An amendment to the fine structure of galactoxyloglucan from Tamarind (Tamarindus indica L.) seed

Journal Pre-proof An amendment to the fine structure of galactoxyloglucan from Tamarind (Tamarindus indica L.) seed

Hui Zhang, Taolei Zhao, Junqiao Wang, Yongjun Xia, Zibo Song, Lianzhong Ai PII:

S0141-8130(19)40740-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.284

Reference:

BIOMAC 14589

To appear in:

International Journal of Biological Macromolecules

Received date:

30 December 2019

Revised date:

28 January 2020

Accepted date:

28 January 2020

Please cite this article as: H. Zhang, T. Zhao, J. Wang, et al., An amendment to the fine structure of galactoxyloglucan from Tamarind (Tamarindus indica L.) seed, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.01.284

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© 2020 Published by Elsevier.

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An amendment to the fine structure of galactoxyloglucan from Tamarind (Tamarindus indica L.) seed Hui Zhang1, Taolei Zhao1, Junqiao Wang2, Yongjun Xia1, Zibo Song3, Lianzhong Ai1,* 1

Shanghai Engineering Research Center of Food Microbiology, School of Medical

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Instruments and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China

Nanchang, Jiangxi 330047, China

Yunnan Maodouli Group Food Co., Ltd., Yuxi, 653100, China

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State Key Laboratory of Food Science and Technology, Nanchang University,

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2

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*Correspondence should be addressed to Prof. Dr. Lianzhong Ai. Address: University of Shanghai for Science and Technology, 516 Jungong Road,

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Shanghai 200093, China

Tel.: 0086-21-55897302; Fax: 0086-21-55897302 Email: [email protected]

Email address for other authors: Hui Zhang: [email protected] Taolei Zhao: [email protected] Junqiao Wang: [email protected] Yongjun Xia: [email protected] Zibo Song: [email protected]

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Abstract: A polysaccharide from tamarind seeds (TSP) was characterized in terms of backbone and side chain structural features, as well as conformational property using methylation and GC-MS analysis, 2D NMR, MALDI-TOF MS, and high performance size exclusion chromatography (HPSEC). Results showed that TSP was a

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galactoxyloglucan (GXG) consisting of glucose, xylose, and galactose in a molar ratio of 3.1: 1.7: 1.0. The Mw was determined to be 524.0 kDa with radius of gyration (Rg)

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of 55.6 nm. The chemical structure was confirmed as a classical β-(1→4)-glucan with

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short side chains of T-β-Galp-(1→2)-α-Xylp-(1→ and T-α-Xylp-(1→ attached to O-6

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position of glucose. MALDI-TOF MS analysis indicated that TSP mainly composed

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of nonasaccharide (XLLG) and octasaccharide (XLXG or XXLG) blocks in periodic

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or interrupted sequence in a ratio of 3: 2, occasionally interrupted by heptasaccharide (XXXG), hexasaccharide (XLG or XXGG), or even hendesaccharide blocks.

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Conformational study indicated that TSP was in a random-coil shape with relative extended stiff chain in aqueous solution. This study provided more evidences to make an amendment to the fine structure of tamarind GXG.

Keywords: Tamarind seed; Polysaccharide; Fine structure; NMR spectroscopy; MALDI-TOF MS

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1. Introduction Tamarind is a long-lived, tropical, and leguminous tree belonging to the member of the dicotyledonous family Fabaceae (Leguminosae). It can be grown in the dry poor soils because of its nitrogen fixing capability and ability to withstand long periods of drought, exhibiting an important ecological function in preventing soil

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erosion [1, 2]. The fruit of tamarind contains about 55% pulp, 34% seed, and 11% shell in a pod with rich dietary fiber, reducing sugar, organic acid, minerals, vitamins,

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and pigments, making the tamarind tree an important economic plant [2]. The fruit

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pulp is the major edible part of tamarind as chief souring agent for curries, sauces, and

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certain beverages. The seed embedded in the pulp is considered as by-products due to

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its unsuitability for consumption, but there is a perspective to make it more useful [3].

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Many ingredients, such as polysaccharide, protein, unsaturated fatty acids, and tannin, etc. have been found in the seeds of tamarind. The proper use of these

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ingredients would not only bring great economic benefits to the pulp processing industries, but also help the people who cultivate the tree. Polysaccharide, which accounted for over 50% of tamarind seed by weight, were the major component with widely applications. It was firstly used as sizing material in the textile, paper, and jute industries in India [4]. In recent years, more commercial interest in tamarind seeds polysaccharide (TSP) focused on its unique gum properties like thickening, filming, pH-resistant, and thermal stability properties [5-8]. These properties made TSP an important alternative material for food additives such as thickener, stabilizer and gelling agent, as well as an ideal potential hydrogel material for drug delivery and

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matrix of joints and organs [6, 9, 10]. As for the chemical features, TSP was identified as galactoxyloglucan (GXG) which composed of a cellulosic skeleton of β-D-(1→4)-glucan and branches of galactose and xylose to form a nonasaccharide repeating unit [11, 12]. Some others also found arabinose in the branch chains and varied ratios of monosaccharides in TSP, indicating the structural diversity [13, 14]. GXGs were commonly found in plant cells

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acting as tethers between the cellulose microfibrils to increase the cell walls’ rigidity.

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They enabled the cell to change its shape in growth and differentiation zones and to

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retain its final shape after cell maturation [15]. The cellulosic β-D-(1→4)-glucan

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backbone of GXGs with different degree of glycosyl substitutions played an

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important role in its tethering function with microfibrils and forming a stiff

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extended-chain structure, thus resulting in efficient volume occupancy and enhancement of viscosity [16]. The oligosaccharide units obtained by cleavage of

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Endo-(1→4)-β-glucanase could be applied to evaluate the degree of glycosyl substitutions and considered as the fingerprint features to distinguish the GXGs from different plants [17].

In current study, we tried to characterize the full chain structure and hydrolyzed oligomer units of tamarind GXG via 1D & 2D NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). High performance size-exclusion chromatography coupled with multi-angle laser light scattering (HPSEC-MALLS) was further conducted to investigate the conformational properties. This study aimed to provide more evidences to make an amendment to the

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fine structure of tamarind GXG. 2. Materials and methods 2.1 Materials Tamarind seeds were provided from Yunnan Maodouli Group Food Co., Ltd. (Yunnan, China). Monosaccharide standards of D-glucose, D-xylose, D-galactose, L-fucose,

D-mannose,

D-arabinose,

D-glucuronic

acid,

and

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L-rhamnose,

D-galacturonic acid were purchased from Sigma-Aldrich Co. (MO, USA). Deuterium

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oxide (99.9% D) and sodium borodeuteride (98% D) were from Sigma-Aldrich Co.

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LLC (Switzerland). Xyloglucanase (GH5) (Paenibacillus sp.) was purchased from

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Megazyme International (Ireland). All other reagents used were of analytical grade

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unless otherwise specified.

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2.2 Isolation and component analysis of polysaccharide sample The extraction process of TSP was conducted according to previous study [8].

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Briefly, the tamarind seed powder was extracted by citric acid aqueous solution (pH 3.5) at 80 °C for 30 min, the extracted supernatant was then precipitated with ethanol at a ratio of 1: 1 (v/v) for two hours. The obtained precipitates were washed twice with 50% ethanol, finally dehydrated by isopropanol and dried in vacuum to obtain polysaccharide sample. The total sugar content of TSP was determined following the phenol sulfuric acid assay using glucose as standard [18]. Uronic acid content was measured by the m-hydroxybiphenyl method using D-galacturonic acid as the standard [19]. Total protein content was analyzed by Kjeldahl method with protein conversion factor of 6.25.

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2.3 High performance anion-exchange chromatography assay The monosaccharide composition analysis was carried out using a high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex ICS-5000, USA) equipped with a CarboPac™ PA20 analytical column (4 mm x 250 mm) [20]. TSP (10 mg) was hydrolyzed with 0.5 mL of 12 M

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H2SO4 at room temperature for 30 min, then diluted to 2 M H2SO4 with water following by hydrolysis at 100 °C for 2 h. The hydrolysate was diluted with pure

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water and injected for analysis directly after filtration with 0.22 μm membrane.

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2.4 Methylation and GC-MS analysis

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measurements were repeated three times.

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Monosaccharide standards were used for qualitative and quantitative analysis. All

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Methylation procedure was conducted according to the method of Ciucanu and Kerek [21] with some modifications [22]. Briefly, 3 mg dried samples of TSP and

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enzymatic hydrolysates were dissolved in anhydrous DMSO with constant stirring overnight. Dried powder of NaOH (20 mg) was added to the solution with 3 h stirring at room temperature to ensure complete dissolution. Methyl iodide (0.3 mL) was carefully added to the mixture with another 2.5 h of constant stirring. The methylated sample was extracted with 1 mL of CH2Cl2, and the CH2Cl2 extract was washed three times with an equal volume of water and passed through a Na2SO4 column (0.5×15 cm) to remove water. The final CH2Cl2 extract was evaporated in a stream of nitrogen, and then hydrolyzed by trifluoroacetic acid (TFA), reduced using sodium borodeuteride and acetylated with acetic anhydride to produce partially methylated

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alditol acetates (PMAA). Analysis was carried out on a GC-MS system (THERMO 1310 GC-ISQ LT MS; Thermo Fisher, USA) equipped with a TG-200MS capillary column (30 m×0.25 mm, 0.25 mm film thickness; Thermo Fisher, USA) programmed from 160 to 210 °C at 2 °C/min, and then from 210 to 240 °C at 5 °C/min. 2.5 NMR spectroscopy analysis

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Sample (50 mg) was exchanged with deuterium by lyophilizing against deuterium oxide (D2O) for three times, and finally dissolved in D2O at room

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temperature for 3 h before NMR analysis. High-resolution of 1H and 13C NMR spectra

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were recorded at 600.10 and 151.01 MHz, respectively, on a Bruker AVIII 600 NMR

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spectrometer (Brucker, Rheinstetten, Germany) at 298 K. The homonuclear 1H/1H

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correlation (DQF-COSY), heteronuclear single-quantum coherence (HSQC) and

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heteronuclear multiple-bond correlation (HMBC) experiments were conducted using the standard Bruker pulse sequence

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2.6 Specific enzymolysis and MALDI-TOF MS analysis TSP sample (40 mg) was treated with 1 mL of xyloglucanase (Paenibacillus sp., 20 U) at pH 5.5 (adjusted by acetic acid) and 40 °C for 24 h. After the reaction was complete, the reaction mixture was heated at 100 °C for 10 min to inactivate the enzyme and centrifuged at 8000 rpm for 15 min to remove insoluble materials. The supernatant (1 mL) was pipetted and mixed with an equal volume of matrix solution (10 mg/mL of 2,5-dihydrobenzoic acid in 0.1% trifluoroacetic acid, DHB). The mixture was then spotted on a stainless steel MALDI plate and allowed to air-dry at room temperature. An AB SCIEX TOF/TOF™ 5800 System (AB SCIEX, USA)

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equipped with a neodymium-doped yttrium aluminium garnet (Nd:YAG) laser was applied for analysis. Spectra were acquired in the Reflector Positive ion mode for MS and MS-MS 2 KV Positive ion mode for tandem MS, respectively. Each sample was recorded for triplicate in the range of m/z 0 to 5000. 2.7 HPSEC-MALLS analysis

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The molecular weight and distribution of TSP was conducted on a high performance size-exclusion chromatography system (HPSEC) equipped with multiple

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detectors (Wyatt Technology, USA): a multi-angle laser light scattering detector

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(DAWN HELEOS-II, MALLS), a differential pressure viscometer (ViscoStar III, DP),

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and a refractive index detector (Optilab T-Rex, RI). An Agilent 1260 Infinity II HPLC

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system (Agilent Technologies, USA) with two columns in series, a OH-pak SB-803

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HQ column and a OH-pak SB-805 HQ column (8 mm x 300 mm, Shodex, Tokyo, Japan), were used. The columns, DP and RI detectors were maintained at 35 ºC. The

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eluent was 0.1 M NaNO3 solution (containing 0.02% NaN3) at a flow rate of 0.6 mL/min. Samples were prepared at a concentration of 1.0 mg/mL. ASTRA 7.1.3 software (Wyatt Technology, USA) was used to obtain and analyze the data according to previous method [23]. 2.8 Statistical analysis All the measurements were conducted in triplicate and shown in means ± standard deviation (SD), within significance P < 0.05 after subjecting the data to an analysis of variance (ANOVA). 3. Results and discussion

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3.1 Chemical components and monosaccharide composition The neutral sugar content in TSP was measured as 90.3% (w/w) with 1.0% of protein, indicating that TSP was mainly a neutral polysaccharide with a few impurities. HPAEC-PAD results showed that TSP composed of glucose, xylose and galactose in a molar ratio of 3.1: 1.7: 1.0. This result is similar with previous reports but with

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different ratios such as 2.8: 2.25: 1 [11], and 2.61: 1.43: 1 [24]. These discrepancies were attributed to the difference of breed, geographical origin or physiological stage

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of tamarind seed, as well as the extraction method. Arabinose was also detected with

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low content previously [13]. However, it could be considered to be from the arabinan

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or arabinoxylan mixtures which were commonly found in plant seeds [11, 25, 26].

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pure galactoxyloglucan.

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The disappearance of arabinose in current study revealed that the TSP sample was a

3.2 Methylation and GC-MS analysis

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The methylation and GC-MS analysis results of TSP are shown in Table 1 (listed in the order of retention time). Five linkage patterns were detected from the GC-MS analysis of PMAAs. The dominate sugar residue found in TSP was →4,6)-Glcp-(1→ with relative percentage of 42.9%. The total percentage of terminal sugar residues (T-Xylp and T-Galp) was 30.2%, the ratio of branched portion to terminal residues was 1.4. According to Hawker, Lee, & Fréchet [27], the degree of branching (DB) of TSP was calculated as 73.1%, which indicated that TSP was hyper-branched. The other linkages were identified as →4)-Glcp-(1→ and →2)-Xylp-(1→ with relative percentages of 13.3% and 13.6%, respectively. It has been reported that

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polysaccharides from tamarind seeds had a common structural elements of cellulosic-type β-(1→4)-glucan backbone with side chains composed of xylose and galactose, but possessed varied ratios of glucose, xylose, galactose residues [11]. In order to deduce the backbone and branch chain features of TSP, further analysis were done using 1D & 2D NMR and MALDI-TOF MS.

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3.3 NMR analysis of polysaccharide from tamarind seeds NMR technology, including 1D and 2D NMR spectrum, was conducted for the

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elucidation of the structural features of TSP. Both 1H (Fig. 1A) and 13C NMR (Fig. 1B)

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showed at least three anomeric signals with overlapped signals, respectively. HSQC

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was conducted to distinguish the chemical shifts of different sugar residues as

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indicated by methylation and GC-MS results, and five anomeric carbon/proton

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correlation signals were assigned as shown in Fig. 2A. The corresponding chemical shifts were determined as δ 107.35/4.56, 105.36/4.56, 105.36/4.56, 101.64/5.18, and

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101.81/4.96 ppm. The chemical shifts of non-anomeric protons of different residues were fully assigned according to DQF-COSY (Fig. 2B), and the corresponding non-anomeric carbons were then determined with the help of HSQC (Fig. 2A). The full chemical shift assignments of different sugar units are listed in Table 2. Specifically, the anomeric carbon/proton signals of δ 101.64/5.18 and 101.81/4.96 ppm were identified as those originating from O-2 substituted Xylp (residue A: Xyl2) and terminal Xylp (residue B: XylT), respectively [28, 29]. The low field of anomeric proton signals indicated an α-configuration for D-Xylp. The H2-H5 signals of residues A and B were assigned step-by-step from the DQF-COSY

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spectrum (Fig. 2B). The corresponding carbon signals were then deduced by HSQC (Fig. 2A) and HMBC (Fig. 3). The low field of C2 of residues A (δ 82.33 ppm) indicated that the O-2 position of residues A was substituted. The anomeric carbon/proton signals of δ 107.35/4.56 ppm were assigned to T-β-D-Galp (residue E: GalT), and δ 105.36/4.56 ppm were deduced as the anomeric signals of

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β-(1→4,6)-D-Glcp (residue C: Glc46) and β-(1→4)-D-Glcp (residue D: Glc4) which overlpped [28-31]. The carbon/proton signals of O-4-substituted position of residue C

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and D were assigned as δ 82.97/3.69 and 81.58/3.69 ppm, respectively. The C6/H6a

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signals of residue C were determined to be δ 71.43/3.93 ppm from the HSQC, and the

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H6b signal was derived as δ 3.73 ppm from the HMBC spectrum.

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Based on the assignments of 1H and 13C chemical shifts of all sugar residues, the

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sequences of different sugar residues were obtained by observing inter- and intra-residual connectivities in HMBC spectrum (Fig. 3). Connectivities between H1

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of residue C and C4 of residue D (δ 4.56/81.58 ppm), and H1 of residue D and C4 of residue C (δ 4.56/82.97 ppm), along with intra-residual couplings between H1 of residue C and its own C4 (δ 4.56/82.97 ppm), C1 of residue C and its own H4 (δ 105.36/3.69 ppm), H1 of residue D and its own C4 (δ 4.56/81.58 ppm), and C1 of residue D and its own H4 (δ 105.36/3.69 ppm) were found, indicating that the backbone of TSP was of β-(1→4)-D-glucan consisting with linkages of Glc46(1→4)Glc46, Glc46(1→4)Glc4, Glc4(1→4)Glc46 and Glc4(1→4)Glc4 as shown below:

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The O-6 position of residue C was found to be substituted by residue A and residue B according to the correlations between C1 of residue A and H6 of residue C (δ 101.64/3.73 ppm), and C1 of residue B and H6 of residue C (δ 101.81/3.73 ppm).

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Besides, residue of residue E was deduced to terminate the side chain of residue A by the connectivity between C1 of residue E and H2 of residue A (δ 107.35/3.68

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ppm) as shown in Fig. 3. In this way, the side chain of TSP could be proposed to be

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consisted of fragments of X and L as shown below:

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The chemical structure of TSP consisted of the fragments of X and L, as well as non-substituted β-D-Glcp (designated as G). These fragments were linked via β-(1→4)-linkage to form a glucan backbone. These features were similar with previous reported structure of TSP [11, 13]. However, the distribution of side chain fragments to the backbone was still not clear and needed more evidences to confirm. 3.4 MALDI-TOF MS and MS/MS analysis of enzyme hydrolysates In order to investigate the side chain distribution of TSP, the polysaccharide was treated with a xyloglucan-specific endo-glucanase (XEG), which specifically cleaves β-(1→4)-glucosidic linkages of xyloglucan backbone next to an unbranched glucose residue (Fig. 4). The enzyme hydrolyzed oligosaccharide mixture was analyzed by 12

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MALDI-TOF MS. The results indicated the presence of at least two major components (nona- and octasaccharide) and three minor components (hepta-, hexa-, and hendesaccharide) as shown in Fig. 4. The detailed information of the composition and m/z of enzymatically produced oligosaccharides are shown in Table 3. The nonasaccharide fraction with the most intense signal was deduced to be one of the

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fragments of XLLG, XLXGG, and XXLGG, and the octasaccharide fraction was one of the fragments of XLXG, XXLG, and XXXGG. The ratio of nona- and

fragments

such

as

XXXG

(heptasaccharide,

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other

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octasaccharide was about 3: 2 according to the intensity signals of MS spectrum. The 7.2%),

XLG/XXGG

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(hexasaccharide, 2.7%), and XLLXG/XLXLG/XXLLG (hendesaccharide, 1.9%) were

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also distributed as the blocks of TSP but with low intensity. Previous studies have

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reported oligosaccharides of XXXG, XXLG/XLXG and XLLG as the blocks of tamarind GXG from Megazyme [32]. However, hexasaccharide and hendesaccharide

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fragments were firstly found in current study. The MALDI-TOF MS/MS analysis was further conducted to study the fragment ions of corresponding parent ions. The MS/MS spectra of [M+Na]+ ions of m/z 1409, 1247, and 1085 are illustrated in Fig. 5 and the corresponding fragment ions are concluded in Table 3. The singly charged [M+Na]+ ion of m/z 1409, upon collision, yielded several daughter ions characteristic of these oligosaccharides (Fig. 5A): the most intense ions of m/z 1247 (resulting from a loss of m/z 162 by glycosidic bond cleavage of β-(1→2)-linkage between galactose and xylose), and less intense ion of m/z 1277 (resulting from a loss of 132 by glycosidic bond cleavage of

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cleavage of β-(1→2)-linkage from m/z 1277. The parent ion of m/z 1247 showed series of daughter ions (Fig. 5B): the most intense ion of m/z 1115 (a loss of m/z 132,

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xylose) → second fragment ion of m/z 953 (a loss of m/z 162, galactose) → third

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fragment ion of m/z 659 (a loss of m/z 294, xylose and glucose), and a less intense ion

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of m/z 1085 (a loss of m/z 162, galactose) → second fragment ion of m/z 953 (a loss

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of m/z 132, xylose) → third fragment ion of m/z 659 (a loss of m/z 294, xylose and

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glucose). These results demonstrated that the [M+Na]+ ion of 1247 was derived from the possible oligomer of XXLG or XLXG. However, due to the low amount of parent

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ion of m/z 1085, only one fragment ion of m/z 953 (a loss of xylose, m/z 132) was collected from the tandem mass analysis (Fig. 5C), whereas no successful collection of fragment ions was obtained from [M+Na]+ ions of m/z 953 and 1703. 3.5 Methylation analysis of zymolytic oligosaccharides According to the methylation results of TSP as aforementioned, the percentage ratio of T-Galp/T-Xylp was 1.6: 1. In this way, the combination of XLLG fragments and XLXG/XXLG fragments was the most possible to form the TSP chain, which was also identified by the MS/MS analysis. In order to confirm the proposal, zymolytic oligosaccharides were further conducted for methylation and GC-MS analysis. The

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results shown in Table 1 indicated that the linkage patterns of the oligosaccharide were similar with those of TSP, except a new detected linkage of →6)-Glcp-(1→ which was derived from the hydrolysis of (1→4)-linkage of β-D-(1→4,6)-Glcp residue. The percentage of β-(1→4,6)-D-Glcp residue reduced to 30.3% due to the enzymatic cleavage. The percentage ratio of T-Galp/T-Xylp in oligosaccharides was

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1.2: 1, which was smaller than that of TSP. By combining the results of monosaccharide composition, methylation analysis

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of TSP and oligosacchairdes, NMR and MALDI-TOF MS studies, the chemical

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structure of TSP was confirmed to be a combination of nonasaccharide (XLLG) and

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octasaccharide (XLXG or XXLG) blocks (periodic or interrupted sequence) in a

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na

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percentage ratio of 3: 2 as shown below:

The oligomer sequence would be occasionally interrupted by a heptasaccharide (XXXG), hexasaccharide (XLG or XXGG), or even hendesaccharide. This result presented an amendment to the fine structure of TSP as a well-known XLLG repeating polymer [11]. 3.6 Molecular weight and conformational properties of TSP HPSEC-MALLS technique was conducted to determine the molecular weight and other molecular parameters of TSP. The major single peak as shown in Fig. 6A indicated that TSP was homogeneous on molecular weight distribution, in spite of a small peak found from the MALLS detector which was probably attributed to a 15

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small amount of aggregates [23]. Calculated by ASTRA software, the weight average molecular weight (Mw) and number average molecular weight (Mn) were determined as 524.0 kDa and 344.5 kDa, respectively, with polydispersity index (Mw/Mn) of 1.52. The radius of gyration (Rg) was determined to be 55.6 nm for TSP. Intrinsic viscosity ([η]) is a critical parameter which is related with the chemical

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structure and molecular size of polysaccharide as well as the solvent property. A high intrinsic viscosity value relates to a more extended structure for a given chain length.

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From the HPSEC, a differential pressure viscometer was applied to determine the [η],

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and the result showed as 4.92 dL/g for TSP in 0.1 M NaNO3 solution. The relative

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high value of [η] indicated the extended stiff chain of TSP [34].

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The chain conformational information of TSP could be further derived by

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establishing the relationship of Mw with [η] from the HPSEC-MALLS results. The double logarithmic plot of the [η] vs Mw can be well described using the

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Mark-Houwink equation:

[η] = kMwɑ

(1)

where k and α are related to the three-dimensional conformation of polymer chains in the solvent environment: α values less than 0.5 reflect a rigid sphere in an ideal solvent; those from 0.5~0.8 indicate a random coil in a good solvent; and from 0.8~2.0 a rigid or rod like conformation (stiff chain) [35]. The α value was determined to be 0.71 by double logarithmic plot of [η] against Mw in the Mw range of 158.0~1054 kDa (Fig. 6B), indicating that TSP was present as a random coil with relative extended and stiff chain in 0.1 M NaNO3 solution. It has been reported that

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TSP from Japan (Glyloids 3s, Dainippon Pharmaceutical Co., Osaka) was of high chain stiffness and easily formed hyper-entanglement/aggregation in non-ionised environment (distilled water) [11, 36]. The current result revealed that the increase of ionic strength would decrease the stiffness and increase the flexibility of TSP. 4. Conclusion

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In the present study, TSP was characterized as a β-(1→4)-glucan branched at O-6 position of glucose with Mw of 524.0 kDa and Rg of 55.6 nm. Short side chains of

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T-β-Galp-(1→2)-α-Xylp-(1→and T-α-Xylp-(1→ were distributed along the glucan

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backbone to form the major subunits of nonasaccharide (XLLG) and octasaccharide

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(XLXG or XXLG) in a ratio of 3: 2. Heptasaccharide (XXXG, 7.2%), hexasaccharide

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(XLG/XXGG, 2.7%), and hendesaccharide (1.9%) were also distributed occasionally

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as the blocks of TSP but with low intensity. This structure was significant different with other GXGs from plants as reported previously [17]. The high degree of glycosyl

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substitutions to the cellulosic backbone of TSP lead to a relative stiff extended-chain conformation in aqueous solution, which then resulting in unique rheological properties of TSP [8]. Besides, the distribution of galactose in the side chain was also identified to play an important role on the gelling ability of TSP [37]. Further work will focus on the study of gum properties of TSP and elucidation of relationship between structure and properties to distinguish the different functional GXGs. Acknowledgements The financial supports for this study by the National Natural Science Foundation of China (No: 31601428), and Shanghai Agriculture Applied Technology

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Development Program, China (No. 2019-02-08-00-07-F01152), and Key Research & Development Project of Shandong Province (No. 2018YYSP003) are gratefully acknowledged.

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[1] P.M.C.L. Reis, C. Dariva, G.Â.B. Vieira, H. Hense, Extraction and evaluation of antioxidant potential of the extracts obtained from tamarind seeds (Tamarindus

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indica), sweet variety, Journal of Food Engineering 173 (2016) 116-123.

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[2] N. Shankaracharya, Tamarind-chemistry, technology and uses-a critical appraisal,

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Critical reviews in food science and nutrition 48(1) (2008) 1-20. [5] N. Ibrahim, M. Abo-Shosha, E. Allam, E. El-Zairy, New thickening agents based on tamarind seed gum and karaya gum polysaccharides, Carbohydrate Polymers 81(2) (2010) 402-408. [6] J. Joseph, S. Kanchalochana, G. Rajalakshmi, V. Hari, R.D. Durai, Tamarind seed polysaccharide: A promising natural excipient for pharmaceuticals, International Journal of Green Pharmacy 6(4) (2012). [7] J. Kochumalayil, H. Sehaqui, Q. Zhou, L.A. Berglund, Tamarind seed xyloglucan–a thermostable high-performance biopolymer from non-food feedstock,

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Carbohydrate Polymers 151 (2016) 538-545. [30] M. Hoffman, Z. Jia, M.J. Peña, M. Cash, A. Harper, A.R. Blackburn, A. Darvill, W.S. York, Structural analysis of xyloglucans in the primary cell walls of plants in the subclass Asteridae, Carbohydrate Research 340(11) (2005) 1826-1840. [31] B. Ray, C. Loutelier-Bourhis, C. Lange, E. Condamine, A. Driouich, P. Lerouge, Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif, Carbohydrate Research 339(2) (2004) 201-208. [32] S.G. Grishutin, A.V. Gusakov, A.V. Markov, B.B. Ustinov, M.V. Semenova, A.P.

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properties, and applications, CRC Press, Boca Raton, Florida, 2005, pp. 162-214.

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[37] M. Shirakawa, K. Yamatoya, K. Nishinari, Tailoring of xyloglucan properties using an enzyme, Food Hydrocolloids 12(1) (1998) 25-28.

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Figure captions: Figure 1. 1D NMR spectra of TSP recorded at 298 K: (A) 1H NMR spectrum (600.10 MHz); (B) 13C NMR spectrum (151.01 MHz). Figure 2. 1H /1H and 1H /13C correlation spectra of TSP recorded at 298 K: (A) 1H/13C HSQC correlation spectrum with partial assignments; (B) 1H/1H DQF-COSY correlation spectrum with partial assignments.

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Figure 3. The 1H /13C HMBC spectrum of TSP recorded at 298 K.

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Figure 4. Enzymatic hydrolysis of TSP and its oligosaccharide analysis by

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MALDI-TOF MS

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Figure 5. MS/MS recording of different zymolytic oligomers from TSP. A, B, and C

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are the secondary MS spectra of [M+Na]+ ion of m/z 1409, 1247, and 1085,

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respectively. The lowercase of a, b, and c in the figures mean the possible cleavage of glycosidic bond in the order of a, b, and c.

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Figure 6. HPSEC chromatography (A) and Mark-Houwink plot (B) of TSP in 0.1 M NaNO3 solution. The curves in red, black, and blue color of (A) are the signals from MALLS detector at the angle of 90°, DP detector, and RI detector, respectively.

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Journal Pre-proof Tables Table 1 Methylation analysis of TSP and its enzyme hydrolysates PMAAs

Linkage pattern

Percentage in TSP (%)

Percentage in hydrolysates (%)

Major m/z

6.77

2,3,4-Me3-Xylp

Xylp-(1→

11.7

14.3

45, 87, 88, 101, 102, 117, 118, 161, 162

9.94

2,3,4,6-Me4-Galp

Galp-(1→

18.5

16.5

45, 87, 88, 101, 102, 118, 129, 145, 161, 162, 205

10.85

3,4-Me2-Xylp

→2)-Xylp-(1→

13.3

14.5

45, 88, 101, 117, 130, 190

14.12

2,3,6-Me3-Glcp

→4)-Glcp-(1→

13.6

8.4

14.41

2,3,4-Me3-Glcp

→6)-Glcp-(1→

n.d.1

19.90

2,3-Me2-Glcp

→4,6)-Glcp-(1→

42.9

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16.0

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30.3

na

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n.d.: not detected

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1

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RT (min)

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45, 87, 99, 102, 118, 129, 162, 173, 233 45, 87, 99, 102, 118, 129, 130, 159, 162, 189, 233 45, 99, 102, 118, 127, 159, 201, 261

Journal Pre-proof Table 2 13C/1H NMR chemical shifts (ppm) of different sugar residues from TSP (in D2O at 298 K). C2/H2

C3/H3

C4/H4

C5/H5,H5’

C6/H6,H6’

101.64/5.18

82.33/3.68

74.8/3.94

72.5/3.67

68.9/3.84

-

101.81/4.96

74.4/3.55

73.4/3.74

72.4/3.68

68.7/3.83

-

105.36/4.56

75.84/3.40

74.33/3.68

82.97/3.69

74.33/3.63

71.43/3.73,3.93

105.36/4.56

75.46/3.41

74.33/3.68

81.58/3.69

74.76/3.63

64.40/3.73,3.56

107.35/4.56

74.32/3.63

76.80/3.72

72.27/3.97

76.27/3.75

63.87/3.78,3.56

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A: Xyl →2)-α-D-Xylp(1→ B: XylT T-α-D-Xylp(1→ C: Glc46 →4,6)-β-D-Glcp(1→ D: Glc4 →4)-β-D-Glcp(1→ E: GalT T-β-D-Galp(1→

C1/H1

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2

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Sugar residue

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Table 3 MALDI-TOF MS and MS/MS analysis of oligosaccharide fragments from the enzymatic hydrolysates of TSP m/z from MS

Ion

m/z of MS/MS fragment ions

953

[M+Na]+ +

[M+K]

-

1085

[M+Na]+

953

[M+K]

-

1247

[M+Na]+

1187, 1115, 1085,953, 659

1263

[M+K]+

-

1409

[M+Na]+

1349, 1277, 1247, 1115

1425

[M+K]+

-

1703

[M+Na]+

-

7.2

XLXG/XXLG/XXXGG

34.5

XLLG/XLXGG/XXLGG

53.7

XLLXG/XLXLG/XXLLG/ XLXXGG/XXLXGG/XXXLGG

1.9

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G: a backbone glucosyl residue without substitution, X: additional xylosyl residue on the O-6

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1

XXXG

-p

[M+K]

2.7

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1719

XLG/XXGG

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1101

+

Relative percentage (%) 2

-

969

+

Possible structure 1

position of G, L: X side-chain with additional galactosyl residue. The fragments labeled in bold are

The percentages of different oligomer fragments were calculated according to the intensity signal

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2

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the most possible oligomer structure.

of MS by normalization method.

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Figures

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B

Figure 1 (International Journal of Biological Macromolecules, Zhang H., et al.)

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A

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B

Figure 2 (International Journal of Biological Macromolecules, Zhang H., et al.)

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Figure 3 (International Journal of Biological Macromolecules, Zhang H., et al.)

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Figure 4 (International Journal of Biological Macromolecules, Zhang H., et al.)

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A

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C

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B

Figure 5 (International Journal of Biological Macromolecules, Zhang H., et al.)

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Journal Pre-proof Define Peaks LS

A

dRI

DP

Relative Scale

1.0

0.5

1

0.0 10.0

1

20.0

30.0

40.0

Mark-Houwink Plot

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tsp-1

-p

1000.000 900.00 800.00 700.00 600.00

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500.00 400.00 300.00

3.0x10

5

Mark-Houw ink-Sakurada properties K = (4.681 ± 0.004) e-2 mL/g, a = (7.106 ± 0.001) e-1

5

5

5

4.0x10 6.0x10 8.0x10 1.0x10 Molar Mass (g/mol)

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2.0x10

5

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Intrinsic Viscosity (mL/g)

B

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time (min)

6

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Figure 6 (International Journal of Biological Macromolecules, Zhang H., et al.)

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Author statement Hui

Zhang: Conceptualization,

Methodology,

Project

Writing-Original draft preparation. Taolei Zhao: Project administration, Formal analysis. Junqiao Wang: Formal analysis, Methodology.

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Yongjun Xia: Validation. Zibo Song: Validation.

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Lianzhong Ai: Supervision.

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administration,

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Graphical abstract

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Highlights 

A classic cellulosic backbone of β-(1→4)-glucan was confirmed for TSP.



Short branches consisted of Xylp and Galp were distributed along the backbone.



TSP composed of XLLG and XLXG/XXLG units with slight XXXG, XLG/XXGG, and hendesaccharide blocks.

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TSP showed as relative extended stiff random coil in aqueous solution.

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

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