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Polysaccharides modification through green technology: Role of endodextranase towards improving physicochemical properties of (1-3)(1-6)-?-D-glucans Chao Huang, Ming Miao, Srinivas Janaswamy, Bruce R. Hamaker, Xingfeng Li, and Bo Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00472 • Publication Date (Web): 02 Jul 2015 Downloaded from http://pubs.acs.org on July 5, 2015

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Journal of Agricultural and Food Chemistry

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Polysaccharides modification through green technology: Role of endodextranase

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towards improving physicochemical properties of (1→3)(1→6)-α-D-glucan

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Chao Huanga, Ming Miaoa,*, Srinivas Janaswamy a,b, Bruce R. Hamaker a,b, Xingfeng

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Lic, Bo Jianga,*

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a

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of Food Safety and Nutrition, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu

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214122, P. R. China

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b

State Key Laboratory of Food Science & Technology, Synergetic Innovation Center

Whistler Center for Carbohydrate Research, Department of Food Science, Purdue

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University, 745 Agriculture Mall Drive, West Lafayette, IN 47907-2009, USA

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c

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Technology, No.70 Yuhuadonglu, Shijiazhuang, Hebei 050018, P.R. China

College of Bioscience and Bioengineering, Hebei University of Science and

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*

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Technology, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, P. R. China. Tel: +86 (0)510

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853 27859; Fax: +86 (0)510 859 19161.

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E-mail address: [email protected] (M. Miao)

Corresponding author. Address: State Key Laboratory of Food Science &

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ACS Paragon Plus Environment


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ABSTRACT

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The structure and properties of bioengineered (1→3)(1→6)-α-D-glucan subjected to

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endodextranase treatment were investigated. Upon enzyme treatment, OD220 and Mw

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decreased substantially during the first 60 min, and thereafter slowed down as the

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modification progressed. Compared to the native glucan, the modified sample solution

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had a lighter opalescent, bluish-white color. The morphological analysis revealed that

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bioengineered glucan produced quite a few little particles after hydrolysis. The

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molecular weight distribution curve gradually shifted to the low Mw region with a

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significant broadening distribution, and the chain hydrolysis reaction followed a

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combination of 0th- and 1th-order processes. The NMR results showed some specific

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α-1,6 linkages of glucan chains were cleaved with enzyme treatment. The viscosity of

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modified glucan solution was markedly reduced and the Newtonian plateaus were also

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observed at the high shear rates (10-100 1/s). The above results suggested that the

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modified (1→3)(1→6)-α-D-glucan showed a tailor-made solution character similar as

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Arabic gum and would be used as a novel food gum substitute to design the

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artificially carbohydrate-based foods.

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KEYWORDS: α-D-glucan; branched structure; enzymatic hydrolysis; kinetic model; molecular weight; rheology

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INTRODUCTION

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In recent years, the increased demand for natural biopolymers with significant

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commercial value has led to a renewed interest in bacterial exopolysaccharides.1-6

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Many microorganisms synthesize extracellular polysaccharides that are primarily

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involved in cell adhesion and protection. These biopolymers can remain attached to

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the cell walls in the form of capsules or to be secreted as unbound “slime” materials.1

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Moreover, most of exopolysaccharides display wide range of biological functions,

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such as antioxidant, immunological and prebiotic properties.5 In general, they can be

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classified into two groups: homopolysaccharides contain a single type of sugar

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monomers (e.g. glucose or fructose), whereas heteropolysaccharides have a

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combinations of sugars (e.g. glucose, galactose, fructose and rhamnose). Due to their

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structural and functional diversity, considerable efforts are being pursued towards

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elucidating the composition, structure, biosynthesis pathway and functional properties.

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For example, xanthan, gellan, dextran and hyaluronan are the successful examples in

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food, pharmaceutics and biomedical industrial applications.1 Meanwhile, these are

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also high molecular weight exopolysaccharides (> 106 Da) with poor solubility, high

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viscosity or unstable physicochemical properties, which in-turn limits their

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widespread potential utility.3,5,7 Thus, there is a pressing need for modifying the

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functional properties of bacterial exopolysaccharides, especially for taking advantage

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of their versatile functionalities in the design and development of novel food

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products.

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Physical, chemical, and enzymatic modification are the common approaches

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employed to tailor the polysaccharide properties in gaining the desired attributes

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including solubility, viscosity, emulsification and digestibility.7,8-11 Among them,

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enzymatic treatment has gained wider acceptability as it proceeds in milder conditions

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and the reactions are highly controllable with few undesirable by-products.9,12

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Endodextranase (1, 6-α-D-glucan 6-glucanohydrolase, EC 3.2.1.11) is an endo-acting

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hydrolase that specifically cleaves α-1, 6 glycosidic linkages in dextran polymers, and

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results in shorter isomaltosaccharides.13,14 Currently, endodextranase is routinely used

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to reduce the viscosity for dextran contamination in the sugar-processing industry.15

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However, its potential in polysaccharide processing is less explored, to the best of our

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knowledge.

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In our previous study, a water-soluble extracellular polysaccharide was obtained

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from Leuconostoc citreum SK24.002. Based on the NMR results, we deduced this

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exopolysaccharide consists of a backbone chain of alternating α-1,3 and α-1,6

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linkages with a branched point at the C6 of the 1,3,6-linked D-glucopyranose unit 5.

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The molecular weight of this glucan was 4.62×107 Da with relatively high viscosity

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as well as poor solubility at a concentration of 10% (w/v) or higher 16. In this work,

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the above α-D-glucan has been chosen as a model system to elucidate the advantage

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of endodextranase for modifying the structural and physicochemical properties. From

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the data, the mechanism of chain hydrolysis reaction and solution properties have

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been revealed, which could lead to insights into the fundamental basis for

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structure-function relationship and develop potential industrial applications as a low

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viscosity and soluble filler ingredient for designing novel carbohydrate-based foods.

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MATERIALS AND METHODS

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Materials. Endodextranase (Cat. No. D0443, 500 U/mL) from Chaetomium

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erraticum, polyethyleneglycol (average Mw 2,000) and deuterium oxide (isotopic

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purity 99.99 atom % D) were purchased from Sigma-Aldrich Co. (St. Louis, MO,

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USA). Arabic gum was kindly provided by Kerry Group (Shanghai, China). All other

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chemicals were reagent grade and were obtained from Sinopharm Chemical Reagent

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Co., Ltd. (Shanghai, China).

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Leuconostoc citreum SK24.002, with high alternansucrase-producing ability, was

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obtained from Chinese traditional pickled vegetables. Man-Rogosa-Sharpe (MRS)

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medium, comprised of 40 g sucrose, 10 g yeast extract, 10 mL tween 80, 20 g

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K2HPO4, 0.02 g CaCl2, 0.2 g MgSO4·7H2O, 0.01 g NaCl, 0.01 g MnSO4·H2O, 0.01 g

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FeSO4·7H2O per liter (pH 6.9), was used for alternansucrase production.5

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Method. (1→3)(1→6)-α-D-glucan biosynthesis. Leuconostoc citreum SK24.002

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was propagated in liquid MRS medium with shaking (160 r/min) at 30 oC for 48 h.

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The cells and other insoluble components were removed from medium by

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centrifugation. Two-phase partition (water- polyethyleneglycol) was used to extract

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the soluble alternansucrase from the supernatants, taking advantage of the

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dextranosyl-enzyme complex form in which the enzyme would be present in enriched

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precipitate. The harvested alternansucrase was washed and dissolved in sodium

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acetate buffer (50 mM, pH 4.5) for further measurements and the activity was

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determined by measuring the initial rate of fructose production using the

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3,5-dinitrosalicylic acid method.17

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The alternansucrase solution was supplemented with 20% (w/w) sucrose in a

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final volume of 2 liter. The biosynthesis of water soluble α-D-glucan was carried out

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at 40 °C with 1 U/mL of enzyme for 48 h. The polysaccharide was then recovered

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from the precipitate by adding one volume of ethanol at 4 °C followed by

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centrifugation at 10,000 g for 10 min. The precipitate was resuspended in the

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deioinised water and the above procedure was repeated for three times, and in the end

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with 50% (v/v) ethanol precipitation to discard mono- and oligosaccharides, if any.

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The final product in supernatant was freeze-dried for further use.

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Enzymatic modification of (1→3)(1→6)-α-D-glucan. One gram of glucan was

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dissolved in 20 mL of sodium acetate buffer solution (pH 4.5, 50 mM). The

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temperature was adjusted to 55 °C and the endodextranase (0.2 mL) was added. The

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process with six different flasks was carried out for 10 min, 30 min, 1 h, 2 h, 4 h or 8

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h, respectively. Subsequently, the reaction was stopped by heating the solution in the

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boiling water for 30 min, cooled to room temperature and precipitated in 3 volumes of

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90% ethanol (v/v). The material was centrifuged at 5,000 g for 10 min, resuspended in

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ethanol, filtrated twice and dried. It was then ground to fine powder (approximately

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100 mesh) and stored in a desiccator for further analysis. These samples were referred

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as DG1, DG2, DG3, DG4, DG5, and DG6, respectively, for brevity, in the rest of the

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manuscript.

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UV absorption analysis. The method described by Kobayashi, Utsugi and

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Matsuda18 was adapted to measure the polysaccharide solution absorption using the

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UV/Visible Spectrophotometer (UV-2102PC, Unico Instrument Co., Ltd., Shanghai,

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China). The glucan exhibited a strong absorption at 220 nm, and thus the optical

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density (OD220) was measured at 220 nm for estimating the extent of structural

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modification.

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Solution appearance analysis. The glucan sample was dissolved in water with a

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concentration of 6% (w/v) at room temperature. The solution was stirred for 30 min

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until the polysaccharide was completely dissolved and then was transferred to a glass

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tube. The appearance of glucan solution was recorded using a digital camera (Canon,

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Shanghai, China).

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Scanning electron microscopy (SEM). The surface morphology was examined by

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a Quanta-200 scanning electron microscopy (FEI company, Eindhoven, The

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Netherlands). The dried sample was coated with a thin gold film (10 nm) and mounted

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on an aluminum stub using a double-sided stick tape. The instrument was operated at

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an accelerating voltage of 5.0 kV.

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Molecular weight distribution (MWD) analysis. The sample (0.5 mg/mL) was

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passed through a cellulose acetate filter (0.45 µm, Whatman, Maidstone, UK) and

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injected into a high-performance size-exclusion chromatography (HPSEC) system

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coupled with the DAWN HELEOS-II multi-angle laser light scattering detector

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(MALLS) and an Optilab® T-rEX refractive-index detector (RI, Wyatt Technology,

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Santa Barbara, CA, USA). The MALLS was equipped with the He-Ne Laser of 658.0

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nm. A Shodex OH-pak SB-806 HQ column (8 × 300 mm, Showa Denko K.K., Tokyo,

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Japan) with an OH-pak SB-G guard column was used at 25 °C. The mobile phase was

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0.1 M NaNO3 along with 0.02% of sodium azide, and a flow rate of 0.5 mL/min was

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used. The data were processed with the Wyatt Astra software (Version 5.3.4.14,

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Wyatt Technology, USA).

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Spectroscopic characterization. The FT-IR spectrum was recorded using the

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Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Co., Waltham, MA, USA)

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at room temperature. The polysaccharide powder was blended with KBr at a ratio of

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1:100 and pressed into tablets. The scanning was carried out in the region 400-4,000

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cm-1 at 4 cm-1 resolution using 32 scans.

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The 1H NMR spectrum was recorded at 80 °C with an AVANCE III 400 MHz

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Digital NMR spectrometer (Brucker Co., Billerica, MA, USA). The glucan sample

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(approximately 60 mg) was exchanged with deuterium by lyophilising it three times

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with deuterium oxide and then dissolving it in 0.45 mL of 99.99% deuterium oxide.

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Chemical shifts (δ) were expressed in ppm and referenced internally with acetone (δH

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2.225).

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Solution properties. The viscosity was measured at 25 °C using the Brookfield

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digital viscometer (type DV-II+ Pro, Brookfield Engineering Labs, Inc., MA, USA),

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with a RV3 spindle. The shear thinning was analysed using the stress-controlled

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rheometer (AR-G2, TA Instrument, DE, USA). The instrument was equipped with a

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stainless steel cone of 40 mm diameter and 2° angle along with the solvent trap to

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minimize water evaporation during the analysis. The temperature was maintained at

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25 °C using a circulating bath and controlled peltier system. The apparent viscosity of

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the polysaccharide was estimated by using a range of shear rates of 0.01-100 1/s.

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Statistical analysis. Data were analyzed using one-way analysis of variance

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(ANOVA) procedure using the Origin 8.5 (OriginLab Inc., USA). A level of 0.05 was

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set to determine statistical significance. The results were expressed as the mean value

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± standard deviation.

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RESULTS AND DISCUSSION

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Optical density. According to Kobayashi, the UV absorbance profile of glucan

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solution exhibits a strong intensity at 220 nm, which reveals a quantitative

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determination method for amount of high Mw glucan without any pre-treatments or

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reagents.18 The OD220 values of the glucan solution as a function of time of enzyeme

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treatment is shown in Table 1. The bioengineered glucan solution exhibits a value of

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2.52. Upon endodextranase hydrolysis, the OD220 decreased substantially to 1.51

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during the first 60 min and then incrementally reached to a final limiting OD220 of

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1.03 after 480 min or more time. Moreover, the observations of glucan solution

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appearance also confirmed the structural change, in which the control sample solution

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has a more opalescent, bluish-white color than the modified one, especially for DG 6

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(see the Figure S1 in Supplementary Material). The larger the molecular size, the

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deeper were the opalescent color of glucan solution, revealing that the enzyme

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treatment affected the depth of solution color. A similar trend was also observed for

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alternan from Leuconostoc mesenteroides strain NRRLB-21297.19 They also reported

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that a rapid reduction of UV absorption at 225 nm after adding 5 U/mL or greater

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enzyme solution. Overall, these results clearly highlight the significant role of enzyme

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treatment in reducing the Mw of polysaccharides. For this bioengineered

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(1→3)(1→6)-α-D-glucan, some α-1, 6 linkages were speculated to be randomly

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hydrolyzed per endodextranase attack, resulting in a reduction of higher Mw

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fragment.

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Surface morphology. The scanning electron microscope is a potent tool to

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elucidate the surface morphology of polymer for predicting its physical properties as

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suggested by Wang and coworkers.20 Figure 1 portrays the surface morphologies of

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the (1→3)(1→6)-α-D-glucan after enzyme treatment. The native glucan appears like a

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cementitious material with a web-like network. The strong attractions between the

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surface functional groups appear to result in the aggregation of the glucan chains. This

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type of network arrangement also prevails in the purified exopolysaccharide made

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from Leuconostoc dextranicum NRRL B-114621 and Lactobacillus plantarum KF520.

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After enzyme hydrolysis for 480 min, the modified glucan (DG6) had a looser

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structure with considerable small fragments. These results suggest that the

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bioengineered glucan after endodextranse modification might produce some particles

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with smaller size, in accord with the results of molecular weight analysis.

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Molecular weight distribution. The MWD of the (1→3)(1→6)-α-D-glucan was

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measured by using HPSEC-MALLS-RI and the related parameters are given in Table

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1. The Figure 2(A) highlights the HPSEC chromatogram of the native glucan

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appeared as a single symmetrical peak (2.41×107 g/mol), indicating that the

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bioengineered glucan was a homogeneous polysaccharide as suggested by our

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previous study.5 Upon enzymatic hydrolysis of native glucan, the distribution

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gradually shifted to the smaller MWD region with a significant broadening. In

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addition, a low Mw peak (3.02×105 g/mol) appeared after the modification, for

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example in DG6, indicating the heterodisperse characteristic of the treated sample.

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Leathers, Nunnally and Côté also found the similar behavior in alternan wherein the

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high peak of 106 -107 Da in the bioengineered polysaccharide moves to peaks of 5-10

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×105 Da and 1-5 ×104 Da after modification.22 Simultaneously, the peak (1-5 ×104 Da)

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did not change over time and only its relative proportion increased as the process took

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a long time up to 24 h. Moreover, Tayal, Kelly and Khan23 observed significant

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reduction in molecular weight during the course of the enzymatic degradation of a

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water-soluble guar, with the peak maximum shifting by approximate 2 orders of

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magnitude, reflecting the scission of backbone linkage by enzyme, which was

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comparable with our results.

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As shown in Table 1, the weight-average Mw decreased sharply from 24.1 ×106

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(control) to 10.2 ×106 g/mol (DG3), and thereafter slowed down and finally settled at

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7.5 ×106 g/mol at 480 min (DG6). This trend is in congruence with the OD220 decrease.

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In particular, a considerable reduction in Mw (approximately 60% Mw reduction)

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took place at the initial short time, indicating enzymatic hydrolysis of the

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(1→3)(1→6)-α-D-glucan was a complex reaction where the mechanism analysis

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could be a combination of two or more reactions with diverse orders as described by

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Tayal et al.23 In order to understand the degradation kinetics of enzymatic hydrolysis,

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the inverse Mw (1/Mw) as a function of process time has been analyzed and

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highlighted in Figure 2 (B). A non-linear relationship between 1/Mw and time was

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observed for enzymatic hydrolysis of either 5.0% or 2.5% (w/v) α-D-glucan solution.

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The slope of 1/Mw vs time decreased as glucan concentration increased from 2.5% to

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5.0%, which indicated that the apparent rate constant was depended on the initial

236

concentration and hydrolysis reaction appeared an nth-order.

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According to Basedow, Ebert and Ederer, the degradation reaction using enzyme

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belongs to an nth-order process: dB/dt =-kBn, wherein B is the total number of

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hydrolysable linkages, k is the rate constant, and n is the order of reaction.24 Our data

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were rationalized as a combination of 0th- and 1th-order processes, where the 0th-order

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reaction preceded initially for time t’, followed by a 1th-order reaction up to time t.

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The two kinetic equations:

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1 1 k t' − = ' M t ' M 0 mN 0

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and

(1)

k 1 1 - ' = 1 (t - t ' ) M t M t' m

(2)

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were used to calculate the experimental kinetics of enzymatic hydrolysis, where M0,

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Mt’, and Mt were the Mws at time 0, t’ and t, respectively, k and k1 were the rate

247

constants for 0th- and 1th-order reactions, respectively, m was Mw of the monomer

248

(glucose) and N0 was the total number of molecules. The data in Figure 2(C) were

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modeled as a combination of two reactions using the above equations (1) and (2). The

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analysis indicates the correlation coefficient (R2) and rate constant (k) of 0.858,

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4.91×10-3 g/(mol·min) and 0.989, 6.22×10-4 g/(mol·min) for 0th- and 1th-order

252

reactions of 5.0 % glucan solution, respectively. In some previous reports, a linear

253

relationship between the 1/Mw and time was used to model the enzymatic degradation

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of natural polymer.25-27 However, our data indicated 0th- and 1st-order kinetics were

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the better representations.

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FT-IR analysis. The FT-IR spectra of the native glucan and its derivatives are

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compared in Figure 3. The control and enzyme treated samples essentially displayed

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similar spectra, indicating the intact chemical glucan structure and non-destructive

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nature of the enzyme treatment. In both the samples, characteristic bands of ν(O-H)

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3,400 cm-1, ν(C-H) 2,930 cm-1, δ(HOH) 1,640 cm-1, δ(O-H) 1,420 cm-1, δ(C-H) 1,370

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cm-1, a complex band ν(C-O) and ν(C-C) 1,200-1,000 cm-1, and γ(C-H) 1,000-700

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cm-1 are observed. The broad peak at 3,393 cm-1, indicate the presence of hydroxyl

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groups of glucan and water. The bands at 2,929 and 1,636 cm-1 are due to the C-H

264

stretching and water-bending, respectively. The spectra also indicate C-H deformation

265

at 1,415 and 1,347 cm-1. In the fingerprint region (1,200-1,000 cm-1), there are three

266

characteristic peaks at 1,150, 1,080 and 1,024 cm-1. The wavenumbers 1,150 and

267

1,080 cm-1 are attributed to the C-O stretching of the anhydroglucose. The sharp peak

268

at 1024 cm-1 could most likely be due the C-O stretching in the C-O-C linkage across

269

the α-1,6-glucosidic bonds. The vibration bands at around 926, 843, 793 cm-1 are

270

attributed to the mixed C-C-H deformations coupled with C-C-O, O-C-O and C-O-C

271

bending. The peak at 793 cm-1 is from the skeletal mode vibrations of the

272

α-1,3-glucosidic linkage.28 All these results clearly suggest that both the control and

273

enzyme treated glucans exhibit similar chemical structure comprised of both α-1,3 and

274

α-1,6 linkages. The similarities in the γ(C-H) range further suggest that there are no

275

differences in the D-glucopyranose conformation that prefer 4C1 chair conformation.6

276

NMR analysis. The linkage changes in the (1→3)(1→6)-α-D-glucan after

277

enzyme treatment are compared in Figure 4 and the related data are presented in

13



278

Table 1, respectively. In the anomeric proton region (4.5-5.4 ppm), the peaks at 4.98

279

and 5.32 ppm indicate the H-1 at α-1,6 and α-1,3 linkage, respectively.29 The

280

percentage of α-1,6 linkages in the control sample was approximately 60%, and agree

281

with previous observations.5 As expected, after enzyme treatment this number

282

decreased to 56.3% of DG6 suggesting that the chain fragments with α-1,6 linkages

283

are broken partially.

284

The main domain of endodextranase is a right-handed parallel β helix connecting

285

to a sandwich domain at the N terminus.14 In the enzyme-glucan complex, the

286

glycosidic oxygen of the glucose unit from the subsite +1 forms a hydrogen bond to

287

the catalytic active site (Asp395) through a single displacement mechanism. Generally

288

glucans from Leuconostoc citreum SK24.002 are considered to be resistant to the

289

enzyme action.5 However, the bioengineered glucan used in this study displayed some

290

short stretches of consecutive α-1,6 linkages in the limited regions, which could be

291

primary factor for the observed structural changes. Overall, it can be concluded that

292

the enzymatic treatment of (1→3)(1→6)-α-D-glucan is influenced by the unique

293

biopolymer structure, and some specific α-1,6 linkages of the glucan chains were

294

broken with enzyme treatment.

295

Rheological analysis. Figure 5 (A) shows the concentration dependence of

296

solution viscosities for the control and its enzymatic derivatives. The viscosity of

297

bioengineered glucan at 5 g/100 mL concentration is around 18 mPa·s, suggesting a

298

thin liquid for such a high Mw polymer. However, at the higher polymer

299

concentrations, in the range of 5-10 g/100 mL, the viscosity increased exponentially

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and finally formed a gel-like dispersion. On the other hand, the viscosity of enzyme

301

treated glucans has decreased considerably, mainly due to the chain degradation,

302

similar with solution behaviour of the commercial gum arabic (9%, w/v), which could

303

be dissolved in water to give solutions of 30 g/100 mL or greater.

304

The profiles of viscosity versus shear rate are shown in Figure 5 (B). The

305

bioengineered glucan displays a non-Newtonian pseudoplastic behavior (shear

306

thinning) in the shear rate range of 0.01-100 1/s, indicating a branched nature of

307

glucan as suggested by Majumder and Goyal.21 The viscosity decreased with

308

increasing the shear rate, but with minimal changes at high shear rates. A similar

309

behavior is noticed from glucans synthesised by Agrobacterium sp. ZX09.30 As

310

shown in Figure 5 (B), the gum Arabic (9%, w/v) showed a flat curve at the high

311

shear rates (10-100 1/s), indicating it more or less behaves like a Newtonian liquid.

312

Interestingly, Newtonian plateaus are also observed from the enzyme treated glucan

313

solutions. In general, polymer chains in solution could exhibit considerable stiffness

314

and thus making themselves more susceptible to orient under suitable shear

315

conditions.30 The shear thinning property observed in our measurements appears to be

316

the outcome of the orientation effect under shear. In other words, the long chains of

317

(1→3)(1→6)-α-D-glucan after enzymatic hydrolysis became aligned in the direction

318

of flow with increasing the shear rate, resulting in less interaction between adjacent

319

chains for modified glucan.

320

In summary, an environmental friendly and reproducible enzymatic modification

321

has been used to reduce the Mw and improve the solutions properties of the high Mw

15



322

(1→3)(1→6)-α-D-glucan from Leuconostoc citreum SK24.002. The enzymatic

323

degradation followed both 0th- and 1th-order kinetics. Some specific α-1,6 linkages of

324

glucan chains were cleaved with enzyme treatment. The viscosity of modified glucan

325

solution was markedly reduced and the Newtonian plateaus were also observed at the

326

high shear rates (10-100 1/s). The enzymatic modified glucans showed comparable

327

solution viscosity and shear thinning behaviour of commercial gum arabic, indicating

328

that the modified (1→3)(1→6)-α-D-glucan as a substitute of gum arabic could find

329

potential applications as low viscosity biomaterials and soluble fillers in the design

330

and development of novel carbohydrate-based foods. Optimising the processing

331

parameters for biosynthesis modification, glucan retrogradation during storage as well

332

as assessing the bioavailability and bioefficency of the modified glucans will be the

333

future study focuses.

334

Supporting Information. Figure S1 Comparison of aqueous solutions of native and

335

enzyme treated glucans. This material is available free of charge via the Internet at

336

http://pubs.acs.org.

337

Funding

338

The research was financially supported by the National Natural Science

339

Foundation of China (31000764, 31230057), International Cooperative Program of

340

Jiangsu Province (BZ2012031) and Science & Technology Pillar Program of Jiangsu

341

Province (BE2013647, BE2014703).

342

REFERENCES

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lactic acid bacteria and exopolysaccharide characterization. J. Agric. Food Chem.

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modification of maize starch for increasing slow digestion property. Food

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Hydrocolloid. 2014, 38, 180-185.

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from Penicillium minioluteum: Reaction course, crystal structure, and product

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complex. Structure 2003, 11, 1111-1121.

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of

a

high

molecular

weight

bioengineered

α-D-glucan

(18) Kobayashi, M.; Utsugi, H.; Matsuda, K. Intensive UV absorption of dextrans and its application to enzyme reactions. Agri. Biolog. Chem. 1986, 50, 1051-1053.

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of exopolysaccharide produced by Lactobacillus plantarum KF5 isolated from Tibet

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Kefir. Carbohyd. Polym. 2010, 82, 895-903.

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from Leuconostoc dextranicum NRRL B-1146. Food Res. Int. 2009, 42, 525-528.

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(22) Leathers, T.D.; Nunnally, M.S.; Côté, G.L. Optimization of process conditions for

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enzymatic modification of alternan using dextranase from Chaetomium erraticum.

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during enzymatic degradation of a water-soluble polymer. Macromolecules. 1999, 32,

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of dextran. Macromolecules, 1978, 11, 774-781.

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(25) Masson, C.R. The degradation of Carrageenan I. Kinetics in aqueous solution at

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pH 7. Can. J. Chem, 1955, 33, 597-603.

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(26) Vink, H. Degradation of some polymers in aqueous solutions. Die

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Makromolekulare Chemie. 1963, 67, 105-123.

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(27) Thoma, J.A. Models for depolymerizing enzymes. Application to α-amylases.

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Biopolymers. 1976, 15, 729-746.

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410

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correlation between structures of dextrans from strains of Leuconostoc mesenteroides

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and of D-glucans from strains of Streptococcus mutans. Carbohyd. Res. 1980, 86,

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(29) Seymour, F.R.; Knapp, R.D.; Bishop, S.H. Correlation of the structure of

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dextrans to their 1H-n.m.r. spectra. Carbohyd. Res. 1979, 74, 77-92.

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Salecan as a new source of thickening agent. Food Hydrocolloid. 2011, 25,

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1719-1725.

20


difference-spectrometry:

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Page 21 of 29


419

List of Table Legends

420

Table 1 UV-absorbance at 220 nm (OD220), molecular weight (Mw) and percentage of

421

α-1,6 linkages of the enzyme treated (1→3)(1→6)-α-D-glucan.

422

List of Figure Captions

423

Figure 1. Surface morphology of the enzyme treated (1→3)(1→6)-α-D-glucans.

424

Figure 2. Molecular weight distribution profiles of the native and enzyme treated

425

glucans for 5% solution(A), comparison of Mw-1 as a function of hydrolysis time for

426

5% and 2.5% solution (B) and (Mt-1-M0-1) as a function of hydrolysis time for 5%

427

solution (C).

428

Figure 3. FT-IR spectra of the enzyme treated (1→3)(1→6)-α-D-glucans.

429

Figure 4. 1H NMR spectra of the enzyme treated (1→3)(1→6)-α-D-glucans.

430

Figure 5. Comparison of the solutions properties of the enzyme treated glucans; (A)

431

viscosity and (B) apparent viscosity.

21



432

Table 1 UV-absorbance at 220 nm (OD220), molecular weight (Mw) and percentage of

433

α-1,6 linkages of the enzyme treated (1→3)(1→6)-α-D-glucan. OD220

434

Mw (106 g/mol)

Percentage of α-1,6 linkages (%)

Control 2.52±0.04 24.1±1.51 DG1 2.19±0.12 17.5±0.39 DG2 1.92±0.08 15.5±0.04 DG3 1.51±0.02 10.2±0.07 DG4 1.27±0.17 9.7±0.11 DG5 1.10±0.06 8.6±0.02 DG6 1.03±0.09 7.5±0.08 Mean ±standard deviations of triplicate analysis.

22


59.5±0.51 59.2±1.64 58.1±1.21 57.4±0.91 57.0±0.80 56.5±1.42 56.3±1.17

Page 22 of 29

Page 23 of 29


429

Figure 1 control

DG6

430

23



431

Page 24 of 29

Figure 2

A 7

2.41×10

DG1

Refractive Index (MV)

Control

DG6 5

3.02×10

16

18

20

22

24

26

28

Time (min) 432

1.5 B

(1/Mw)×10

7

1.2

0.9

0.6 5.0% 2.5%

0.3

0.0 0

100

200

300

Time (min) 433 434

24


400

500

30

Page 25 of 29


0.75 C

(1/Mt-1/M0)×10

7

0.60

0.45

0.30

0.15

0.00 0

100

200 300 Time (min)

435 436

25


400

500


Figure 3 1024.62

2.0

1.4

926.15

1347.69

1636.92

0.8

1415.38

1.0

2929.23

Absorbance

1.2

0.6

541.54

1150.77 1080.00

1.6

3393.85

1.8

843.08 793.85

437

Page 26 of 29

Control 0.4

DG1

0.2

DG6

0.0

3000

2000

1500 Wavenumbers (cm-1)

438

26


1000

500

Page 27 of 29


439

Figure 4

DG6

DG1

Control

440

27



441

Page 28 of 29

Figure 5

1000

Viscosity (mPa⋅s)

A

100

Control DG1 DG2 DG3 DG4 DG5 DG6 Gum arabic

10

1 0

5

10

15

20

25

30

Concentration (g/100mL) 442

10

B

Viscosity (Pa⋅s)

1

0.1

0.01

1E-3 1E-3

Control DG1 DG2 DG3 DG4 DG6 Gum arabic

0.01

0.1

1

Shear rate (1/s) 443

28


10

100

Page 29 of 29


TOC Graphic

29


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