Isolation and structural characterization of a polysaccharide derived from a local gum: Zedo (Amygdalus scoparia Spach)

Accepted Manuscript Isolation and structural characterization of a polysaccharide derived from a local gum: Zedo (Amygdalus scoparia Spach) Roxana Seyfi, Mohammad Reza Kasaai, Mohammad Javad Chaichi PII:

S0268-005X(18)30004-3

DOI:

10.1016/j.foodhyd.2018.09.017

Reference:

FOOHYD 4654

To appear in:

Food Hydrocolloids

Received Date: 1 January 2018 Revised Date:

11 July 2018

Accepted Date: 10 September 2018

Please cite this article as: Seyfi, R., Reza Kasaai, M., Javad Chaichi, M., Isolation and structural characterization of a polysaccharide derived from a local gum: Zedo (Amygdalus scoparia Spach), Food Hydrocolloids (2018), doi: https://doi.org/10.1016/j.foodhyd.2018.09.017. 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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Graphical Abstract

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Isolation and Structural Characterization of a Polysaccharide Derived from a Local Gum:

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Zedo (Amygdalus scoparia Spach)

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Roxana Seyfi1. Mohammad Reza Kasaai1,*. Mohammad Javad Chaichi2 1

Department of Food Science and Technology, Sari Agricultural Sciences and Natural Resources

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University

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Khazar Abad road, Km. 9, P.O. Box, 578, Sari, Mazandaran, Iran

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Faculty of Chemistry, Mazandaran University, Babolsar, Iran *

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e-mail: [email protected]

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Short title: Structural Characterization of a polysaccharide from zedo gum

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Abstract

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In this study, a novel polysaccharide was isolated from a local gum, zedo (Amygdalus scoparia

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Spach). Chemical composition of the gum and elemental analysis of the isolated polysaccharide

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were determined. The polysaccharide and its fragments were examined by gas chromatography-

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mass spectrometry, GC-MS, viscometry, 1H NMR, and13CNMR spectroscopy. Results obtained

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from different methods, revealed that a hetero-polysaccharide is the main component of the gum.

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The amounts of moisture, minerals, protein and lipid were 9.42, 2.23, 1.05, and 0.03 (w/w %),

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respectively. The amounts of carbon, hydrogen, oxygen and nitrogen obtained from elemental

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analysis were 38.5, 5.8, 35.3, and 0.3 (w/w %), respectively. The presence of arabinose,

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galactose, rhamnos, mannose, β- D- glucose, fructose, galacturonic and glucuronic acids residues

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were measured by GC-MS analysis. However, conversion of glucose into fructose probably

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occurred during the analysis, because the temperature of the analysis is high enough. Both

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carboxylic groups and minerals/ salts had a major influence on the rheological properties of the

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gum. The gum exhibited polyelectrolyte behavior, and its solutions did not follow Newtonian

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flow behavior. It can be naturally occurred as an anionic polysaccharide. The gum was a highly

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branched polysaccharide and a member of arabinogalactans family. The main chain contains α-

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L-arabinofuranosyl, α- L-Araf, and β-D-galactopyranonyl, β-D-Galp, units. The following

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residues were identified: → 3,4) β-D-Galp (1→ ; → 3,4,6) β-D-Galp (1→; →3)- α- L-Araf-

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(1→; → 4) β-D-GlcpA (1→.; glucosyl residues; and α-L- Rhap, (1→. Terminal residues were:

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α-L- Rhap; and α- L-Araf.

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Keywords: Zedo gum; Polysaccharide; Characterization; Chemical composition; Molecular

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Structure. 2

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1. Introduction

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Gums are a wide range of hydrophilic biopolymers including polysaccharides, proteins, and

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complex polysaccharides. They possess different chemical structures: linear; branched; highly

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branched; homogeneous; heterogeneous; neutral; and ionic, with various physico-chemical,

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biological, rheological, and functional properties (Belitz, Grosch, & Schieberle, 2009; Ouellette

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& Rawn, 2014; Saeidy et al., 2018; Stylianopoulos, 2013). The ionic polysaccharides may be

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anionic (pectin, alginic acid, alginates carboxymethyl cellulose, hyaluronic acid, heparin, and

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chondroitin sulfate) (Funami et al., 2008; Xiao et al., 2011), or cationic (chitin, chitosan)

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(Yudovin-Farber, Azzam, Metzer, Taraboulos, & Domb, 2005).The anionic groups play a

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significant role in their dissolutions and applications. The polysaccharides as alkali salt forms are

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soluble in the neutral medium or alkaline pH range (Belitz, et al., 2009). The structural features

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of polysaccharides, such as steric configuration, degree of substitutions, linkages of

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monosaccharides, and molecular weight and its distribution play critical roles on their physico-

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chemical and biological properties (Liu, Willfor,& Xu,, 2015; Ngo & Kim, 2013).

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Biological activities of polysaccharides are influenced by their chemical structure, chain

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conformation, and molecular size. As the molecular size of a polysaccharide decreases, the

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number of its reducing end groups increases, and its biological activities increases. Low

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molecular weight polysaccharides and oligosaccharides exhibit a wide range of biological

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activities (MacGregor, 2002). Bioactive polysaccharides from different sources could offer

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numerous biological properties and benefit humanity in health care (Liu et al., 2015; Ngo &

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Kim, 2013). Polysaccharides particularly oligosaccharides participate in many biological

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processes (Dube & Bertozzi, 2005; Varki, 1993).

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Polysaccharides from different natural resources have been used in different industries, such as

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food, feed, biomedical, cosmetics, medicine and pharmaceutics, because they are non-toxic,

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biocompatible, and biodegradable (Liu et al., 2015). In food processing, a variety of

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polysaccharides are employed to produce thickening of solutions, stabilizing of emulsions,

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promote gelling and affect numerous related improvements in functionality (Stephen &

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Merrifield, 2005). The amounts of polysaccharides incorporated in food preparations may be

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very large or as low as fractions of 1%. Some polysaccharides applied in food industries, because

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they improve rheological properties, extend shelf- life, preserve flavor, create elasticity, retain

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moisture, stabilize and emulsify food systems, and add value to food products (Ibanez &

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Ferrerob, 2003; Tabatabaee Amid, Mirhosseini, 2012). Branched polysaccharides such as

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derived from arabic gum produce Newtonian solutions with a low solution viscosity and cannot

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be used to improve rheological properties of foods as well (Nie et al., 2013a, b).

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A wild peanut tree with the scientific name of Amygdalus scoparia Spach is a tree or shrub of

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Rosaceae family. The plant is a small tree with several stems and branches. Every square meter

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of tree produces 20-50 gr. zedo gum. The plant is distributed in different regions of Iran

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especially in the lime and rocky mountain (Abbasi & Mohammadi, 2013; Abbasi & Rahimi,

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2014; Azarikia & Abbasi, 2016). The gum exudates from stems, trunk, and branches of the plant,

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is called “Persian gum” in some cases, "Shirazi gum" and locally it is called “Zedo.” (Alijani,

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Balaghi, & Mohammadifar, 2011; Azarikia & Abbasi, 2016; Ghasempour, Alizadeh, & Bari,

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2012; Kashki & Amirabadizadeh, 2011). The gum is gradated, based on chemical composition,

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climate conditions and particle size of soils, where the plant is grown. The gum may be occurred

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in different color (white, yellow, orange and red), due to different chemical compositions. The

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gum color is brighter in the tropical region than that of the cold region. Figure 1 shows some

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images of the plant at different developmental stages of growth.

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Figure 1: Different images of wild peanut trees at different developmental stages/ seasons of

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growth. Zedo gum exudates from stems, trunk, and branches of the plant.

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Zedo gum is used in food and pharmaceutical industries. Its combination with tragacanth or

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arabic gum has been also used in pharmaceutical industries as an emulsifier and suspending

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agents (Abbasi & Mohammadi, 2013; Ghasempour et al., 2012). The gum has been used in

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traditional medicine to stimulate appetite. Arabic, gahatti, and Karaya gums (similar to zedo

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gum) are structurally branched and exudate from different trees (Belitz, et al., 2009). The main

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chains of these gums carry side chains, covalently linked via the side chains and may be also

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covalently linked with a protein via an amino acid. These gum are suitable as binders or

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adhesives and can be used as emulsifiers and stabilizers.

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Studies on properties, characteristics and applications of the zedo gum have been already

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reported as follow: use of zedo gum for stabilization of milk-orange juice mixture (Abbasi &

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Mohammadi, 2013); and botanical sources, physicochemical, rheological and functional

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properties, structural characterization, its interaction with other proteins and polysaccharides, and

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its traditional applications in medicine and pharmaceutics and possible applications in foods,

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beverages, cosmetics, and pharmaceutics (Abbasi, 2017; Abbasi & Rahimi, 2014). In the present

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study, structure and chemical composition of zedo gum were investigated using elemental

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analysis, gas chromatography-mass spectrometry, GC-MS, rheological properties, and magnetic

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resonance spectroscopy of hydrogen and carbon, 1H NMR, and 13C NMR.

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2. Materials and methods

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2.1. Materials

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Zedo gum was purchased from a local plant market (Marvdasht, Fars Province, Iran). The gum

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granules were initially cleaned to remove their impurities (dust and sand). The granules were

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then converted into powder using a laboratory mill machine (Bullet Magic, Model MB 1001,

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Germany). The powder passes through a sieve with a mesh number of 100. A fine powder with a

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mesh number of smaller than 100 (149 µm) was then packed in a plastic bag, sealed and stored at

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-18ºC, except for the short interval step for the isolation of polysaccharide. The following

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chemicals were purchased from different companies as follows: ethanol (96%), n- hexane,

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chloroform and hydrochloric acid (37%) (Merck company, Darmstadt, Germany); sodium

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borohydride and different standard sugars (Sigma-Aldrich, St. Louis,MO, USA); Filter paper

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(ash-less, grade 42, diameter 125 mm) (Whatman, England), and a regenerated cellulose dialysis

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tube (Serva, Electrophoresis GmbH, Heidelberg, Germany), with a cut- off of 3.5kDa. De-

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ionized water was also used in this study.

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2.2. Determination of chemical composition of the original gum

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The total moisture content was measured by heating the zedo gum at 105°C to achieve a constant

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weight (AOAC, 2000). Ash content was measured based on dry weight of the gum (AOAC,

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1990). The total nitrogen content (N2) was measured using an automatic Kjeldahl apparatus

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(Behr, Germany) (Anderson & Farquhar, 1982), and the amount of protein was estimated by

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using the conversion factor, 6.25. The lipid was extracted using a mixture of two solvents,

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hexane and chloroform (1:1 v/v) and a soxhlet apparatus (Brummer, Cui, & Wang, 2003).

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Measurements for each component (moisture, ash, protein, or lipid) were replicated three times.

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The calculation was performed based on the dry weight of the zedo gum. The results were

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expressed as a mean ± standard deviation (SD).

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2.3. Isolation of the polysaccharide from the crude gum

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The polysaccharide was initially isolated from the crude gum as follows: 10 g of the gum powder

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with 100 mL of a mixture of two solvents, hexane and chloroform (1: 1 v/v) was refluxed in a

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soxhlet apparatus for 3h. The solvent was then separated by distillation. The solvent residues

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were removed using a ventilated oven at 30°C for 3h (Memmert, Germany). In the second step, a

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separation procedure by ethanol was then performed using a soxhlet apparatus for 24h, in order

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to separate impurities (proteins, enzymes, pigments, resins and other compounds). The white

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powder was then dried in an oven at 30°C for 24h. The white dried powder was dispersed in

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deionized water (1% w/v of the dried powder in deionized water). The dispersed aqueous

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solution was then filtered using vacuum created by a water aspirator pump and a filter paper

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(ash-less, grade 42, circle diameter 125 mm). The clear solution was dialyzed against deionized

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water for 48h by using a regenerated cellulose dialysis tube with a cut- off of 3.5kDa. The

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deionized water was replaced every 24 hours to achieve a better results. After dialysis, the gum

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solution was lyophilized (Operon company, FDu-8624, Korea), to obtain a polysaccharide, zedo,

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ZG.

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2.4. Elemental analysis

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Carbon, hydrogen, nitrogen and oxygen contents of the original/ isolated ZG, were determined

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by an elemental analyzer (ECS 4010 CHNSO Analyzer, Costech Analytical Technologies Co.,

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Italy), as described elsewhere (Vinod, Sashidhar, Sarma, & Vijaya Saradhi, 2008).

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2.5. Partial acid hydrolysis of the isolated polysaccharide

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The isolated sample, ZG, was dissolved in deionzed water and pH was adjusted to 2.0 by adding

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1M HCl and the final polysaccharide concentration was 4.8%. The solution was heated at 80°C

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for 24 h. The solution was cooled down and neutralized by sodium boron hydride (NaBH4). The

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suspended particles remaining in the solution were separated by centrifugation (4500 rpm, 15

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min) (Hermle Z 200 A, Germany). The clear solution was placed in a dialyze tube and the

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dialysis procedure was performed against deionized water for 24h. The solution obtained from

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the inside of the tube was dried by lyophillization. The dried sample was called, ZG-H1. Similar

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to the above-mentioned procedure was performed, except the solution was heated at 100°C for

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4h. The resulting dried sample was ZG-H2. The samples (ZG-H1 and ZG-H2), were used for

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their structural characterization.

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2.6. Monosaccharide analysis of gum by gas chromatography- mass spectroscopy

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Trimethylsilyl (TMSi) derivative of the original/ isolated sample (ZG) was prepared using ZG,

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bistrimethysilyl trifluoro acetoamid (BSTFA), and

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described elsewhere (Vinod et al., 2008). The resulting monosaccharide derivatives were

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analyzed by a gas chromatography-mass spectrometer, GC-MS (Agilent 6890,Agilent

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Technologies, Palo Alto, USA), equipped with a silica capillary column (ACP SIL 8 CB,

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30m×0.25mm, film thickness 0.25 µm). The oven temperature was initially maintained at 50°C

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for 2 min, the temperature was then raised by a rate of 10°C/min, and finally maintained at

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280°C for 5 min. The temperatures of electron ionization (EI) detector and quadrupole were set

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at 230°C/ and 150°C, respectively. Helium was used as a carrier gas with a flow rate of 1.0

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mL.min.-1 The resulting monosaccharide derivatives were identified by the National Institute for

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Standards and Technology (NIST) and Wiley database supplied by Agilent (Agilent

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Technologies, USA), along with the instrumental software. The retention times for the resulting

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fragments were also compared with the corresponding values of TMSi derivatives of sugar

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standards (Sigma-Alderich, MO, USA).

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2.7. Nuclear magnetic resonance spectroscopy

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Proton magnetic resonance, 1H NMR, and carbon magnetic resonance, 13C NMR, for the original

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polysaccharide and their hydrolyzed samples (ZG, ZG-H1 and ZG-H2) were recorded (Bruker,

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Model AC-400 MHz spectrometer, Germany). The 1H NMR spectrum for each sample in D2O at

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400 MHZ and two temperatures (25 and 50℃), were obtained with the number of scans and the

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pulse repetition delay 16 and 6s, respectively. The chemical shifts were expressed in ppm

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relative to 4,4-di-methy l-4- Sylapanten- 1 - sulfonic acid (DDS) as an internal standard. The 13C

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NMR spectrum for each sample in D2O at 25℃, at 100 MHz was also obtained with 3000 scans

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and a delay time of 2s.

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acetonitrile substances, and a method

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2.8. Viscometry

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Solution viscosities of the original gum were measured using a viscometer (DV-II+ Pro, S61-

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Model LV, Brookfield, USA), under low shear rate at 25℃. Deionized water was used as a

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solvent. Solution concentrations were less than 4 gr.100 mL-1 (4.0 %, w/v). The viscometer

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initially was calibrated using standard samples. The viscosity of deionized water at 25℃, was

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0.89 centipoise (c.P).

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3. Results and discussion

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3.1. Chemical composition and elemental analysis

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The chemical composition of the original gum was found as follows: moisture (9.42%); minerals

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(2.23%); protein (1.05%); and a negligible amount of lipid (0.03%). Moisture content of the gum

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(9.42%) was greater than that of the corresponding value for guar gum (8.60%) and smaller than

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that of xanthan (10.20%) (Cui & Mazza, 1996) and arabic (15.00%) gums (Yebeyen, Lemenih,

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& Feleke, 2009). Since the gum was carefully cleaned in the preparation stage, and was free

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from any impurities such as sand and soil, therefore, the total ash content of gum (2.23%) is an

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indicative of physiological minerals (Pachuau, Lalhlenmawia & Mazumder, 2012). The amount

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of protein (1.05%) in the ZG was smaller than that of the reported protein for arabic (2.31%)

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(Yebeyen et al., 2009), xanthan (5.40%); guar (8.20%) gums (Cui & Mazza, 1996), and

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assafoetida (Saeidy et al., 2018) gums. A negligible amount of lipid (0.03%) was observed in the

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ZG, which is very smaller than the lipid in the fenugreek seed (7.24%) (Brummer et al., 2003).

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Plant cell walls are complex materials essentially made of polysaccharides. These

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polysaccharides are either alone or covalently linked to proteins or lipids. In this study, no lipid

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was observed in the zedo gum. Hence, the polysaccharide may be covalently attached to the

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protein and formed a glycoprotein. Glycoproteins play a variety of roles in various aspects of

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plant growth and development (Berg, Tymoczko, & Stryer, 2002; Boudet, 2003; Ellis, Egelund,

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Schultz, & Bacic. 2010; Gaspar, Johnson, McKenna, Bacic1 & Schultz, 2001; Showalter. 1993;

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Tan, et al., 2012). These materials usually include the hydroxyproline-rich glycoproteins

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(OʾNeill & York, 2003). Four types of interactions can be taken place between polysaccharides

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and proteins. The most common interaction is the chemical reaction between amino groups from

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proteins and carboxylic groups from the polysaccharides (as N-linked glycans) (Margnin &

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Dumitriu, 2005). In glycoproteins, sugars are attached either to the amide nitrogen atom in the

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side chain (classified as N-linked glycans) or to the oxygen atom in the side chain (classified as

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O-linked glycans) (Berg et al., 2002; Kumar et al., 2013; Stephen & Merrifield, 2005).

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The results of elemental analysis (C, H, O, and N) for the isolated ZG are presented as follows:

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carbon (38.53%); hydrogen (5.82%); oxygen (35.26%); and nitrogen (0.26%). The amounts of

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carbon (38.53%) and nitrogen (0.26%) for the ZG were smaller than that of these elements:

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carbon (41.95%), and nitrogen (0.47%), for arabic gum (Yadav, Igartuburu, Yan, & Nothnagel,

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2007), and greater than that of these elements, carbon (34.97%) and nitrogen (0.23%) for the

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Kondagogu (a polysaccharide derived as an exudate from the bark of cochlospermum gossypium)

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(Vinood et al., 2008) gums. While the hydrogen content (5.82%) of the ZG was greater than that

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of hydrogen in arabic (5.78%) and Kondagogu (5.58%) (Vinood et al., 2008; Yadav et al., 2007)

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gums.

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3.2. Monosaccharide residues analysis of gum by GC−MS

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The results of trimethyl silylation of the isolated ZG and different monosaccharide residues

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found in the GC-MC chromatogram are presented in Table 1. The monosaccharide derivatives

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were identified by comparing their retention times, RTs, with their corresponding sugar

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standards (arabinose, rhamnose, mannose, fructose, galactose and β-D-glucose) (See Table 1).

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These results indicate that the gum was a complex polysaccharide containing neutral and acidic

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monosaccharide residues with various glycosidic linkages. Galactose and arabinose were found

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as the major neutral residues and formed quantitatively 47.36% of the total residues. These

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results suggest that the polysaccharide could be a member of arabinogalactans family. The gum

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may be comprised arabinogalactan-proteins AGPs, principally consisted of carbohydrate

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building blocks. Small proportions of the macromolecules may be comprised both carbohydrate

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and protein components. Generally, the polysaccharide portion (more than 90% of the molecule)

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is rich in arabinose and galactose, while the protein moieties have diverse amino acid sequences,

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they are often enriched in Hyp, Ala, and Ser (Bacic et al., 2000; Gaspar, et al., 2001; Nothnagel,

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1997; OʾNeill & York, 2003; Showalter, 1993). They are widely distributed in plants and

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typically comprised less than 10% protein (dry weight) (Showalter, 1993). The amount of protein

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in the zedo gum was very low (1.05%) in comparison with other member of the AGPs family

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such as assafoetida gum (6.8%) (Saeidy et al., 2018). Structural investigation on protein moiety

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(deglycoslating of the crude gum, amino acid composition, sequence analysis, its linkage with

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the polysaccharide) was out of this study.

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Two acidic monosaccharide residues, glucuronic and galacturonic acids were also found among

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monosaccharide residues. The backbone may be ramified by two acidic monosaccharide residues

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galacturonic and glucoruonic acids. Most of monosaccharide residues identified in the ZG

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and kondagogu gums were identical (Vinod et al., 2008). However, the amounts of

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monosaccharide residues for the zedo were different from the corresponding residues of the

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kondagogu gum.

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Fructose was also identified by GC-MS., however, conversion of glucose into fructose probably

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occurred during the analysis, because the temperature of the analysis is high enough.

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Isomerization of glucose into fructose is influenced by several parameters (temperature, reaction

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time, solvent/ medium, catalyst loading). The temperature is one of the major parameter. Free or

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semi-free form of aldehyde groups (open-chain form of the aldehyde form) or hemi-acetal form

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of glucose readily oxidized at a high temperature. A low energy is needed for the conversion of

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glucose into fructose (Berg et al., 2002).

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Table 1: Data for different monosaccharide sugars

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obtained from GC-MS chromatogram for ZG gum

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RT(i) RT(ii) Peak area (min) (min) (%) Arabinose 16.44 16.49 25.32 Rhamnose 18.49 18.45 9.01 Mannose 18.78 18.74 11.54 Fructose 18.89 18.90 10.82 Galactose 19.36 19.32 22.04 Glucuronic acid 19.75 19.75 3.85 Galacturonic acid 20.21 20.21 5.88 20.54 20.54 5.16 β-D-glucose (i) Retention time for sugars obtained from the chromatogram (ii) Retention time for standard sugars

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3.3. Viscometry

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The variation of solution viscosity as a function of solution concentration was shown in Figure 2.

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Solution viscosity increased with an increase in solution concentration. The relationship did not

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follow linear fashion, similar to the Newtonian fluids. We have shown the relationship as linear

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plots having different slopes. The slope of the initial linear portion was smaller than that of

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further portions. In the literature, the variation of viscosity versus concentration has been

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illustrated as (linear, bi-linear, and exponential) fashions (Graessley, 1974; Vinogradov, &

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Martin, 1980).

Non-Newtonian flow behavior is associated with both increase in solution

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concentration and increases in molecular weight of macromolecules in solution. The

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entanglement network density increases with an increase in molecular weight and concentration

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of macromolecules. In dilute solutions, entanglements between macromolecules chains are

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limited and the solution viscosity increases linearly with an increase in macromolecule

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concentration. Entanglements begin to occur when the concentration exceeds a certain value

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(Graessley, 1974; Hager & Berry, 1982; Kasaai, Charlet & Arul). As the polymer concentration

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increases, overlapping of macromolecular chains becomes important and the relative viscosity of

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solution increases significantly with an increase in concentration, up to a critical concentration.

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This region called the semi-dilute regime (Graessley, 1974). Above the critical concentration, the

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entanglement between macromolecular chains increase sharply, it may be called concentrated

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regime (Vinogradov, & Martin, 1980). Zedo gum with two acidic monosaccharide residues,

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galacturonic and glucoruonic acids can be considered as an anionic polysaccharide. Due to the

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presence of significant amount of minerals (2.23%) in the original gum, metal ions of the

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minerals (salts) may be linked to carboxylic groups of the anionic polysaccharide. Thus, the

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carboxylic groups of the polysaccharide can be occurred as salt forms (carboxylate forms). In

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other word, the gum partially exhibits poly-electrolyte behavior. In conclusion, both carboxylic

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groups and minerals (salts and metal ions) had a major influence on the rheological properties of

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the polysaccharide. That is why, the solution of polysaccharide did not follow Newtonian flow

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behavior. Generally, highly branched gums in solutions such as arabic gum at low concentrations

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exhibit Newtonian flow behavior.

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Concentration (g.100 mL-1)

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Figure 2: Solution viscosity of the original gum as a function of solution concentration at 25℃

314

under a low shear rate.

315 316

3.3. NMR spectroscopy

317

1

318

polysaccharides at 80℃ (ZG-H1-25), and 100℃ (ZG-H2-25) were illustrated in Figure 3. Figure

H NMR spectra examined at 25℃ for the original polysaccharide (ZG-25), the hydrolyzed

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4 showed 1H NMR spectra recorded at 50℃ for the hydrolyzed polysaccharides at 80℃ (ZG-H1-

320

50) and 100℃ (ZG-H2-50). 13C NMR spectra recorded at 25℃ for the original (ZG-25) and the

321

hydrolyzed polysaccharide at 80℃ (ZG-H1-25) was illustrated in Figure 5. Figure 6 shows

322

NMR spectrum examined at 25℃ for the hydrolyzed polysaccharide at 100℃ (ZG-H2-50).

323

3.3.1. General aspects

324

NMR spectra with low resolutions are commonly observed for macromolecules having large

325

molecular weights. Generally, in 13C NMR spectra, some of carbon signals cannot be observed/

326

or may be observed with low intensities, particularly for the samples with high molecular

327

weights, this is because: (i) natural abundance of

328

lower sensitivities in comparison with 1H NMR spectra. The spectra of 1H NMR of different

329

samples at a temperature are similar and no change was observed in the position of different

330

peaks. A negligible difference between the chemical shifts (ppm) for each peak of NMR spectra

331

is within the experimental uncertainty (Kasaai, 2010; Kasaai, 2011). The intensity of peaks for

332

the hydrolyzed samples are shaper and greater than that of ZG sample. In order words, the

333

hydrolyzed samples possessing smaller molecular weights than that the original one, resulted in

334

clearer and a higher resolution of NMR spectra in comparison with the original one.

335

The intense deuterated water signal, HOD, was eliminated from each 1HNMR spectrum or the

336

signal was observed as a sharp vertical line in each 1HNMR spectrum (suppressed /fixed at 4.70

337

ppm) (please see Figures 3 and 4) (Hård, Zadelhoff, Moonen, Kamerling, and Vilegenthart,

338

1992; Mandal, et al., 2009). Figure S1 shows a typical 1HNMR spectrum examined at 50˚C,

339

where the deuterated water signal can be seen at 4.72 ppm in the spectrum.

340

Some changes (position of signals, intensity of signals, and the number of signals), were

341

observed in the spectra, when the temperature was changed from 25 to 50˚C. The major

C

C is low; and (ii)

13

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advantage of the analyzing at 50 ˚C was “some functional groups such as acetyl group were

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distinguished”. In general, as the temperature of 1HNMR analysis increased from 25 to 50˚C, the

344

signals were shifted slightly to greater chemical shifts. The amount of shift for different signals

345

was up to 0.03 ppm (except for some methyl and acetyl protons, the amount of shift was up to

346

0.30 ppm). The HOD signal was shifted 0.03 ppm, from 4.69 ppm at 25 ˚C to 4.72 ppm at 50˚C.

347

Two anomeric regions for protons, (4.3-5.0 ppm), and (5.00−5.65 ppm) are belonged to β- and

348

α- sugar residues, respectively (de Pinto, Martinez, & Sanabria, 2001; Duan, Wang, Dong, Fang,

349

& Li, 2003; Vinod et al., 2008; Yin et al., 2012a, b). Several peaks were observed in the

350

anomeric regions of both 1HNMR and 13CNMR spectra (Figures 3-6). These results indicate that

351

the isolated polysaccharide possesses a complex molecular structure having multiple

352

monosaccharide residues and various glycosidic linkages. Two relatively high signals in

353

amoneric proton regions (between 4.3-4.5, and 5.0- 5.31 ppm (Figure 3B) and two anomeric

354

carbons between 96-110 ppm (Figure 6) cab be seen in the NMR spectra. The results obtained

355

from the NMR spectroscopy (multiple monosaccharide residues), are consistent with GC-MS

356

results.

357

3.3.2. Arabian residues

358

A signal at 5.15-5.16 ppm in Figure 3 (Nie et al, 2013a; Polle, Ovodova, Shashkov, & Ovodov,

359

2002; Saeidy et al., 2018) and a small signal at 109.72 ppm in Figure 5A (109.39 ppm in Figure

360

6) (Das et al., 2009; Fischer et al., 2004: Kang et al., 2011a, b,c; Saeidy et al., 2018),.are ascribed

361

to H-1 and C-1 of α-L- arabinofuranosyl units, →3) α-L-Araf (1→ (Nie et al, 2013a; Polle, et

362

al., 2002; Saeidy et al., 2018). A complete series of carbon and protons signals at 109.39/ 5.15,

363

79.75/ 4.14, 76.56/ 3.85, 84.04/4.08, 59.97/ 3.76 ppm are attributed to C1/ H-1, C-2/H-2, C-3/

364

H-3, C-4/H-4 and C5/ H-5, to →3) α-L-Araf (1→ (Figure 3B and Figure 6) (Cui, Phillips,

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Blackwell & Nikiforuk, 2007; Kang et al., 2011b, c; Nie et al., 2013a; Saeidy et al., 2018; Sims

366

& Furneaux, 2003).

367

Anomeric proton and carbon signals observed at 5.11 and 107.50 ppm could be assigned to →5)

368

α-L-Araf (1→ (Cornelsen et al., 2015; Kang et al., 2011c; Saeidy et al., 2018), and the signals at

369

5.46 (or5.65) and 108.80 ppm probably are attributed to H-1 and C-1 of →2) α-L-Araf (1→

370

residues (Figures 3B, 4, 6) (Kang et al., 2011b, c; Nie et al., 2013a; Sims & Furneaux, 2003).

371

3.3.3. Galactan residues

372

The signals within 4.3-4.5 ppm can be assigned to highly branched β-D-Gal (Nie et al., 2013a;

373

Saeidy et al., 2018). Two relatively high and close each other of anomeric carbon signals within

374

103.1- 103.5 and two relatively high and close each other of anomeric protons within 4.30- 4.40

375

ppm can be seen in both 1H NMR and

376

instance, one can observe them at 4.37 and 4.39 ppm in Figure 3B and 3C.and at103.15 and

377

103.30 ppm in Figure 6. These signals are assigned to β-linkages and could be belonged to →3,

378

4), and →3,4,6) of β-D-Galp (Kang et al., 2011 b, c; Saeidy et al., 2018), respectively. A

379

complete carbon and proton peaks observed at (103.15/4.37, 70.26/3.38, 76.11/3.60, 75.04/3.96,

380

73.69/3.78, 61.19/3.74 and 3.72 ppm) (Nie et al., 2013; Saeidy et al., 2018), and a complete

381

carbon and proton signals at 103.30/4.39, 71.89/3.44, 72.53/3.78, 69.17/3.92, 68.60/4.03,

382

70.70/3.89, 3.82 ppm) were assigned to (C1/H1, C2/H2, C3/H3, C4/H4, C5/H5, C6/H6, and H6')

383

(Kang et al., 2011c) (Figures 3B, and 6), and are corresponded to → 3,4) β-D-Galp (1→ , and →

384

3,4,6) β-D-Galp (1→ residues, respectively. Two above-mentioned residues have been also

385

already reported for branched galactans and arabionogalactans (Cui et al., 2007; Kang et al.,

386

2011 b, c; Nie et al., 2013a; Saeidy et al., 2018; Sims, & Furneaux, 2003).

387

3.3.4. Uronic acid residues

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CNMR spectra (Figure 3, 5, 6, and Figure 4A). For

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The anomeric protons appeared at 4.24 and 4.27 ppm (Figure 4B); two very small signals

389

observed at 178.22 and 180.81 ppm in the

390

the spectrum was not shown); are attributed to carboxylic acid groups of uronic acid residues (C-

391

6) (Anderson, & Wang, 1991; de Paula, Santana, & Rodrigues, 2001; Kang et al., 2011b;

392

Larrazabal, Martınez, Sanabria, de Pinto, & Herrera, 2006; Vinod et al., 2008). The uranic acids

393

can be either D-galacturonic acid or D-glucuronic acid (Blackwood & Chaplin, 2000). A small

394

signal at 2.46 ppm in Figure 4B is related to the acetyl group. This signal was not observed in

395

25C. Sims and Fumeaux (2003) reported that some peaks in a NMR spectrum were observed at

396

higher temperatures, whereas at smaller temperatures these peaks were not observed.

397

literature data indicate that a signal appeared between 2.08-2.24ppm is belonged to the acetyl

398

group (Mandal et al., 2009; Nie et al., 2013; Wu, Wu, Zhou, & Pan, 2005).The difference

399

between our results with literature data could be due to the change in the temperature of analysis.

400

Due to the presence of significant amount of minerals (2.23%) in the original gum, the

401

polysaccharide can be occurred naturally as a carboxylate form (as a salt form).

402

3.3.5. → 4) β-D-GlcpA (1→

403

The signals at 4.44/ 4.46 ppm (Figure 4A or 4B) and 102.58 ppm (Figure 6) can be ascribed to

404

H-1 and C-1 resonances of → 4) β-D-GlcpA (1→ residues) (Cui et al., 2007; Kang et al., 2011b,

405

c).

406

3.3.6. Glucosyl residues

407

Anomeric proton and carbon signals observed at 4.90 and 96.50 ppm are attributed to β--glucose

408

residues (Figurers 3A, 3B, and 6). These anomeric signals have been already reported for β-

409

glucose residues (Kang et al., 2011a; Vinod et al., 2008). Signals at 5.50 and 99.7 ppm were also

410

observed. These signals were assigned to 1,4-linked-α- D-glucose residues (Figurer 4A, 4B, and

C NMR spectrum of the original ZG (This part of

The

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6) (Nie et al., 2011). General information on NMR spectra of α- and β--glucose residues have

412

been also available in the literature (Agrawal, 1992).

413

As described in section 3.2 (lines 262-269), isomerization of glucose (α- and β-) into fructose,

414

probably occurred during GC-MS measurement, where the GC-MS analysis was performed at a

415

high temperature. These results suggest that the original polysaccharide sample was composed of

416

glucose residues. The glucose was converted into fructose during GC-MS analysis.

417

3.3.7. Rhamnose residues

418

A proton signal centered at 1.20 ppm examined at 25 ˚C (Figure 3) or 1.49 ppm at 50 ˚C (Figure

419

4) (Chandra, Ghosh, Ojha, & Islam, 2009; Gutierrez de G., Martınez, Sanabria, de Pinto, &

420

Igartuburu, 2005; Roy et al., 2007; Tao, Biao, Yu, & Ning, 2008); and an anomeric carbon signal

421

at 100.30 ppm (C-6) are assigned to the methyl groups of rhamnose (α-L- Rhap ) residues

422

(Agrawal, 1992; Defaye & Wong, 1986; Sims & Furneaux, 2003). Two closed// unresolved

423

signals at 1.49 and 1.50 ppm (Figure 4B) were observed in the 1H NMR spectra recorded at 50˚C

424

(see Figure 4). The intensities of the similar signals at ZG-50 spectrum (Figure S1 in Appendix)

425

are significantly greater than that of the corresponding signals observed in ZG-H1-50 and ZG-

426

H2-50 (Please see Figure 4). The intensities of the signals gradually were decreased as the

427

hydrolysis process has been progressed, i.e., the signals in the 1H NMR spectra of ZG-50 and

428

ZG-H2-50 samples are the highest and the smallest, respectively. These changes indicate that

429

chain scission (methyl groups), occurred during the hydrolysis process. This study suggests that

430

α-L- Rhap units probably present as branch linkages or as terminal residues in the structure of

431

the original polysaccharide. A resonance peak at 5.00 ppm is assigned to the anomeric proton of

432

α-L- Rhap residues (see Figure 4) (Agrawal, 1992; Wu et al., 2005). The signals at 71.89 and

433

70.26 ppm (Figure 6) could be assigned to C-4 and C-5 of α-L- Rhap residues (Kang et al., 2011

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a, b). In contrast to our evaluation and conclusion on the location of rhamnose residues in the

435

structure of the polysaccharide, Abbasi (2017) recently reported that rhamnose is most likely

436

located in the backbone of this polysaccharide. This disparity may be partly due to differences in

437

the origin of the gum: structural modification, reorganization, synthesis and insertion of new

438

materials into the existing of the plant components may be occurred during plant growth and

439

developments (OʾNeill & York, 2003, and references therin).

440

Based on the NMR spectroscopy results: (i) the isolated polysaccharide is a member of

441

arabinogalactan family: (ii) the main chain contains α- L-arabinofuranosyl, α- L-Araf, and β-D-

442

galactopyranonyl, β-D-Galp, units; (iii) the following residues were identified: → 3,4) β-D-Galp

443

(1→ ; → 3,4,6) β-D-Galp (1→; →3)- α- L-Araf-(1→; → 4) β-D-GlcpA (1→.; and α-L- Rhap,

444

(1→; and (4) Terminal residues were: α-L- Rhap; and α- L-Araf.

445

Figure 3A

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450 451 452

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Figure 3B

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456

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457 458 459 22

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460 461 462

Figure 3C

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465

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466 467

Figure 3: 1H NMR spectra examined at 25℃ for: (A) the original ZG (ZG-25); the

468

hydrolyzed polysaccharides at (B) 80℃ (ZG-H1-25), and (C) 100 ℃ (ZG-H2-25).

23

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469 470 471

Figure 4A

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475 476 477 24

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478 479

Figure 4B

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483 484

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485

Figure 4: 1H NMR spectra examined at 50 ℃ for the hydrolyzed polysaccharides

486

at: (A) 80 ℃ (ZG-H1-50); and (B) 100 ℃ (ZG-H2-50)

487

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Figure 5A

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Figure 5B

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492

Figure 5:

CNMR spectra (100 MHz, D2O, 25℃), for: (A) the original zedo (ZG-25) (above

493

spectrum); and (B) for the hydrolyzed sample, ZG-H2-25 (below spectrum).

494 495

26

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496 497 498

Figure 6

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Figure 6: 13CNMR spectrum (100 MHz, D2O, 25℃), for the hydrolyzed sample, ZG-H1-25.

503 27

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504 505

4. Conclusions

507

Chemical composition of the zedo gum, ZG, isolated from a plant material was: 9.42% moisture,

508

2.23% ash, 1.05% protein, and a negligible amount of lipid (0.03%). These results suggest that

509

the major component of the gum is carbohydrate (87.3%). The amounts of carbon, hydrogen,

510

oxygen and nitrogen in the ZG were found to be: 38.5, 5.8, 35.3, and 0.3%, respectively. The

511

results of ZG analysis by GC-MS showed that the polysaccharide consisted of arabinose

512

(25.5%), galactose (22.1%), rhamnose (9.0%), mannose (11.5%) fructose (10.8%), β-D-glucose

513

5.2%, galacturonic acid (5.9%) and glucuronic acid (3.9%). However, conversion of glucose into

514

fructose probably occurred, because the temperature of the analysis is high enough. The amounts

515

of total neutral sugars and uronic acids were 84.1 and 9.8%, respectively. Arabinose and

516

galactose residues were major components of the polysaccharide.

517

The gum was a highly branched polysaccharide and a member of arabinogalactans family. The

518

main chain contains α- L-arabinofuranosyl, α- L-Araf, and β-D-galactopyranonyl, β-D-Galp,

519

units. The following residues were identified: → 3,4) β-D-Galp (1→ ; → 3,4,6) β-D-Galp (1→;

520

→3)- α- L-Araf-(1→; → 4) β-D-GlcpA (1→.; glucosyl residues; and α-L- Rhap, (1→; and (4)

521

Terminal residues were: α-L- Rhap; and α- L-Araf. The structure of the polysaccharide was

522

found to be analogous with highly branched hydrocolloids such as arabic and ghatti gums.

523

The original gum having carboxylic groups can be naturally occurred as an anionic (carboxylate)

524

form, due to the presence of carboxylic acid groups and minerals/ salts. The presence of

525

carboxylic groups and minerals/ salts had a major influence on the rheological properties of the

526

gum. The gum partially exhibits poly-electrolyte behavior. A branched polysaccharide having

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partially anionic nature, generally produces a higher solution viscosity than that of other

528

branched polysaccharides having similar structures. The gum solutions did not follow Newtonian

529

flow behavior, where highly branched gums in solutions such as arabic gum at low

530

concentrations exhibit Newtonian behavior. The presence of protein in the zedo gum improves

531

emulsifying properties. Arabinogalactans such as zedo gum can be used in pharmaceutics and

532

food industries as a tablet binder and as an emulsifier for water-in-oil or oil-in-water emulsions.

533

Arabinogalactans-proteins enhance physico-chemical and functional properties in comparison to

534

polysaccharides and proteins alone.

535

Further investigations are needed to elucidate clearly the structure of the new gum as follows: (1)

536

structural investigation on protein moiety (amino acid composition, sequence analysis); (2)

537

whether the polysaccharide (s) is occurred alone or covalently linked to protein (s) and formed a

538

glycoprotein?; (3) determination of type of linkages between (s) protein (s) and polysaccharide

539

(s); and (4) determination of other components of gum (analysis of minerals, salts, metal ions,

540

and other impurities).

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Acknowledgement

544

The authors are grateful to Dr. Satar Saberi for his assistance in NMR spectroscopy. The

545

financial support of Sari Agricultural Sciences and Natural Resources University, and technical

546

support of department of chemistry of Ferdowsi and Mazandaran are gratefully acknowledged.

547 548

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624

2D-NMR spectroscopy studies of the polysaccharide gum from Spondias purpurea var. lutea.

625

Food Hydrocolloids, 19, 37–43.

626

Hager, B. L., & Berry, G. C. (1982). Moderately concentrated solutions of polystyrene. 1.

627

Viscosity as a function of concentration, temperature, and molecular weights. Journal of

628

Polymer Science, Polymer Physics, 20, 911-928.

629

Hård, K.; Zadelhoff, G. V.; Moonen, P.; Kamerling, J. P.; Vilegenthart, J. F. G. (1992). The Asn-

630

linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male: Novel sulfated

631

and novel N-acetyl-galactosamine-containing N-linked carbohydrate chains. European Journal

632

of Biochemistry, 209, 895–915.

633

Ibanez, M., & Ferrerob, C. (2003). Extraction and characterization of the hydrocolloid from

634

Prosopis flexuosa DC seeds. Food Research International. 36. 455–460.

635

Kang, J., Cui , S, W., Chen, J., Phillips, G, O., Wu, Y., & Wang, Q. (2011a). New studies on

636

gum ghatti (Anogeissus latifolia) part I. Fractionation, chemical and physical characterization of

637

the gum. Food Hydrocolloids, 25, 1984-1990.

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33

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Kang, J., Cui, S. W., Phillips, G. O., Chen, J., Guo, Q., & Wang, Q. (2011b). New studies on

639

gum ghatti (Anogeissus latifolia) part II. Structure characterization of an arabinogalactan from

640

the gum by 1D, 2D NMR spectroscopy and methylation analysis. Food Hydrocolloids, 25, 1991-

641

1998.

642

Kang, J., Cui, S. W., Phillips, G. O., Chen, J., Guo, Q., & Wang, Q. (2011c). New studies on

643

gum ghatti (Anogeissus latifolia) part III: structure characterization of a globular polysaccharide

644

fraction by 1D, 2D NMR spectroscopy and methylation analysis. Food Hydrocolloids, 25, 1999-

645

2007.

646

Kasaai, M. R. (2011). The use of various types of NMR and IR spectroscopy for structural

647

characterization of chitin and chitosan, In: Se-Kwon Kim, Ed., Chitin, Chitosan,

648

Oligosaccharides and Their Derivatives: Biological Activities and Applications, (pp. 159-170),

649

Boca Raton: Taylor & Francis Group.

650

Kasaai, M.R. (2010). Determination of the degree of N-acetylation for chitin and chitosan by

651

various NMR spectroscopy techniques: A review, Carbohydrate Polymers, 79, 801-808.

652

Kasaai, M.R., G. Charlet, G., & Arul. J. (2000). Master curve for concentration dependence in

653

semi-dilute solutions of chitosan homologues: Martin equation. Food Research International, 33,

654

63-67.

655

Kashki, M, T., Amirabadizadeh, H. (2011). Approach to plant communities in desertregions of

656

Khorasan province in Iran, 2, 42–46.

657

Larrazabal, M., Martınez, M., Sanabria, L, de Pinto, G.L., & Herrera, J. (2006) Structural

658

elucidation of the polysaccharide from Sterculia apetala gum by a combination of chemical

659

methods and NMR spectroscopy. Food Hydrocolloids, 20, 908–913.

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EP

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SC

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638

34

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Liu, J., Willfor, S., & Xu, C. (2015). A review of bioactive plant polysaccharides: Biological

661

activities, functionalization, and biomedical applications. Bioactive Carbohydrates and Dietary

662

Fibre, 5, 31-61.

663

MacGregor, E.A. (2002). Biopolymers. In: Encyclopedia of Physical Science and Technology,

664

R.A. Meyers, Ed., 3rd Ed., New York: Academic Press.

665

Mandal, S., Sarkar, R., Patra, P., Nandan, C. K., Das, D., Bhanja, S. K., and Islam, S.S. (2009).

666

Structural studies of a heteropolysaccharide (PS-I) isolated from hot water extract of fruits of

667

Psidium guajava (Guava). Carbohydrate Research, 344 (11), 1365-1370.

668

Ngo, D.H., & Kim, S.K. (2013).Sulfated polysaccharides as bioactive agents from marine algae.

669

International Journal of Biological Macromolecules, 62, 70–75.

670

Nie, S, P., Cui, S, W., Phillips, A.O., Xie, M,-Y., Phillips, G, O. Al-Assaf, S., Zhang, X-L.

671

(2011). Elucidation of the structure of a bioactive hydrophilic polysaccharide from Cordyceps

672

Sinensis by methylation analysis and NMR spectroscopy. Carbohydrate polymers, 84, 894-899.

673

Nie, S, P., Wang, C., Cui, S, W., Wang, Q., Xie, M, Y., & Phillips, G, O. (2013a). The core

674

carbohydrate structure of Acacia seyal var. seyal (Gum arabic), Food Hydrocolloids, 32, 221-

675

227.

676

Nie, S, P., Wang, C., Cui, S, W., Wang, Q., Xie, M, Y., & Phillips, G, O. (2013b). A further

677

amendment to the classical core structure of gum arabic (Acacia senegal), Food Hydrocolloids,

678

31, 42-48.

679

Nothnagel, E.A. (1997). Proteoglycans and related components in plant cells. International

680

Review of Cytology, 174, 195–291.

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M AN U

SC

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660

35

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681

OʾNeill , M. A., & York, W.S. (2003). The composition and structure of plant primary cell walls.

682

In: The plant cell wall: Annual Plant Reviews, J.K. Rose, Ed., (Vol. 8, pp. 1- 54), Carlton

683

(Victoria, Australia): Blackwell Publishing.

684

Ouellette, R, J., &

685

Mechanism, and Synthesis, (pp. 907-951.), Amsterdam: Elsevier.

686

Polle, A. Ya., Ovodova, R. G., Shashkov, A. S., & Ovodov, Yu. S. (2002). Some structural

687

features of pectic polysaccharide from tansy, Tanacetum vulgare L. Carbohydrate Polymers, 49,

688

337–344.

689

Popescu, C. M., Larsson, P. T., Olaru, N., & Vasile, C. (2012). Spectroscopic study of acetylated

690

kraft pulp fibers. Carbohydrate Polymers, 88, 530–536.

691

Roy, S. K., Chandra, K., Ghosh, K., Mondal, S., Maiti, D., Ojha, A. K., et al. (2007). Structural

692

investigation of a heteropolysaccharide isolated from the pods (fruits) of Moringa oleifera

693

(Sajina). Carbohydrate Research, 342, 2380-2389.

694

Saeidy, S., Nasirpour, A., Keramat, J., Desbrières, J., Le Cerf, D., Pierre, G., Delattre, C.,

695

Laroche, C., De Baynast, H., Ursu, A-V., Marcati, A., Djelveh, G., & Michaud, P. (2018).

696

Structural characterization and thermal behavior of a gum extracted from Ferula assa foetida L.

697

Carbohydrate Polymers, 181, 426-432.

698

Showalter. A.M. (1993). Structure and function of plant cell wall proteins. The Plant Cell 5(1),

699

9–23 (doi:10.1105/tpc.5.1.9).

700

Sims, I.M., & Furneaux, R.H. (2003). Structure of the extudates gum from Meryta sinclairii.

701

Carbohydrate Polymers. 52, 423-431.

AC C

EP

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SC

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Rawn, J. D. (2014). Carbohydrates Organic Chemistry: Structure,

36

ACCEPTED MANUSCRIPT

Stephen, A.M., & Merrifield, E.H. (2005). Carbohydrates. In: Encyclopedia of Analytical

703

Science. P. Worsfold, A., Townshend, A., & C. Poole, (Vol. 1, pp. 392- 414), Elsevier:

704

Amsterdam.

705

Stylianopoulos. C. (2013). Carbohydrates: Chemistry and Classification. In: Reference Module

706

in Biomedical Sciences Encyclopedia of Human Nutrition, 3rd Ed., (pp. 265-275). Amsterdam:

707

Elsevier.

708

Tabatabaee Amid, B., Mirhosseini, H. (2012). Emulsifying Activity, Particle Uniformity and

709

Rheological Properties of a Natural Polysaccharide-Protein Biopolymer from Durian Seed.

710

Food Biophysics, 7( 4), 317–328.

711

Tao, F., Biao, G, Z., Yu, J, Z., & Ning, Z, H. (2008). Isolation and characterization of an acidic

712

polysaccharide from Mesona Blumes gum. Carbohydrate Polymers, 71, 159–169.

713

Varki, A.(1993). Biological roles of oligosaccharides: All of the theories are correct.

714

Glycobiology, 3 (2), 97–130.

715

Vinod, V. T. P., Sashidhar, R. B., Sarma, V. U. M., & Vijaya Saradhi, U. V. R.(2008).

716

Compositional analysis and rheological properties of gum Kondagogu (Cochlospermum

717

gossypium): A tree gum from India. Journal of Agricultural and Food Chemistry, 56, 2199–

718

2207.

719

Vinogradov, G.V., Markin, A.Y. (1980). Rheology of Polymers, Moscow: Mir Publisher

720

Wu, M., Wu, Y., Zhou, J., & Pan, Y. (2005). Structural characterization of a water-soluble

721

polysaccharide with high branches from the leaves of Taxus Chinensis var. mairei. Food

722

Chemistry, 113, 1020-1024.

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SC

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702

37

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Xiao, Z, Tappen, B.R, Ly M, Zhao W, Canova, L.P, Guan, H, & Linhardt, R.J. (2011). Heparin

724

mapping using heparin lyases and the generation of a novel low molecular weight heparin.

725

Journal of Medicinal Chemistry, 54, 603– 610.

726

Yebeyen, D., Lemenih, M. & Feleke, S. (2009). Characteristics and quality of gum arabic from

727

naturally grown Acacia senegal (Linne) Wild trees in the central Rift Valley of Ethiopia. Food

728

Hydrocolloids, 23, 175–180.

729

Yin, J, Y., Lin, H, X., Nie, S, P., Cui, S, W., & Xie, M, Y. (2012a). Methylation and 2D NMR

730

analysis of arabinoxylan from the seeds of Plantago asiatica L. Carbohydrate Polymers, 88,

731

1395– 1401.

732

Yin, J., Lin, H., Li, J., Wang, Y., Cui, S, W., Nie, S., & Xie, M. (2012b). Structural

733

characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

734

Carbohydrate Polymers, 87, 2416– 2424.

735

Yudovin-Farber, I, Azzam, T, Metzer, E, Taraboulos, A, & Domb, A.J. (2005). Cationic

736

polysaccharides as antiprion agents. Journal of Medicinal Chemistry, 48, 1414–1420.

739

Figure S1

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Figure S1: 1H NMR spectrum examine at 50℃ for the original zedo (ZG-50).

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TE D

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EP

Nie, S, P., Cui, S, W., Phillips, A.O., Xie, M,-Y., Phillips, G, O. Al-Assaf, S., Zhang, X-L. (2011). Elucidation of the structure of a bioactive hydrophilic polysaccharide from Cordyceps

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Nie, S, P., Wang, C., Cui, S, W., Wang, Q., Xie, M, Y., & Phillips, G, O. (2013b). A further amendment to the classical core structure of gum arabic (Acacia senegal), Food Hydrocolloids, 31, 42-48.

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TE D

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EP

Roy, S. K., Chandra, K., Ghosh, K., Mondal, S., Maiti, D., Ojha, A. K., et al. (2007). Structural

AC C

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ACCEPTED MANUSCRIPT

Showalter. A.M. (1993). Structure and function of plant cell wall proteins. The Plant Cell 5(1), 9–23 (doi:10.1105/tpc.5.1.9). Sims, I.M., & Furneaux, R.H. (2003). Structure of the extudates gum from Meryta sinclairii.

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Food Biophysics, 7( 4), 317–328.

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EP

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AC C

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naturally grown Acacia senegal (Linne) Wild trees in the central Rift Valley of Ethiopia. Food

Yin, J, Y., Lin, H, X., Nie, S, P., Cui, S, W., & Xie, M, Y. (2012a). Methylation and 2D NMR analysis of arabinoxylan from the seeds of Plantago asiatica L. Carbohydrate Polymers, 88, 1395– 1401.

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Yin, J., Lin, H., Li, J., Wang, Y., Cui, S, W., Nie, S., & Xie, M. (2012b). Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L. Carbohydrate Polymers, 87, 2416– 2424.

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Yudovin-Farber, I, Azzam, T, Metzer, E, Taraboulos, A, & Domb, A.J. (2005). Cationic

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polysaccharides as antiprion agents. Journal of Medicinal Chemistry, 48, 1414–1420.

ACCEPTED MANUSCRIPT

> Zedo gum exudates from a tree, Amygdalus scoparia Spach, of Rosaceae family. > Based on chemical analysis, zedo contains a high amount of carbohydrates.

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> Galactose and arabinose units are the major units of the polysaccharide.

> The gum is a branched polysaccharide and a member of arabinogalactan family.

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> Zedo consists of a backbone,β-D-galactopyranosyl units and several branches.