Isolation, fine structure and morphology studies of galactomannan from endosperm of Gleditsia japonica var. delavayi

Accepted Manuscript Title: Isolation, fine structure and morphology studies of galactomannan from endosperm of Gleditsia japonica var. delavayi Authors: Mingzhe Sun, Yumeng Li, Tianxin Wang, Yanwei Sun, Xiyuan Xu, Zesheng Zhang PII: DOI: Reference:

S0144-8617(17)31400-5 https://doi.org/10.1016/j.carbpol.2017.12.003 CARP 13065

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

10-10-2017 14-11-2017 3-12-2017

Please cite this article as: Sun, Mingzhe., Li, Yumeng., Wang, Tianxin., Sun, Yanwei., Xu, Xiyuan., & Zhang, Zesheng., Isolation, fine structure and morphology studies of galactomannan from endosperm of Gleditsia japonica var.delavayi.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.12.003 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.

Isolation, fine structure and morphology studies of galactomannan from endosperm of Gleditsia japonica var. delavayi Mingzhe Sun 1 , Yumeng Li1, Tianxin Wang1,Yanwei Sun1, Xiyuan Xu1, Zesheng 2, 

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Zhang1,

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Graphical abstract

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Highlights

A high purity polysaccharide from G. japonica var. delavayi seeds was obtained.

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EGSP was identified a galactomannan (Mw, 1913kDa) with an M/G ratio of 2.54-2.66.

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A molecular structure model of general galactomannan was established. The apparent morphology of EGSP was observed by different methods. 1

Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China. 2 Tianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center, 300457, Tianjin, China.  Corresponding author.

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Abstract The water-soluble polysaccharides extracted from endosperm of Gleditsia japonica var. delavayi seeds (EGSP) were identified as galactomannan having the M/G ratio of 2.54 to 2.66 and a weight average molecular weight (Mw) of 1913kDa.

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The molecular structure of EGSP was determined by periodate oxidation, Smith degradation, methylation, FTIR and NMR spectroscopy. The main chain is composed β-1,4-D-mannopyranose

and

the

branches

composed

of

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of

single

α-1-D-galactopyranose. We had also established a model to speculate the fine

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structure of galactomannan molecules and given preliminary results. The I2-KI test

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indicated that there were many branches on the EGSP backbone and no starch in

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EGSP. The CD spectra and Congo red test showed EGSP was random coil conformations in solution and could form a small quantity of helical conformation

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under alkaline conditions. The microstructure of morphology was observed by OM, SEM and AFM. The results showed that the fibers composed of multiple microfibers

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formed a network construction by entangling with each other.

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Keyword: galactomannan, isolation, fine structure, morphology. 1. Introduction

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Galactomannans is a biological macromolecule polymer with wide raw material

source, which mainly comes from plants and microorganisms (Prajapati et al., 2013). The endosperm of the leguminous plant seeds contains a large amount of galactomannan used as an energy reservation for its seed germination, such as Cyamopsis tetragonoloba and Ceratonia silique (Meier & Reid, 1982). Gleditsia is a 2

leguminous plant, distributed in eastern Asia, eastern North America, South America, the southern Caucasus Mountains and India (Gordon, 1966), in which its seed endosperm is also rich in galactomannan. Galactomannan extracted from Gleditsia is diverse (Table 1), which embodies in different ratios of M/G (manose/galactose) and

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Mw (weight-average molecular weight). Table 1. Galactomannan from Gleditsia. Area

M/G

Mw (kDa)

Reference

G. ferox Desf.

Russia

2.54

1660

(Egorov, Mestechkina, & Shcherbukhin, 2004)

G. caspia Desf.

Soviet

2.85

685

G. triacanthos L.

Union

2.40

480

G. triacanthos Linn.

Turkey

-

-

(Cengiz, Dogan, & Karaman, 2013)

G. triacanthos

Portugal

2.82

1620

(Cerqueira et al., 2009)

G. triacanthos

Argentina

2.70

-

(Mazzini & Cerezo, 1986)

G. amorphoides

Argentina

2.70

1390

(Cerezo, 1965; Perduca et al., 2013)

G. sinensis Lam.

Georgia

2.69

1230

(Olennikov & Rokhin, 2010)

3.25

1282

3.31

651.8

2.30

979.3

China

G. melanacantha Uzbekistan

1.10

G. japonica

Uzbekistan

5.00

Note: -, Not detected.

7.9 -

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G. delavayi

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G. microphylla

(V. D. Shcherbukhin, 1992)

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G. sinensis Lam.

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Latin name

(Jiang, Jian, Cristhian, Zhang, & Sun, 2011)

(Rakhmanberdyeva, 2004) (Mirzaeva,

Rakhmanberdyeva,

Kristallovich,

Rakhimov, & Shtonda, 1998)

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The ratios of M/G and Mw are important structure parameters of galactomannan. In addition to this, the general structure of galactomannan is a linear molecule and the

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backbone is composed of D-mannopyranose residue ligation of β-1,4-glycosidic linkages, and some of the hydroxyl groups of mannose residue are attached by single

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D-galactopyranose ligation of α-1,6-glycosidic linkages (Dea & Morrison, 1975). It is worth noting that there will be exceptions. Mazzini’s (Mazzini & Cerezo, 1986) research shows that the galactomannan of G. triacanthos has different structures determined by the side-chains which are attached to the backbone by 1,6-linkages or 1,3-linkages and which contain, on average, two galactose units, either 1, 3-1,6-linked 3

or 1,3-1, 3-linked with α-D and β-D configurations, respectively. At present, the researches on the structure of galactomannan are mainly focused on the ratio of M/G, Mw and glycosidic bond typing, but there are few researches on the fine structure of the side chain substitution sequence and its position. Grasdalen and Painter

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(Grasdalen & Painter, 1980) point out that the split of C4 signal of 13C NMR spectrum indicates the ratio of three mannobiose blocks UM (unmodified mannobiose block: MM

(monosubstituted

mannobiose

block:

(Gal)Man-Man

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Man-Man),

or

Man-Man(Gal)) and DM (disubstituted mannobiose block: (Gal)Man-Man(Gal)).

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However, there is no further discussion about the quantity of these mannobiose blocks.

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The structure of galactomannan determines its nature; hence the studies on its fine

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structure and conformation characteristics are of great significance.

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G. japonica var. delavayi, the varietas of G. japonica, grown in Yunnan province

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of China, is a unique Gleditsia species in the local area. The endosperm has been used as health-care food for hundred years. It contains a lot of high quality galactomannan,

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so it can also be used as a new galactomannan resource. In this paper, we conducted

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the following studies: i, the polysaccharides of EGS was firstly isolated and purified, and named EGSP; ii, the molecular structure of EGSP was determined through physical and chemical methods and confirmed as a galactomannan; iii, the surface

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morphology and conformation characteristics of EGSP were investigated; iv, what’s more, the meaning of fine structure represented by mannobiose blocks was discussed.

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2. Material and methods 2.1 Materials The EGS for this study was supplied by Lianghexian Forestry Bureau from Yunnan, China. The specimens of the plant were kept in Kunming Institute of Botany,

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Chinese Academy of Science, with the registration number 0126893. Sodium periodate, sodium iodate, sodium hydroxide, sodium borohydride, iodine, potassium

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iodide, Congo red, potassium sulfate, copper sulfate, methyl red, bromocresol green,

boric acid, ethanol, trifluoroacetic acid, acetic acid, dimethyl sulfoxide, iodomethane,

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dichloromethane, dideuterium oxide, diethyl ether, sulfuric acid and hydrogen

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2.2 Extraction and purification of EGSP

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chloride used were of Analytical Reagent.

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2.2.1 Water-extraction and alcohol-precipitation method

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Extraction of EGSP was performed as described in Cunha, A. P. (Cunha et al., 2017)with some changes in the water-extraction processes, and the detailed steps were

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list in the supplement.

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2.2.2 Sepharose column chromatography Purification of EGSP was carried out with the GFC (gel filtration

chromatography) methods, in which the column was packed with sepharose CL-2B.

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And UV spectrum scanning was used for the purity identification of purified EGSP. 2.3 Component analysis of EGS and EGSP The component analyses of EGS and EGSP were determined according to the approved AOAC International Method, and test items were Moisture (934.01), crude fat (920.39), total protein (2001.11), crude fiber (942.05) and ash (962.09) 5

respectively. Each trial was performed three times. Carbohydrates were calculated by difference. 2.4 Structure determination of EGSP 2.4.1 Mw determination by gel permeation chromatography

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The Mw of EGSP was determined by GPC (gel permeation chromatography) method using HPLC (high performance liquid chromatography LC-20A, Shimadzu

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Corporation, Japan), equipped with a Water-soluble polymer matrix chromatographic column shodex OHpak SB-805 HQ (8×300mm) and a refractive index detector

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(RID-10A). Deionized water was used as mobile phase with a flow rate of 0.8mL/min.

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Preparation of Mw standards and sample was described in the supplement.

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2.4.2 Monosaccharide analysis

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3mg powder sample of EGSP and 2mL TFA (Trifluoroacetic acid, 2.0M) were

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added into an ampoule bottle. Until EGSP dissolvesd, the ampoule bottle was sealed and placed in an oven of 120°C for 3h. TFA was removed when the hydrolysis was

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completed (Yan, Li, Wang, & Wu, 2010). Monosaccharide composition analysis was on the basis of Blakeney's method (Blakeney, Harris, Henry, & Stone, 1983), details

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see the supplement.

2.4.3 Periodate oxidation and Smith degradation

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Periodate oxidation and Smith degradation were referenced Bobbitt’s method

(Bobbitt, 1956) with some changes and the details was explained in the supplement. 2.4.4 Methylation analysis Methylation of EGSP was performed based on the literature method (Hakomori, 1964), detailed in the supplementary material. Methylated samples were hydrolyzed, 6

reduced and acetylated for GC-MS (Gas Chromatography-Mass Spectrometer) analysis. 2.4.5 FTIR (Fourier transform infrared) spectroscopy FTIR spectrum of EGSP was carried out at room temperature by the potassium

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bromide pellet method on Fourier transform-infrared spectrometer (Nicolet, IS50, America) for FTIR measurements in mid-infrared wavelengths of 4000-400cm−1.

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2.4.6 NMR (Nuclear magnetic resonance) spectroscopy

50mg EGSP was dissolved in 1mL D2O. NMR spectra of EGSP were obtained

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on Bruker AV-III 400M NMR (Switzerland). The temperature of NMR spectrometer

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was set at 298K, and the 1H NMR and 13C NMR spectra were collected at a working

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frequency of 400MHz and 100MHz respectively.

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2.4.7 A structure model of galactomannan

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In this study, the general formula of the galactomannan structure was given by inductive method based on the experimental data of EGSP and the concluding results

out.

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of previous studies. And a possible molecular structural formula of EGSP was inferred

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2.5 Conformation and morphology characterization of EGSP 2.5.1 Conformation studies of EGSP

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I2-KI analysis and Circular Dichroism (CD) spectra of EGSP were performed

with common methods, and the Congo red analysis of EGSP was referenced Palacios and Rout’s method (Palacios, García-Lafuente, Guillamón, & Villares, 2012; Rout, Mondal, Chakraborty, & Islam, 2008). Details were described in the supplement.

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2.5.2 Observation by OM (optical microscope) A drop of EGSP solution (1mg/mL) was placed on a clean slide and placed in a refrigerator to pre-freeze at -80°C. After being freeze-dried, the slide to which the sample solids were attached was observed under a microscope (Olympus Corporation,

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CX41, Japan). 2.5.3 Scanning electron microscope (SEM)

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The external morphology of sample was observed by scanning electron

microscope (Hitachi, SU1510 SEM, Japan) with an accelerating voltage of 5kV under

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a high vacuum condition. The dried samples were evenly sprinkled on a metal sample

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table with double-sided adhesive tape. Then they were plated with a 10nm gold alloy

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2.5.4 Atomic force microscopy (AFM)

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with the ion sputtering apparatus and prepared for SEM imaging.

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Place the EGSP solution (1μg/mL) on a clean mica sheet, spread evenly and dry at room temperature for atomic force microscopy. The mica sheet loaded with the

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sample was fixed on a sample stage and measured with a non-contact method by an

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atomic force microscope (JEOL, JSPM-5200 AFM, Japan). Scanning range is 5×5μm with a scanning frequency of 1Hz, and finally we can get 512 × 512 resolution image. 2.6 Data analysis

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All parallel test data were analyzed by the SPSS 19.0 statistical analysis system,

and results were expressed as the mean ± standard deviation (SD).

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3. Result and discussion 3.1 Extraction and purification of EGSP The extraction rate of crude EGSP from EGS was 67.5% (w/w). The results of purification (supplementary data figure 1 A) showed a single symmetrical peak which

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indicated that the EGSP obtained from the water extracts procedure appeared to be

composed of one fraction. And the absorption spectrum of EGSP at 190-500nm

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(supplementary data figure 1 B) showed that there is no obvious absorption peak at

260-280nm, which means that this polysaccharide contains little protein and is of high

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purity.

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3.2 Components analysis of EGS and EGSP

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The results of composition of EGS and EGSP were shown in Table 2, from

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which we can see that the carbohydrate content of EGS is pretty high (92.16% dry

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weight), while the total amount of ash, crude fat, total protein, crude fiber together accounted for 7.84% (dry weight).

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Table 2. Composition of ECG samples (g/100 g on dry basis). Moisture

Ash

Crude fat

Total protein

Crude fiber

Carbohydrate*

EGS

12.00±0.02

0.84±0.04

0.81±0.04

4.20±0.07

1.05±0.03

81.10

0.32±0.02

0.17±0.03

0.28±0.06

0.42±0.08

90.37

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Sample

EGSP

08.44±0.02

Note: *, Calculated by difference.

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From the results, it can be seen that the content of impurities (ash, crude fat, total

protein and crude fiber) in EGSP is particularly small (1.19% dry weight), and the calculated total carbohydrate content is as high as 98.7% (dry weight). This result echoes the UV spectrum of EGSP.

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3.3 Structural information of EGSP 3.3.1 Molecular weight determination GPC chromatogram of EGSP (supplementary data figure 1C) showed a single symmetrical peak was consistent with the results of GFC. It indicated that EGSP

7.779min, and the calculated molecular weight was 1913kDa.

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3.3.2 Monosaccharide composition

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components are single and with a high degree of uniformity. Its retention time was

After the full hydrolysis and derivatization, GC analysis results of EGSP

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(supplementary data figure 2 A and B) were obtained. And the molar ratio (M/G) of

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EGSP was calculated as 2.66; in addition the glucose and arabinose take a small

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proportion, suggesting that EGSP is mainly composed of galactomannan. It is singular

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that galactomannan from G. japonica has an M/G ratio of 5.00 (table 1), which is very

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different from the current research of G. japonica var. delavayi (varietas of G. japonica). The M/G ratio of galactomannan has a great difference in different species,

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different growth conditions of the same species and different extraction methods (Jian, Cristhian, Zhang, & Jiang, 2011). The difference M/G ratio of galactomannan

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between G. japonica and G. japonica var. delavayi may be caused by the differences in their geographical location and growth environment.

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3.3.3 Periodate oxidation, Smith Degradation The consumption of periodate was calculated according to periodate standard

curve as 0.50mmol. Formic acid in the oxidation product was determined by titration with sodium hydroxide was 0.11mmol. The GC analysis results of Smith degradation products were shown in supplementary data figure 2C, which revealed that the ratio 10

between glycerol and erythritol is 1: 2.67. The results of the periodate oxidation showed not only the consumption of periodate, but also the formation of formic acid, Indicating that EGSP contains (1→), (1→6) linkages; and the amount of periodate consumption is more than twice the

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amount of formic acid production, indicating that EGSP also has (1→2), (1→2,6), (1→4), (1→4,6) linkages (X. T. Wang et al., 2016). And the ratio of periodate

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consumption to formic acid production was 4.56 (> 2), indicating the ratio between

the (1→), (1→6) linkages and the (1→2), (1→2,6), (1→4), (1→4,6) linkages is 1:

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2.56. Smith degradation results in the formation of glycerol and erythritol, indicating

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the presence of (1→), (1→6), (1→2), (1→2,6) linkages and (1→4), (1→4,6) linkages

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in EGSP, respectively (Yu et al., 2010); and the ratio of the two kind of linkage above

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is 1: 2.67. Similar to the results of the composition of monosaccharides (1: 2.66),

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suggesting that galactose in C1 position into a bond, mannose in the C1, C4 or C6

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position.

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A: Gas chromatogram; B: Mass spectra.

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Fig. 1. Results of methylated EGSP GC-MS analysis.

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3.3.4 Methylation and GC-MS analysis

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The GC-MS gas chromatogram of EGSP methylation is shown in figure 1A, in which the three peaks are the methylated sugar residues. And mass spectra of the three

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sugar residues were shown in figure 1B, the data of mass spectra are listed in

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supplementary data (Table 1). According to the results, the three sugar residues were Gal-1→, →4-Man-1→ and →4,6-Man-1→, respectively.

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3.3.5 FTIR spectrum analysis The FTIR spectrum of EGSP was shown in figure 2, 3423.5cm-1 absorption peak

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is O-H and N-H stretching vibration peak; 2922.0cm-1 and 2852.5cm-1 are absorption peaks of C-H resonance absorption. These two categories are characteristic absorption peaks of polysaccharides. The absorption peaks from 1600 to 1650cm-1 are caused by C-O asymmetric stretching vibration (Jia et al., 2015), which may be related to the bending mode of absorbed water (Zhang, Pang, Dong, & Liu, 2015). The absorption 12

peak of EGSP at 1632.1cm-1 may be due to its hydration vibration. The absorption peaks of 1030.9cm-1, 1077.8cm-1 and 1150.2cm-1 indicate that the sugar rings in EGSP are pyranose rings, which are the absorption peaks generated by the vibrations of the ether bond C-O-C; and there are only two strong absorption peaks of the furan chains

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in this interval (Jia et al., 2015). The absorption peak at 874.5cm-1, 813.0cm-1 suggests that the presence of both mannose and α- and β- anomers in EGSP is inferred as

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α-D-galactopyranose and β-D-Mannose sugar (Figueiró, Góes, Moreira, & Sombra,

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M

A

N

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2004; Prado, Kim, Özen, & Mauer, 2005; Yuen, Choi, Phillips, & Ma, 2009).

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Fig. 2. FTIR spectrum of EGSP.

3.3.6 NMR spectra analysis In this experiment, the 1D NMR peak was clearly overlapped, and the H2-H6

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signal peak was difficult to be clearly assigned. Besides, the 2D COSY spectrum was so blurred and indistinct. Thus, the signals of the C atoms were assigned with 1D 1H spectrum,

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C spectrum, 2D H-HCOSY and HSQC spectra, and compared with free

β-mannopyranose and α-galactopyranose other galactomannans (Table 3), the NMR 13

spectra results were presented in supplementary data figure 3. Table 3. Position and interpretation of the signals of 13C NMR spectrum of EGSP 13C

Chemical shift (δ), ppm

C1

C2

C3

C4

C5

C6

α-D-galactopyranosyl

98.89

68.54

69.42

69.42

71.43

61.36

α-D-galactopyranose*

93.32

70.37

70.26

69.45

71.43

62.22

100.30

70.01

71.57

76.69

75.16

60.67

73.43

66.62

77.09

62.08

4-O-β-D-mannopyranosyl

100.12

70.01

72.91

76.92

4,6-di-O-β-mannopyranosyl 77.04 α-D-mannopyranose*

94.67

72.24

74.11

67.72

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Note: *, Given for a comparison.

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76.92

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Monosaccharide residue in the polymer

H spectrum of the EGSP, which were α-galactopyranosyl and β-mannopyranosyl

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1

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There were two distinct peaks (5.04ppm, 4.76ppm) in the anomeric region of the

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respectively (Li, Chen, & Xu, 2005). The 13C NMR spectrum of EGSP was clear, and three signal peaks (100.30ppm, 100.12ppm, 98.89ppm) were present in the anomeric

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region, which contributed to M1 (→4-Man-1→), M*1 (→4,6-Man-1→) and G1

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(Gal-1→) sugar residues respectively. The integral area ratio was G1: M*1: M1 = 1: 1.08: 1.46, which was M/G = 2.54. It was similar to the monosaccharide composition

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obtained by GC analysis.

Compared with the free galactopyranose, the chemical shift of G1 shifted to the

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weak field by 5.57ppm, which was caused by the α-effect, indicating that the anomeric center of galactose in EGSP is α-configuration. There was no significant change of the chemical shift of G2-G6, indicating that galactose in EGSP only forms glycosidic bonds in the C1. So galactose in EGSP is linked to other sugar residues in the form of terminal sugar residues, which was consistent with the results of the 14

analysis. G6 chemical shift was 61.36ppm, indicating that the mannopyranosyl is pyran type (Chizhov & Shashkov, 1985), and infrared spectrum also shows the presence of the pyranose. The chemical shift of the signal at the C5 of the mannose (M5: 75.16ppm; M*5:

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73.43ppm) shifted 1.93ppm and 3.40ppm to the strong field respectively, indicating that the mannose was β-configuration because the signal of α-configuration here has a

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greater shift to the strong field (Lipkind, Shashkov, Knirel, Vinogradov, & Kochetkov, 1988). The C6 signal (M6: 60.67ppm) was the feature of the pyranose ring residue,

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indicating that the mannose is pyranose. The signal of the other mannose residue M*

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C6 (M*6: 66.62ppm) shifted to the weak field (+4.32ppm), indicating that the hydroxyl

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group at C6 of the mannose residue was substituted, which was confirmed by the

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adjacent C5 signal (M*5: 73.43ppm) with strong field offset (-3.97ppm). The signal of

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C6-substituted mannose (66.62ppm) indicated that the substituent associated with it was α-aomer, because the substituent of the β-aomer will produce a greater shift

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(greater than 70ppm) (V. and N. S. Shcherbukhin, 1987).

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NMR signal of mannopyranose C4 can provide fine structure information of polymer (Grasdalen & Painter, 1980). The signal is divided into several peaks within 0.5ppm depending on whether there are replacement at the C6 of two adjacent

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mannose residues (mannobiose blocks) in the polymer chain or not. In the spectrum, the strong-field peak (77.04ppm) of the C4 signal is UM; the medium-field peak (76.92ppm) is MM; the weak-field peak (76.69ppm) is DM. The intensity of each signal reflects the frequency these blocks appear in the polymer chain. In the

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C

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spectrum, the integral area ratio of these three signal peaks is 1.00: 0.34: 0.68, and the order of substitution of galactose on EGSP mannose main chain can be deduced. 3.4.7 Inference of the molecular structure of galactomannan Scholars had found that the

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C-NMR spectrum data could explain some of the

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fine structure of galactomannan, which relates to the three mannobiose blocks UM, MM and DM. The ratio of the three structures to the polymer may give more

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information of the fine structure of galactomannan, but no deeper and broader

discussions were given. In this paper, an induction about the galactomannan

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molecular formula from special to general was demonstrated, and the concrete

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deduction process is as follows.

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First, the numbers of galactose (x) and mannose (y) and the ratio of the three

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mannobiose blocks (a: b: c) need to be determined. Then we need to define a

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structural unit, as shown in figure 3A, where n, m is a natural number. Assuming that all galactomannans are composed of u such units (figure 3B), this can be represented

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by a general structural formula (figure 3C), where m1 + m2 + ··· + mu = x, n1 + n2

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+ ··· + n(u + 1) = y-x.

Now the question becomes how to determine the numerical value of u. We

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divided this structure form into the following three cases. a. n1 and n(u + 1) are all not zero, combined with experimental data and theoretical

values of the three mannobiose blocks ratio a : b : c = y-x-u-1 : 2u : x-u, which can be derived formula 1. 𝑢1 = 𝑏𝑥 ⁄ (𝑏 + 2𝑐)

(1) 16

b. One of n1 and n(u+1) is not zero: a : b : c = y-x-u-1: 2u-1 : x-u, which can be derived formula 2. 𝑢2 = (𝑏𝑥 + 𝑐) ⁄ (𝑏 + 2𝑐)

(2)

c. n1 and n(u + 1) are all zero: a : b : c = y-x-u-1 : 2u-2 : x-u, which can be derived

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formula 3. (3)

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A

N

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𝑢3 = (𝑏𝑥 + 2𝑐) ⁄ (𝑏 + 2𝑐)

Fig. 3. A, B and C: Molecular structure derivation process of galactomannan; D: Illustrative structural diagrams of EGSP.

Considering the general galactomannan molecular weight is huge above the level

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of million, the galactose residual of a single molecule chain of it will be quite a lot, so the structure model for a relatively large values of x. When x is large enough (x→∞), we do the following in combination of formula 1, 2 and 3: 𝑙𝑖𝑚 𝑢1 = 𝑙𝑖𝑚 [𝑏𝑥 ⁄ (𝑏 + 2𝑐)] = 𝑏𝑥 ⁄ (𝑏 + 2𝑐)

𝑥→∞

𝑥→∞

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𝑙𝑖𝑚 𝑢2 = 𝑙𝑖𝑚 [(𝑏𝑥 + 𝑐) ⁄ (𝑏 + 2𝑐)] = 𝑏𝑥 ⁄ (𝑏 + 2𝑐)

𝑥→∞

𝑥→∞

𝑙𝑖𝑚 𝑢3 = 𝑙𝑖𝑚 [(𝑏𝑥 + 2𝑐) ⁄ (𝑏 + 2𝑐)] = 𝑏𝑥 ⁄ (𝑏 + 2𝑐)

𝑥→∞

𝑥→∞

Then the formula 4 was obtained. 𝑙𝑖𝑚 𝑢1 = 𝑙𝑖𝑚 𝑢2 = 𝑙𝑖𝑚 𝑢3 = 𝑏𝑥 ⁄ (𝑏 + 2𝑐)

𝑥→∞

𝑥→∞

(4)

𝑥→∞

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And the conclusion is that when x is large enough, the effect on the substituent of

that is 𝑢 = 𝑏𝑥⁄(𝑏 + 2𝑐)

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molecular chain terminal will become smaller. So the three cases are classified as one,

(5)

U

The numerical value of u calculated by formula 5 represents the number of continuous

N

substituted fragment on mannose backbone, and the number of galactose in each

A

fragment is between 1 and x-u.

M

According to the numerical value of u, we can calculate the theoretical value of

ED

the mannose in one molecule of galactomannan, computational formula as follows: 𝑦 ∗ = (2𝑎 ⁄𝑏 + 1)𝑢 + 𝑥 + 1

(6)

PT

The structure model can be verified by comparing the number of mannose from

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calculation (y*) and experimental determination (y). In order to investigate the applicability of this structure model, we had selected

some published galactomannan data for verification, and the results were shown in

A

table 4. The results showed that the difference between y* and y is not significant, which indicates that the molecular structure calculated according to this model conforms to the actual structure. The size of the numerical value of u indicates the number of unit (figure 3A), which can directly represent the degree and location of 18

the side chain substitution of galactomannan molecular chain. However, the model cannot give the number of mannose and unsubstituted mannose in each unit. More precise structure determination of molecular chain requires further study. Table 4. Numerical value calculation of u and y*. Galactomannan source

M/G

x1

y2

a

b

c

u

y*

EGSP

2.54

100

254

1.00

0.34

0.68

20

239

G. ferox Desf.

2.54

100

254

1.00

1.27

0.43

60

255

(Egorov et al., 2004)

S. alopecuroides L.

1.48

100

148

1.00

1.56

3.24

19

144

(Guo et al., 2016)

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reference

(Mestechkina, 1.24

100

124

1.00

9.84

20.41

19

124

Smirnova,

&

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A. Lehmannianus Bunge

Anulov,

Shcherbukhin, 2000)

1.86

100

186

1.00

3.10

1.60

49

S. nux-vomica

2.70

100

270

1.00

1.00

0.38

57

S. innocua

2.90

100

290

1.00

0.77

0.38

50

S. potatorum

2.50

100

250

1.00

1.35

0.50

Artificially determined to be 100;

2,

57

(V.

D.

Shcherbukhin,

1992)

272 281

(Corsaro et al., 1995)

242

Calculated by the ratio of M/G.

N

Note:

1,

182

U

I. tinctoria L.

A

About EGSP, the Mw of EGSP is 1913kDa and its ratio of M/G is 2.54-2.66. The

M

polymer is composed of 1,4-β-D-mannopyranose as the backbone, with a branch of

ED

1-α-D-galactopyranose linked at the C6 hydroxy of portion mannopyranose and the ratio of fine structure UM, MM, DM is 1.00: 0.34: 0.68. Synthesize these provided the

PT

values of x, y, a, b and c is 3280, 8528, 1.00, 0.34 and 0.68, respectively. With the

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molecular model above, we got the u and y* of EGSP (651 and 7812). Then we compared the number of y and y* was very close, this indicated that the number of the

A

u (651) was correctly. Finally, the illustrative structural diagram of EGSP was displayed in figure 3D. 3.4 Conformation and morphology of EGSP 3.4.1 I2-KI analysis The results of the I2-KI test (supplementary data figure 4A) showed the 19

maximum absorption was mainly concentrated on 351nm and there was no absorption peak at 565nm, indicating that there are many branches on the EGSP molecular backbone (Yamada, Yanahira, Kiyohara, Cyong, & Otsuka, 1985). This result is consistent with the M/G ratio in the monosaccharide composition analysis. In addition,

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EGSP and iodine reagent reaction also shows that EGSP does not contain starch. 3.4.2 CD spectra and Congo red analysis of EGSP

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From the results of the CD spectra (supplementary data figure 5), no obvious CD signal was observed in the absence of NaOH, indicating that EGSP did not complex

U

with Congo red in neutral aqueous solution and showed mainly random coil. When

N

NaOH is present, two CD signals appeared at 200nm and 213nm, showing a positive

A

COTTON effect, indicating that the spatial conformation of EGSP has changed to an

M

ordered α-helix (Adler, Greenfield, & Fasman, 1973). This coincided with the result

ED

of no bathochromic shift at the NaOH concentration of 0.0M in the Congo red experiment (supplementary data figure 4B).

PT

The λmax of EGSP-Congo red complexes had a bathochromic shift compared

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with Congo red. When the concentration of NaOH is 0.0-0.1M, the λmax is shifted to a longer wavelength, indicating that EGSP can form complexes with Congo red, and have a regular triple-helical conformation. When the NaOH concentration continues

A

to increase, the λmax decreases, indicating that the triple-helical conformation of EGSP disintegrated into an irregular coil (J. H. Wang et al., 2015). That is, EGSP can form an ordered helical conformation in the weak alkaline range. However, the bathochromic shift of EGSP-Congo red complexes was inconspicuous, hinting that 20

there are few helical conformations in EGSP, which reveals that EGSP contains many random coil conformations. 3.4.3 Results of microstructure observation . Through the microscope (figure 4 A and B) it could be visually seen that the

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EGSP solution formed a homogeneous fibrous filament network structure after freeze-drying dehydration, indicating that the polysaccharide purity is high. It is

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speculated that these fibers are formed by the long chain molecules of EGSP intertwined with each other.

U

The observations of SEM (figure 4 C and D) further confirmed the EGSP fiber

N

network structure, and we can see the EGSP fiber is composed of more fine

A

microfibers. In addition, SEM can be employed to make a qualitative identification

M

whether a certain kind of polysaccharide belongs to starch polysaccharides (S. Wang,

polysaccharide.

ED

Cao, Fan, Cao, & Cao, 2011). The SEM image shows that EGSP is a non-starch

PT

The image of EGSP through AFM was shown in figure 4 (E and F). We observed

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that the EGSP had formed the coral-like structure on the mica sheet. Its chain structure is 200-300nm wide and 1.15-1.20nm high. This EGSP molecular chain height is larger than the general height (The molecular size of the polysaccharide

A

chain is about 0.1-1nm (Y. Wang et al., 2010) ), indicating that the molecule chain of EGSP are entangled with each other to form thicker ones.

21

IP T SC R U N A M

ED

Fig. 4. Microstructure observation of EGSP.

OM: Magnification of 40 (A) and 400(B) times; SEM: Magnification of 2,700 (C) and 11,000(D) times;

PT

AFM: The scanning range is 5 × 5um.

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4. Conclusion

In conclusion, the carbohydrate content in EGSP was 98.7% (dry weight), which

A

was identified as galactomannan. The molecular structure of EGSP was consistent with the general structure of galactomannan and we further deduced the order of the galactoside substituent with the methods established in this paper. The solution conformation of EGSP was investigated, the results showed that EGSP has many

22

branches on the backbone and exists in a random coil state in aqueous solution. When the pH was increased, conformational transition occurs, resulting in an ordered helical conformation. The observation of the morphological features showed that the EGSP molecule had linear chains which entangled to form fiber network. In this paper, the

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study of polysaccharide structure of EGSP was carried out, which laid a theoretical

properties and bioactivity of polysaccharide in EGSP. 5. Acknowledgements

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foundation for the research on the relationship among structure, physicochemical

U

This research is supported by the National Science-Technology Pillar Program

N

(2012BAD33B05), the Program for Changjiang Scholars and Innovative Research

M

A

Team in University of the Ministry of Education of the People’s Republic of China (Grant IRT1166), the Foundation of Tianjin University of Science and Technology,

ED

Institute for New Rural Development, P. R. China (No. XNC201511) and Youth

2015lG24).

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