPS from strain YW11 included anomeric

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Page 15 of 37

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the anomeric carbons regions at δ 104.58, 104.05 and 101.21 ppm were corresponded

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to the α-linkages. Although no aminosugars were detected by monosaccharide

306

analysis of the EPS as described above, the

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signals between 50-60 ppm, and several signals at 22.85, 174.82, 175.30 ppm that

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were probably from C-2 and N-acetyl group of the aminosugar, respectively,

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indicating possible presence of this sugar in the EPS of strain YW11.

C NMR spectrum demonstrated two

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The single-bond correlations between the protons and the corresponding carbons

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obtained from 2D 1 H–13 C HSQC spectrum (Fig. 4C) of the EPS from strain YW11

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in D2O demonstrated three crosspeaks in the anomeric region resulted from cross link

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of the C-1 signals at δ 104.58, 104.05 and 101.21 ppm to the proton signals at δ 5.22,

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5.07 and 4.97 ppm, respectively, confirming presence of three monosaccharide

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residues in the repeating unit of the polysaccharide. Detailed chemical structure of the

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EPS from strain YW11 needs to be further studied.

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3.7. Rheological properties of EPS The rheological behavior of the EPS from strain YW11 was studied in water, skim

milk, different salt solutions (NaCl and CaCl2), and at different pHs (4, 6 and 7) and temperatures (25 °C, 35 °C and 45 °C) (Fig. 5). All the EPS solutions showed a shear

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thinning behavior, a decrease of viscosity with increasing shear rates that was caused

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mainly by breakdown of structural units in the EPS by hydrodynamic forces generated

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during shear (Kavita, Singh, Mishra, & Jha, 2014). This property was considered to be

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important for yielding desired sensory properties such as mouth-feel and flavor 16

Page 16 of 37

release properties, as well as some processing operations such as stirring, pouring,

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pumping, spray drying (Zhou et al., 2014). The EPS in skim milk was shown to be

327

more viscous than in water over the whole shear rate range (Fig. 5A). However, no

328

obvious difference in viscosity was observed with the EPS in 0.1 M NaCl solution or

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in 0.1 M CaCl2 solution at all the shear rates tested (Fig. 5B). The higher viscosity of

330

the EPS solution at acidic pHs (4, 6) than at a neutral pH (7.0) (Fig. 5C) would be

331

beneficial to the use of this EPS-producing strain to improve texture of fermented

332

milk that usually has pH values between 4.0 and 4.5. Ahmed, Wang, Anjum, Ahmad,

333

and Khan (2013) also reported that an EPS produced by L. kefiranofaciens ZW3 was

334

more viscous at acidic pHs than alkaline pHs. Studies on the effect of temperature

335

(Fig. 5D) showed that increasing temperature from 25 °C to 35 °C did not affect the

336

viscosity of the EPS solution over the whole shear rate range, but further increasing to

337

45 °C resulted in decreased viscosity. Similarly, an acidic EPS produced by

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Streptococcus phocae PI80 had decreased viscosity with increasing temperature from 25 °C to 45 °C (Kanmani et al. 2011). This decrease in viscosity of EPS solution might be attributed to the decreased interactions between the EPS molecules when temperature was increased, thus leading to a loosened polymer structure. Therefore,

342

the rheological behavior of the EPS produced by L. plantarum YW11 as described

343

above would make it particular suitable as a potential stabilizer used in dairy foods.

344

3.8. Scanning electron microscopic (SEM) analysis of EPS

345

The microstructures of the EPS from strain YW11 and xanthan gum as a reference 17

Page 17 of 37

material are represented by SEM (Fig. 6). The EPS showed a relatively stable three

347

dimensional structure that appeared to be a porous web (Fig. 6A), whereas the

348

xanthan gum showed a dispersive structure with irregular lumps of different size (Fig.

349

6C). At a higher magnification (Fig. 6B and 6D), additional details of the

350

microstructure of the EPS and xanthan gum were visible. The EPS had a smooth and

351

glittering surface, but xanthan gum presented a coarse surface. Similar porous

352

structures were also reported with the purified EPSs of Bifidobacterium longum subsp.

353

infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 (Prasanna, Bell,

354

Grandisona, & Charalampopoulos, 2012). However, Wang et al. (2010) observed a

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sheet-like compact morphology of the EPS from L. plantarum KF5. The highly

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branched and porous structure of the EPS from strain YW11 as observed in this study

357

may facilitate its application in foods to improve the physical properties, e.g. viscosity,

358

water holding capacity of the product.

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3.9. Atomic force micrograph (AFM) analysis of EPS In recent years exopolysaccharide has been studied extensively by using atomic

force microscopy that provides a powerful tool to characterize the morphological features of polymers (Admed et al., 2013). Images of the strain YW11 EPS deposited

363

from 0.01 mg mL-1 aqueous solution were obtained by AFM (Fig. 7). There was

364

presence of many spherical lumps with the height ranged from 3.0 nm to 13.5 nm.

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Some regions formed fibrous network, whereas other regions were relatively sparse,

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suggesting that the structure of the EPS might be tangled networks, which was in 18

Page 18 of 37

agreement with its fibrous morphology in dry solid form. This structural property of

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the EPS was also observed earlier with another viscous polysaccharide from Mesona

369

blumes gum that had irregular worm-shaped structure (Feng, Gu, Jin, & Zhuang,

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2008). The viscous nature of the EPS from strain YW11 described above might also

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be caused by its capability of binding with water in the aqueous solution.

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3.10.

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Thermogravimetric analysis (TGA)

The thermogravimetric analysis (TGA) of the EPS from L. plantarum YW11 was

374

carried out dynamically between weight loss vs temperatures. As shown in Fig. 8A,

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the EPS showed an initial weight loss of approximately 11.64% between about 25 and

376

100 °C. This initial weight loss may be associated with the loss of moisture (Wang et

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al., 2010), suggesting high content of carboxyl groups in the EPS molecules. Kumar,

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Joo, Choi, Koo, and Chang (2004) reported that the high level of carboxyl group in

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the EPS increased the degradation of the first phase as carboxyl groups were bound to

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more water molecules. A degradation temperature (Td) of 287.7 °C and a large weight loss of approximately 69.93 % could be observed from the differential thermogravimetric mass loss spectrum (Fig. 8B) and thermogravimetric mass loss spectrum (Fig. 8A) for strain YW11 EPS between about 200 and 300 °C. Then the

384

weight loss gradually decreased to leave a final residue of ca. 16.07% of the total EPS.

385

Ahmed, Wang, Anjum, Ahmad, and Khan (2013) and Wang et al. (2010) showed that

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the degradation temperatures of ZW3 EPS, KF5 EPS, xanthan and locust gum were

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299.62 °C, 278.53 °C, 282.65 °C and 278.46 °C, respectively. The degradation 19

Page 19 of 37

temperature of the EPS from strain YW11 in the present study was within the range of

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the KF5 EPS and ZW3 EPS, but slightly higher than xanthan gum and locust gum.

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The different thermal stability and degradation behavior of these EPSs probably

391

attributed to their different carbohydrate compositions. Due to the relatively higher

392

degradation temperature of the EPS from strain YW11, it would be safe for use of this

393

EPS in dairy industry where in most of the processes temperature seldom overpasses

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150 °C.

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3.11.

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DSC analysis

Commercial application of an EPS is crucially dependent on its thermal and

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rheological behavior. DSC analysis of the EPS from strain YW11 showed an

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exothermic peak with heat flow from 10 °C to 400 °C (Fig. 9). The melting point of

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the EPS exothermic peak started at about 143.6 °C, and the enthalpy change (△H)

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phocae PI80 endothermic peak started at 120.09 °C and the enthalpy change was

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about 404.6 J g-1. Thus the EPS from strain YW11 showed a different thermal

406

behaviour from these EPSs.

400 401 402

needed to melt 1 g of EPS was 217.8 J. Different results were reported previously for different EPSs. Wang et al. (2010) showed that the melting point and enthalpy change of KF5 EPS isolated from L. plantarum KF5 were 88.35 °C and 133.5 J g-1,

respectively. Kanmani et al (2011) reported that the melting point of the EPS from S.

20

Page 20 of 37

407

Conclusion In this study, an EPS-producing strain YW11 isolated from Tibet Kefir was

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identified as L. plantarum. The EPS from YW11 was composed of glucose and

410

galactose in a molar ratio of 2.71:1, with possible presence of N-acetylated sugar

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residues in the polysaccharide. The EPS had a molecular mass of 1.1 × 105 Da. It

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exhibited a shear thinning behavior under different conditions and had higher

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viscosity in skim milk, at lower temperature and acidic pHs. The SEM images of the

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EPS demonstrated a porous structure that was highly branched, while the AFM

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images of the EPS revealed presence of many spherical lumps. Furthermore, the EPS

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had a higher degradation temperature. These characteristics of the EPS produced by L.

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plantarum YW11 would make it a promising candidate for its exploitation in food

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industry.

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Acknowledgment

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This work was financially supported by The Key Project of the Educational

Committee of Beijing City (KZ201310011011), Natural Science Foundation of China (31371804), and National Public Benefit Research (Agriculture) Foundation (201303085).

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References

425

Ai, L., Zhang, H., Guo, B., Chen, W., Wu, Z., & Wu, Y. (2008). Preparation, partial

426

characterization and bioactivity of exopolysaccharides from Lactobacillus casei

427

LC2W. Carbohydrate Polymers, 74, 353‒357. 21

Page 21 of 37

Amjres, H., Béjar, V., Quesada, E., Carranza, D., Abrini, J., Sinquin, C., et al. (2014).

429

Characterization of haloglycan, an exopolysaccharide produced by Halomonas

430

stenophila HK30. International Journal of Biological Macromolecules, 72, 117‒

431

124.

ip t

428

Ahmed, Z., Wang, Y., Anjum, N., Ahmad, A., & Khan, S. T. (2013). Characterization

433

of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated

434

from Tibet kefire – Part II. Food Hydrocolloids, 30, 343–350.

cr

432

Bradford, M. M. (1976). A rapid and sensitive method for the quantification of

436

microgram quantities of protein utilizing the principle of protein–dye binding.

437

Analytical Biochemistry, 72, 248–252.

an

us

435

Costa, N. E., Hannon, J. A., Guinee, T. P., Auty, M. A. E., McSweeney, P. L. H., &

439

Beresford, T. P. (2010). Effect of exopolysaccharide produced by isogenic strains

440

of Lactococcus lactis on half-fat Cheddar cheese. Journal of Dairy Science, 93,

441

3469‒3486.

d

M

438

de los Reyes-Gavilán, C. G., Suárez, A., Fernández-García, M., Margolles, A.,

443

Gueimonde, M., & Ruas-Madiedo, P. (2011). Adhesion of bile-adapted

444

Bifidobacterium strains to the HT29-MTX cell line is modified after sequential

446 447 448 449 450

Ac ce p

445

te

442

gastrointestinal challenge simulated in vitro using human gastric and duodenal

juices. Research in Microbiology, 162, 514–519.

de Man, J. C., Rogosa, M., & Sharpe, M. E. (1960). A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology, 23, 130–135.

Dische, Z. (1947). A new specific colour reaction of hexuronic acid. The Journal of Biological Chemistry, 167, 189–198.

451

DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956).

452

Colorimetric method for determination of sugars and related substances.

453

Analytical Chemistry, 28, 350–356.

454

Feng, T., Gu, Z., Jin, Z., & Zhuang, H. (2008). Isolation and characterization of an 22

Page 22 of 37

455

acidic polysaccharide from Mesona Blumesgum. Carbohydrate Polymers, 71,

456

159–169. Gan, D., Ma, L., Jiang, C., Xu, R., & Zeng, X. (2011). Production, preliminary

458

characterization and antitumor activity in vitro of polysaccharides from the

459

mycelium of Pholiota dinghuensis Bi. Carbohydrate Polymers, 84, 997–1003.

ip t

457

Ismail, B., & Nampoothiri, K. M. (2010). Production, purification and structural

461

characterization of an exopolysaccharide produced by a probiotic Lactobacillus

462

plantarum MTCC 9510. Archives of Microbiology, 192, 1049–1057.

us

cr

460

Kanmani, P., Kumar, R. S., Yuvaraj, N., Paari, K. A., Pattukumar, V., & Arul, V.

464

(2011). Production and purification of a novel exopolysaccharide from lactic acid

465

bacterium Streptococcus phocae PI80 and its functional characteristics activity in

466

vitro. Bioresource Technology, 102, 4827–4833.

an

463

Kavita, K., Singh, V. K., Mishra, A., & Jha, B. (2014). Characterisation and

468

antibiofilm activity of extracellular polymeric substances from Oceanobacillus

469

iheyensis. Carbohydrate Polymers, 101, 29–35.

d

M

467

Kimmel, S. A., & Roberts, R. F. (1998). Development of a growth medium suitable

471

for exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus

472

RR. International Journal of Food Microbiology, 40, 87–92.

474 475 476 477 478

Ac ce p

473

te

470

Kumar, C. G., Joo, H. S., Choi, J. W., Koo, Y. M., & Chang, C. S. (2004). Purification and characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp. I-450. Enzyme and Microbial Technology, 34, 673–681.

Liu, C., Chu, F., Chou, C., & Yu, R. (2011). Antiproliferative and anticytotoxic effects of cell fractions and exopolysaccharides from Lactobacillus casei 01. Mutation Research, 721, 157–162.

479

Li, C., Li, W., Chen, X., Feng, M., Rui, X., Jiang, M., et al. (2014b). Microbiological,

480

physicochemical and rheological properties of fermented soymilk produced with

481

exopolysaccharide (EPS) producing lactic acid bacteria strains. LWT-Food 23

Page 23 of 37

482

Science and Technology, 57, 477–485. Lindström, C., Holst, O., Nilsson, L., Öste, R., & Andersson, K. E. (2012). Effects of

484

Pediococcus parvulus 2.6 and its exopolysaccharide on plasma cholesterol levels

485

and inflammatory markers in mice. AMB Express, 2, 66–69.

ip t

483

Lin, M. S., Al-Holy, M., Chang, S. S., Huang, Y., Cavinato, A. G., Kang, D. H., et al.

487

(2005). Rapid discrimination of Alicyclobacillus strains in apple juice by Fourier

488

transform infrared spectroscopy. International Journal of Food Microbiology,

489

105, 369–376.

us

cr

486

Liu, C., Lu, J., Lu, L., Liu, Y., Wang, F., & Xiao, M. (2010). Isolation, structural

491

characterization and immunological activity of an exopolysaccharide produced

492

by Bacillus licheniformis 8–37-0–1. Bioresource Technology, 101, 5528–5533.

an

490

Li, W., Ji, J., Chen, X., Jiang, M., Rui, X., & Dong, M. (2014a). Structural elucidation

494

and antioxidant activities of exopolysaccharides from Lactobacillus helveticus

495

MB2-1. Carbohydrate Polymers, 102, 351–359.

M

493

Li, Wei, Ji, J., Tang, W., Rui, X., Chen, X., Jiang, M., & Dong, M. (2014).

497

Characterization of an antiproliferative exopolysaccharide (LHEPS-2) from

498

Lactobacillus helveticus MB2-1. Carbohydrate Polymers, 105, 334–340.

500 501 502 503 504 505 506

te

London, L. E. E., Price, N. P. J., Ryan, P., Wang, L., Auty, M. A. E., Fitzgerald, G. F.,

Ac ce p

499

d

496

et al. (2014). Characterization of a bovine isolate Lactobacillus mucosae DPC 6426 which produces an exopolysaccharide composed predominantly of mannose residues. Journal of Applied Microbiology, 117, 2, 509–517.

Melo, M. R. S., Feitosa, J. P. A., Freitas, A. L. P., & de Paula, R. C. M. (2002). Isolation and characterization of soluble sulfated polysaccharide from the red seaweed Gracilaria cornea. Carbohydrate Polymers, 49, 491–498. Nikolic, M., López, P., Strahinic, I., Suárez, A., Kojic, M., Fernández-García, M., et al.

507

(2012).

Characterisation

of

the

exopolysaccharide

(EPS)-producing

508

Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative 24

Page 24 of 37

509

strains as potential probiotics. International Journal of Food Microbiology, 158,

510

155–162.

512

Pan, D., & Mei, X. (2010). Antioxidant activity of an exopolysaccharide purified from Lactococcus lactis subsp. lactis 12. Carbohydrate Polymers, 80, 908–914.

ip t

511

Prasanna, P. H. P., Bell, A., Grandison, A. S., & Charalampopoulos, D. (2012).

514

Emulsifying, rheological and physicochemical properties of exopolysaccharide

515

produced by Bifidobacterium longum subsp. infantis CCUG 52486 and

516

Bifidobacterium infantis NCIMB 702205. Carbohydrate Polymers, 90, 533–540.

517

Ramchandran, L., & Shah, N. P. (2010). Characterization of functional, biochemical

518

and textural properties of synbiotic low-fat fermented milks during refrigerated

519

storage. LWT-Food Science and Technology, 5, 819–827.

an

us

cr

513

Salazar, N., Prieto, A., Leal, J. A., Mayo, B., Bada-Gancedo, J. C., de los

521

Reyes-Gavilán, C. G., et al. (2009). Production of exopolysaccharides by

522

Lactobacillusand Bifidobacterium strains of human origin, and metabolic

523

activity of the producing bacteria in milk. Journal of Dairy Science, 92,

524

4158–4168.

te

d

M

520

Sandal, I., Inzana, T., Molinaro, A., De Castro, C., Shao, J., Apicella, M., et al. (2011).

526

Identification, structure, and characterization of an exopolysaccharide produced

527 528 529 530 531

Ac ce p

525

by Histophilus somni during biofilm formation. BMC Microbiology, 11, 186.

Shang, N., Xu, R., & Li, P. (2013). Structure characterization of an exopolysaccharide produced by Bifidobacterium animalis RH. Carbohydrate Polymers, 91, 128–134.

Song, Y. R., Jeong, D. Y., Cha, Y. S., & Baik, S. H. (2013). Exopolysaccharide

532

produced by Pediococcus acidilactici M76 isolated from the Korean traditional

533

rice wine, Makgeolli. Journal of Microbiology and Biotechnology, 23, 681–688.

25

Page 25 of 37

534

Tallon, R., Bressollier, P., & Urdaci, M. C. (2003). Isolation and characterization of

535

two exopolysaccharides produced by Lactobacillus plantarum EP56, Research in

536

Microbiology, 154, 705–712. Vijayendra, S. V. N., Palanivel, G., Mahadevamma, S., & Tharanathan, R. N. (2008).

538

Physico-chemical characterization of an exopolysaccharide produced by a

539

nonropy strain of Leuconostoc sp. CFR 2181 isolated from dahi, an Indian

540

traditional lactic fermented milk product. Carbohydrate Polymers, 72, 300–307.

541

Wang, K., Li, W., Rui, X., Chen, X., Jiang, M., & Dong, M. (2014). Characterization

542

of a novel exopolysaccharide with antitumor activity from Lactobacillus

543

plantarum 70810. International Journal of Biological Macromolecules, 63,

544

133–139.

an

us

cr

ip t

537

Wang, Y., Li, C., Liu, P., Ahmed, Z., Xiao, P., & Bai, X. (2010). Physical

546

characterization of exopolysaccharide produced by Lactobacillus plantarum KF5

547

isolated from Tibet Kefir. Carbohydrate Polymers, 82, 895–903.

M

545

Yang, Z., Li, S., Zhang, X., Zeng, X., Li, D., Zhao, Y., et al. (2010). Capsular and

549

slime-polysaccharide production by Lactobacillus rhamnosus JAAS8 isolated

550

from Chinese sauerkraut: Potential application in fermented milk products.

551

Journal of Bioscience and Bioengineering, 110, 53–57.

553 554 555 556 557

te

Ac ce p

552

d

548

Ye, S., Liu, F., Wang, J., Wang, H., & Zhang, M. (2012). Antioxidant activities of an exopolysaccharide isolated and purified from marine Pseudomonas PF-6. Carbohydrate Polymers, 87, 764–770.

Ye, S., Zhang, M., Yang, H., Wang, H., Xiao, S., Liu, Y., et al. (2014). Biosorption of Cu2+, Pb2+ and Cr6+ by a novel exopolysaccharide from Arthrobacter ps-5. Carbohydrate Polymers, 101, 50–56.

558

Zhang, L., Liu, C., Li, D., Zhao, Y., Zhang, X., & Zeng, X. (2013). Antioxidant

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activity of an exopolysaccharide isolated from Lactobacillus plantarum C88.

560

International Journal of Biological Macromolecules, 54, 270–275. 26

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Zheng, J., Wang, J., Shi, C., Mao, D., He, P., & Xu, C. (2014). Characterization and

562

antioxidant activity for exopolysaccharide from submerged culture of Boletus

563

aereus. Process Biochemistry, 49, 1047–1053. Zhou, F., Wu, Z., Chen, C., Han, J., Ai, L., & Guo, B. (2014). Exopolysaccharides

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produced by Rhizobium radiobacter S10 in whey and their rheological properties.

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Food Hydrocolloids, 36, 362–368.

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570 Figure captions

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Figure 1. Kinetics of growth and EPS production by L. plantarum YW11 in a

573

semi-defined medium at 37 °C showing changes of the bacterial cell counts, EPS

574

yield and pH of the medium during the incubation. Each value represents the average

575

of triplicate measurements.

576

Figure 2. GPC-MALLS-RI chromatogram of EPS from L. plantarum YW11.

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purified EPS from L. plantarum YW11 recorded on Bruker AVANCE 600 MHz

582

spectrometer in D2O.

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Figure 5. Rheological behavior of the EPS from L. plantarum YW11 in distilled

577 578 579

Figure 3. UV spectrum of the purified EPS from L. plantarum YW11 in the range of

190-550 nm (A); FTIR spectrum of the purified EPS in the range of 400-4000 cm-1 (B).

Figure 4. The

1

H NMR (A), 13 C (B) and

1

H–13 C HSQC (C) NMR spectra of the

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water and skim milk (11%, w/v) (A), in 0.1 M NaCl and CaCl2 (B), at different pH

585

values (4, 6 and 7) (C), and at different temperatures (25, 35 and 45 °C) (D).

586

Figure 6. Scanning electron micrograph images of the purified EPS from L.

587

plantarum YW11 (A: 1000 ×; B: 5000 ×) as compared to a reference material xanthan

588

gum (C: 1000 ×; D: 5000 ×).

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Figure 7. Atomic force microscopy images of the purified EPS from L. plantarum

590

YW11 (A) planar, (B) cubic.

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Figure 8. Thermogravimetric mass loss spectrum (A, left hand ordinate ordinate axis),

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and differential thermogravimetric mass loss spectrum (B, right hand ordinate

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ordinate axis) of the purified EPS from L. plantarum YW11.

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Figure 9. DSC thermogram of the purified EPS from L. plantarum YW11.

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Characterization of an exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibet Kefir.

An exopolysaccharide (EPS)-producing strain YW11 isolated from Tibet Kefir was identified as Lactobacillus plantarum, and the strain was shown to prod...
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