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Characterization of Oat (Avena nuda L.) #Glucan Cryogelation Process by Low Field NMR Jia Wu, Linlin Li, Xiaoyan Wu, Qiaoling Dai, Ru Zhang, and Yi Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03948 • Publication Date (Web): 09 Dec 2015 Downloaded from http://pubs.acs.org on December 11, 2015

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Journal of Agricultural and Food Chemistry

Characterization of Oat (Avena nuda L.) β-Glucan Cryogelation Process by Low Field NMR Jia Wu*,†, Linlin Li†, Xiaoyan Wu†, Qiaoling Dai†, Ru Zhang†, Yi Zhang‡ †

College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian

350116, P. R. China ‡

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian

350002, P. R. China

Corresponding Author *Phone: +86 591 22866378. Fax: +86 591 22866378. E-mail: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT: Low field nuclear magnetic resonance (LF-NMR) is a useful method in studying

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the water distribution and mobility in heterogeneous systems. This technique was used to

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characterize water in oat β-glucan aqueous system during cryogelation by repeated freeze-thaw

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treatments. The results indicated that microphase separation occurred during cryogelation, and

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three water components were determined in the cryostructure. The spin-spin relaxation time was

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analyzed based on chemical exchange and diffusion exchange theory. The location of each water

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component was identified in the porous microstructure of the cryogel. The pore size measured

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from SEM image is in accordance with that estimated from relaxation time. The formation of

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cryogel is confirmed by rheological method. The results suggested that the cryogelation process

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of the polysaccharide could be monitored by LF-NMR through the evolution of spin-spin

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relaxation characteristics.

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KEYWORDS: oat, β-glucan, cryogelation, LF-NMR, water distribution, SEM, rheology

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INTRODUCTION

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Cereals such as oat, barley, rye, and wheat are good sources of β-glucan. Oat β-glucan is an

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important kind of soluble dietary fiber, which has received much attention for its function in

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lowering cholesterol,1 reducing the risk of type 2 diabetes,2,3 enhancing gut health,4 and

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improving immunity.5 It is a linear molecule composed of D-glucopyranosyl residues linked by

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single β-(1→3) and consecutive β-(1→4) glycosidic bonds, with 3-O-β-cellobiosyl-D-glucose

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(DP3) and 3-O-β-cellotriosyl-D-glucose (DP4) being the major fragments.6–8 It is considered that

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consecutive cellotriosyl fragments in β-glucan are responsible for the interaction between

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β-glucan molecules. These ordered structures are supposed to form stable junction zones causing

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aggregation of β-glucan molecules in aqueous solution.9,10 Longer cellulose-like segments

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probably also contribute to the interaction between β-glucan molecules and formation of gel

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structure. Lower content of DP3 or cellotriosyl units endows oat β-glucan with better solubility,

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solution stability, and physiological activity. Oat β-glucans extracted from different cultivars

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have molecular weights ranging from 60 × 103 to 3 × 106 g/mol. when oat β-glucan is dispersed

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in hot water with stirring, it could form a viscous solution. If the solution is set at room

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temperature for a period of time, it may form a hydrogel. Or the solution is treated with repeated

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freeze-thaw cycles, it may also gradually transform into a cryogel.8 The solution and hydrogel

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properties of oat β-glucans are influenced by molecular characteristics (concentration, molecular

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weight, and fine structure) and environmental factors (temperature, store time, and shear).11–14

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Due to the health benefit of oat β-glucan, it is increasingly used in food industry. Oat β-glucan

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can be utilized as thickening agent for its high viscosity in aqueous solution. Hydrogel prepared

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from oat β-glucan is capable of modifying the texture and sensory of food.15 Oat β-glucan gel is a

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good form to deliver the fiber to the gastrointestinal tract instead of β-glucan drinks since the 3



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latter cause a slimy mouth feel.14 Cereal β-glucan gel is a potential carrier capable of protecting

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bioactive substances from degradation and controlling the release of functional components.16

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Cryogelation is a physical phenomenon that occurs in many polymer solutions during

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freeze-thaw cycles. Synthesized polymers such as PVA, PEG, and polyacrylamide as well as

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natural polymers like starch, locust bean gum, xanthan, chitosan, gelatin, agarose, and alginate

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are capable of producing cryogel17–26. Food systems containing cereal β-glucan may form

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cryogel or cryoprecipitate when submitted to repeated freezing and thawing process. Although

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the cryostructure could lead to unfavorable appearance of food or drinks, freeze-thaw is a useful

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method to prepare cereal β-glucan hydrogel. Systematic researches indicate that molecular

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weight, concentration, number of freeze-thaw cycles, freezing and thawing temperature, and fine

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structure are the major factors showing significant effect on the formation of cereal β-glucan

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cryogel. 27–31

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Low field nuclear magnetic resonance (LF-NMR) is a useful technique in studying the state

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and mobility of water in heterogeneous system.32–37 The spin-spin relaxation time, T2, of water

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proton is an important parameter reflecting different mobility of water molecules in the aqueous

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system. The T2 of water protons in macromolecular solution is usually lower than that in pure

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water due to the chemical exchange between water protons and labile protons on polymer as well

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as diffusive exchange of water in different microenvironment. So T2 of water protons indirectly

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provides information on the polymer chain dynamics and microstructure of polymer solution and

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hydrogel.38 LF-NMR has been successfully used in characterization of water in polysaccharide

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solution and gel. The coil-helix transition and aggregation of helices in the sol-gel transition of

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carrageenan were monitored through water proton spin-spin relaxation time based on chemical

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exchange theoretical analysis and polymer chain mobility.38 In the study of pullulan aqueous 4


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solution, two groups of protons obtained from signal curve were assigned to inert protons on

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pullulan and the sum of water protons and the labile protons on pullulan.39 The hydration of

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chitosan and the water tightly coordinated with the polysaccharide were investigated in chitosan

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hydrogel.40 The microstructure of native starch granule and the gelatinization of starch were

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elucidated by NMR relaxation and diffusion methods.41,42

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The purpose of this article is identification of different water components by LF-NMR, and

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designation of the microstructures where the water components stay. The formation process of

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oat β-glucan cryogel is also verified by rheological method.

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MATERIALS AND METHODS

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Sample Preparation. Oat β-glucan was extracted from Weiduyou 1 oat cultivar provided by

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Shanxi Plateau Plant Institute by the method proposed by Lazaridou et al. with minor

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modification.43 The obtained β-glucan was designated as original oat β-glucan. Lower molecular

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weight oat β-glucan samples were prepared by hydrolysis of 1% original oat β-glucan solution

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using 0.1 M HCl at 70 °C for 15, 30, 45, 60, and 90 min. The hydrolysates were neutralized with

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2 M NaOH and dialyzed against deionized water to get rid of the salt in solution. All the

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β-glucan samples were lyophilized in FD5-3 freeze dryer (SIM, China) with cold trap

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temperature of -58 °C and pressure at 10 mTorr. The lyophilized samples were stored in a

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desiccator at room temperature for about 3 weeks before used to prepare β-glucan cryogel.

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Composition Analysis. The β-glucan content of the samples was determined by β-glucan

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mixed linkage test kit (Megazyme, Ireland). The moisture content was measured by heating the

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samples in a hot air oven at 105 °C until constant weight. The nitrogen was quantified using a

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Vario EL Cube elemental analyzer (Elementar, Germany) and the nitrogen content was

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converted into protein content with a factor of 5.83 for oat.44 The residual starch in the samples 5



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was determined with total starch assay kit (Megazyme, Ireland). Each analysis was performed

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with 3 replicates.

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Structural Characterization. The molecular weight, polydispersity index, and radius of

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gyration of oat β-glucan were obtained using a HPSEC-MALLS system. Oat β-glucan aqueous

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solution was prepared by heating the polysaccharide in 0.1 M NaNO3 at 85 °C with magnetic

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stirring for 3 h followed by cooling and adjusting the concentration to 1 mg/mL. The solution

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was filtered through a 0.45 µm membrane filter and injected into a HPSEC-MALLS system. The

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size exclusion chromatography system combined with multiple detectors (Wyatt, USA): a

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DAWN HELEOS-II multi-angle laser light scattering detector (with a laser wave length of 664

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nm), and an Optilab T-rEX refractive index detector. A Model 1500 HPLC pump (Scientific

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Systems, USA) with two columns in series, a SB-806 HQ and a SB-804 HQ (Shodex OHpak, 8

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mm × 300 mm, Showa Denko, Japan) were used. The columns and RI detector were maintained

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at 45 °C. The eluent was 0.1 M NaNO3 with 0.02% NaN3 aqueous solution at a flow rate of 0.6

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mL/min. A refractive index increment, dn/dc, of 0.146 mL/g was used for calculation.14 The data

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were obtained and analyzed using ASTRA 6.1 software.

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The intrinsic viscosity of oat β-glucan was determined with a 0.35 mm inner diameter

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Ubbelohde viscometer (Shanghai Sunlex, China) in a water bath at 25.0 ± 0.1 °C. The relative

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viscosity, ηr, was kept between 1.2 and 2.0 to ensure that the flow was basically Newtonian. The

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Huggins plot was used to obtain the intrinsic viscosity [η]. Measurement at each concentration

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was performed at least 3 times.

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The oat β-glucan sample (10 mg) was dissolved in 5 mL of sodium phosphate buffer (20 mM,

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pH 6.5) at 85 °C with magnetic stirring at 240 r/min for 3 h, then the polysaccharide solution was

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incubated with 5 units of lichenase at 50 °C for 90 min. The hydrolysate was filtered (0.45 µm) 6


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and analyzed using Dionex ICS-5000 HPAEC system (Dionex, USA). The oligosaccharide

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fragments were separated on a Carbopac PA1 column (4 × 250 mm) and detected by a pulsed

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amperometric detector. The mobile phase and the gradient were as described by Tosh et al.10

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Chromeleon 6.8 software (Dionex, USA) was used for data analysis.

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Cryogelation of Oat β-Glucan. Oat β-glucan samples with different molecular weight were

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used to make 4% (w/w) β-glucan aqueous solutions. The solutions were prepared the same as

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solubilization of β-glucan for HPSEC-MALLS analysis except that the solvent was deionized

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water. 2 g of each oat β-glucan solution was transferred into a NMR sample tube with internal

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diameter of 15 mm. The tubes were sealed with PTFE plugs and stored in a refrigerator at -18 ºC

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for 21 h. Then the tubes containing oat β-glucan samples were moved into an incubator and

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allowed to thaw at 25 °C for 3 h. The above freezing and thawing process is termed as a

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freeze-thaw cycle. The samples were tested after every cycle for the first 7 freeze-thaw cycles

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and then tested after every 3 cycles until 16 freeze-thaw cycles.

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For rheology analysis, 5 g of oat β-glucan solution was transferred into a cylindrical mold with

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internal diameter of 36 mm. The solutions were treated with repeated freeze-thaw cycles and

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tested after 0, 1, 4, 7, 10, 13, and 16 freeze-thaw cycles.

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LF-NMR Measurements. A MiniMR NMR spectrometer (Niumag, China) operating at 23

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MHz for 1H resonance was used to carry out the LF-NMR experiments. All the NMR tubes

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containing samples were placed into a water bath with the temperature of 32 °C and kept for at

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least 15 min. Then the NMR tubes along with the samples were transferred into the NMR probe

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with constant temperature of 32 °C. The spin-spin relaxation time, T2, was obtained using CPMG

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(Carr-Purcell-Meiboom-Gill) sequence with a 90°-180° pulse spacing of 0.1 ms. The 90° pulse

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was 18 µs and the number of echoes was 18000. It produced a sampling space of about 4.23 s. 7



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Samples prepared under different conditions were tested with at least 3 replicates. Each sample

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was repeated scanned for 8 times with a delay of 10 s.

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Signal collection and analysis were conducted using Niumag NMR analysis software (Niumag, China). The decay curves of relaxation signal were fitted with exponential equation: = exp −

+ 1

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where Ai is the echo amplitude of the ith component at time t, and T2i is the corresponding

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spin-spin relaxation time. A0 is the noise of the curve.

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Scanning Electron Microscopy. The prepared oat β-glucan solution and cryogel were

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lyophilized and a thin layer of the sample was cut with a sharp blade carefully. The

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microstructure of cross section of the sample was obtained with a JSM-7500F scanning electron

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microscope (JEOL, Japan). A small piece of cross section was fixed onto an aluminum stub with

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double-sided conductive tape and sputter-coated with gold. Then it was observed with SEM at an

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acceleration voltage of 3 kV.

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Dynamic Rheometry. The rheological properties of oat β-glucan solution and cryogel were

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investigated with Physica MCR-301 rheometer (Anton Paar, Austria) at 25 °C using parallel

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plate geometry (PP25/P2, 25 mm diameter, 1 mm gap). The solution or cryogel was transferred

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from cylindrical mold to the lower plate of the rheometer before test. G' (storage modulus), G''

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(loss modulus), and tan δ (G''/G') were obtained from oscillatory measurements with 0.1% strain

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and frequency from 0.1 to 10 Hz. Analysis was carried out with at least 3 replicates.

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Statistical Analysis. SPSS 19.0 software was used to evaluate the difference between

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measurements. One-way ANOVA was used with Duncan’s test and the significance level was set

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at 0.05. 8


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RESULTS AND DISCUSSION

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Composition of Oat β-Glucan Samples. The samples have a high content of β-glucan as

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shown in Table 1. Considering that the moisture content is about 5%, the β-glucan purity is over

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90% on a dry weight basis. The major residual components are protein and starch. Pentosan and

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ash are probably included in the other trace compositions.45 There is no significant difference

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between the components of oat β-glucan samples except that starch content decreases with the

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hydrolysis time, probably due to the hydrolysis of starch by acid.

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Molecular Characteristics of Oat β-Glucan. The original oat β-glucan sample has a weight

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average molecular weight, Mw, of 494 × 103 g/mol (Table 2). After hydrolyzed for 15 to 90 min,

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the Mw gradually decreases to 112 × 103 g/mol. The polydispersity (Mw/Mn) of oat β-glucan

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samples is in the range from 1.08 to 1.40, reflecting a relatively narrow distribution of molecular

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weight. Although the Mw of the original sample (494 × 103 g/mol) is significantly lower than that

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of the β-glucan isolates used by Doublier and Wood (1200 × 103 g/mol),45 and Agbenorhevi et al.

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(2800 × 103 g/mol),46 probably due to the different oat cultivars used for extraction of β-glucan,

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the β-glucan hydrolyzed for 90 min has a Mw of 112 × 103 g/mol, which is on the same order of

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magnitude as that of hydrolyzed β-glucan prepared with similar method by Doublier and Wood

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(100 × 103 g/mol, hydrolyzed for 60 min),45 and Agbenorhevi et al. (142 × 103 g/mol, hydrolyzed

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for 90 min).46 Acid hydrolysis causes a reduction in molecular weight of the polysaccharide,

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meanwhile the molar ratio of DP3/DP4 calculated from peak area of DP3/DP4 × 1.321 is not

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affected and shows similar values for both unhydrolyzed and hydrolyzed samples.10 Other

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researches on acid hydrolysis of oat β-glucan got the same results on the change of molecular

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weight and DP3/DP4.14,46

9



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The relationship between intrinsic viscosity and molecular weight of polymer is usually described by Mark-Houwink equation: = 2

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where α is the exponent of the equation, and K is a constant. The value of α was determined to be

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0.79 for oat β-glucan from double logarithmic plot of [η] against Mw (Figure 1). If a polymer

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molecule has a spherical shape, the value of α approaches 0. For a random coil, it is assumed to

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be in the range of 0.5 to 0.8. The value of α = 1 or 2 means a rigid coil or a rod. The obtained α

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of oat β-glucan is 0.79, indicating a random coil conformation of the polysaccharide in aqueous

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solution. The value is in accordance with α = 0.82 and 0.73 for oat β-glucan from other

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sources.47,48

181 182

Radius of gyration, Rg, has been obtained from HPSEC-MALLS test. It is linked with conformation parameter ν by power-law equation: = 3

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where Mw is the weight average molecular weight, and K is a constant. For a hard sphere, the

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value of ν = 0.33. For a random coil polymer, the ν value is between 0.5 and 0.6. A rigid rod has

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a ν = 1. The ν value of oat β-glucan is 0.57 as shown in Figure 1. It is consistent with the results

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from other researches (0.56 to 0.70).49 The results from exponent α of Mark-Houwink equation

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and conformation parameter ν both mean that the oat β-glucan exists as random coil in aqueous

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solution.

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LF-NMR Analysis of Cryogelation. Oat β-glucan samples with molecular weight of 494 ×

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103, 250 × 103, 155 × 103, and 112 × 103 g/mol were solubilized in deionized water at a

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concentration of 4% (w/w) and then treated with freeze-thaw cycles. The above samples are

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designated as OBG494, OBG250, OBG155, and OBG112 according to their molecular weight. 10


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The spin-spin relaxation time, T2i, of the water protons in oat β-glucan aqueous system after

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certain freeze-thaw cycles was obtained using CPMG pulse sequence.

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A typical T2 distribution of oat β-glucan cryogel is shown in Figure 2. Three water

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components are presented in the relaxation modes. As shown in Figure 3a, the fast relaxation

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component (component 1) has a spin-spin relaxation time, T21, of about 10 ms. The inert protons

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on oat β-glucan are not supposed to contribute to component 1 because CPMG pulse sequence

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can hardly collect the rapid decay signal. Component 1 can be reasonably considered as

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stemming from water molecules trapped within the cross-links formed by consecutive DP3 units

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and stable physical entanglement points of oat β-glucan chains. Once the cross-links and stable

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physical entanglements formed through freeze-thaw cycles, they became stable structures and

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could not easily be altered by further freeze-thaw treatment. So T21 basically keeps constant

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through all the cryogelation process, even though the values have some fluctuation. The

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spin-spin relaxation of this group of water protons is mainly modified by chemical exchange

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with labile protons of polysaccharide in the cross-links and stable entanglement points. The

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relatively small value of T21 reflects the low mobility of oat β-glucan chains in the network

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skeleton. These water molecules can be regarded to some extent as an integral part of the

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network skeleton structure.50 The spin density, A21, of component 1 gradually increases with the

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number of freeze-thaw cycles (Figure 3b), reflecting an increase in cross-links and stable

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physical entanglements. Oat β-glucan molecules with smaller size forms network skeleton earlier

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than their larger counterparts. Water associated with network skeleton is presented in OBG112

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and OBG155 sample after only 1 freeze-thaw cycle. But it takes 3 and 5 cycles for OBG250 and

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OBG494 to show component 1 in freeze-thaw treatment. Smaller β-glucans seemed to form more

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network skeleton during cryogelation (Figure 3b). It is obvious that OBG494 produced network 11



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skeleton significantly lower (about 1.5%) than the other samples smaller in size (about 3.5%)

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after 16 freeze-thaw cycles (p < 0.05). It is generally considered that oat β-glucan with smaller

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size has higher mobility of chains, facilitating the formation of cryogel structure. The formation

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and increase of cryostructure deduced from the presence of component 1 and increase in A21 is in

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accordance with the results with traditional methods.28

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After 5 freeze-thaw cycles, component 2 begins to appear in the relaxation modes of OBG112.

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The other oat β-glucan samples with higher molecular weight need more freeze-thaw cycles to

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present component 2, and which is detected in OBG155, OBG250, and OBG494 after 6, 6, and 7

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freeze-thaw cycles respectively (Figure 3c, 3d). The spin-spin relaxation time, T22, distributes

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from 20 to 90 ms, which means higher mobility of this group of water protons than component 1.

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The spin density, A22, is significantly higher than the corresponding A21 during cryogelation. So

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component 2 is considered as water confined in the interstitial between β-glucan chains. Oat

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β-glucan aqueous solutions experience slow freezing process at -18 °C in a refrigerator. During

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the freezing process, ice crystals gradually grow bigger and the concentration of β-glucan gets

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higher in the unfrozen phase, which facilitates the interaction between β-glucan molecules. In the

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following thawing process, the rise of temperature enhances the dynamics of β-glucan

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interactions; meanwhile the temperature is still below the freezing point in a period of time, the

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ice crystals remain in a solid state, keeping the high concentration of oat β-glucan in the liquid

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phase. The freeze-thaw cycles result in the formation of water pools surrounded by thin walls of

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concentrated cryogel, similar to a cell structure. Water molecules trapped in the interstitial

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between aggregates of oat β-glucan chains is assigned to component 2. Water in the holes left by

237

ice crystals after freezing and thawing is bulk water and named component 3. The diffusion of

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water in component 2 is restricted by the aggregates of β-glucan around them. The trapped water 12


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indirectly reflects the aggregation of oat β-glucan chains into a gel microphase. It is worth noting

240

that it takes more freeze-thaw cycles to produce component 2 than component 1. That indicates

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cross-links and stable entanglements formed at first but the quantity of which is not enough for

242

the formation of β-glucan gel microphase and there is little water entrapped within the gel

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microphase. After several more freeze-thaw cycles, additional cross-links and stable

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entanglements make a gel microphase. The spin-spin relaxation time, T22, firstly increases and

245

then shows a falling trend with the increase of freeze-thaw cycles. The increase of T22 maybe an

246

indication of more open association of β-glucan aggregates. And the decrease of T22 is probably

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caused by more dense gel phase compressed by ice crystals. At least, the change of T22 indicates

248

the porosity and heterogeneity during the cryogelation. There is no clear relationship between the

249

molecular weight of oat β-glucan and the corresponding T22. As shown in Figure 3d, spin density,

250

A22, increases with the number of freeze-thaw cycles, reflecting the increase of cryogel

251

microphase. The A22 shows an inverse relationship with the molecular weight of oat β-glucan at

252

the same number of freeze-thaw cycles. It demonstrates that oat β-glucan with small size

253

produces more cryogel microphase comparing to the larger polysaccharide. The result has been

254

verified by other researches as mentioned before. The A22 increases sharply and then levels off

255

(Figure 3d), indicating the quantity of cryogel microphase nearly approaches a maximum and

256

keeps constant after 10 free-thaw cycles. The A21 also shows the same trend (Figure 3b), and 10

257

could be considered as a critical number of free-thaw cycles.

258

The variation of spin-spin relaxation time of component 3, T23 is shown in Figure 3e.

259

Freeze-thaw treatment causes slight decrease of T23 at the first stage, and then T23 comes to a

260

quick increase and finally levels off at the end of the test. The initial values of T23 are

261

approaching that in fresh oat β-glucan solutions, so the component 3 is considered as bulk water 13



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in the aqueous systems. It is surprised that T23 is lower in aqueous system containing oat

263

β-glucan with smaller size at the beginning of freeze-thaw treatment. Although lower molecular

264

weight oat β-glucan aqueous system appears less viscous, the mobility of water therein seems to

265

be lower than that in the more viscous oat β-glucan with larger size. A likely reason for the

266

abnormal result lies in the self-aggregation of oat β-glucan. Oat β-glucan with smaller size is

267

more prone to aggregate than the bigger polysaccharide molecules. The aggregation may result

268

in a suspension or a network,45 which causes the decrease of the mobility of oat β-glucan

269

molecules and the slight lowering of T23 of water interacting with these polysaccharide

270

aggregates after the first several freeze-thaw cycles (Figure 3e). With more freeze-thaw

271

treatments, β-glucans in the bulk water gradually transfer to the gel microphase and lead to the

272

decrease of β-glucan concentration in bulk water. It means less labile protons on the

273

polysaccharide are available to exchange with bulk water protons. That results in an increase of

274

T23, since it is mainly determined by chemical exchange. Higher mobility makes more β-glucans

275

with smaller size enter the gel microphase and further rise of T23. So T23 begins to present a

276

remarkable rise after about 4 freeze-thaw cycles, and it is more significant for lower molecular

277

weight samples. The leveling off of T23 (Figure 3e) as well as T21 (Figure 3a) demonstrates the

278

cryostructure has little change after 10 to 13 freeze-thaw cycles. The spin density, A23, of the

279

bulk water decreases from nearly 100% to about 95%, 91%, 87%, and 85% respectively in oat

280

β-glucan samples with decreasing molecular weight after 10 freeze-thaw cycles (Figure 3f). The

281

reduction of bulk water ratio is due to the increase of cryogel microphase and increasing amount

282

of water entrapped in the gel microphase. In other words, the bulk water gradually converts into

283

entrapped water during cryogelation.

14


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284 285


Attempts were made to determine the cryogelation rate using the maximum increment of T23 as following, !"

$!

= # $'%& (

)*+

4

286

IT23 is the maximum increment of T23. N is the number of freeze-thaw cycles. The dependence of

287

cryogelation rate on oat β-glucan molecular weight is shown in Figure 4. The cryogelation rate

288

calculated from T23 is inversely proportional to the logarithm of β-glucan molecular weight. It is

289

in accordance with the results from rheological method.9,13 IT23 seems to be a good definition of

290

cryogelation rate and an alternative characteristic parameter complementary to rheological

291

definition.

292

The observed spin-spin relaxation time, T21, of the water protons interacting with labile

293

protons on oat β-glucan in the cross-links and stable entanglements through fast chemical

294

exchange is given by the equation,51 ./ 1 1 = + 5 - / + 0/1-

295

where T2w is the intrinsic relaxation time of free water protons, which is about 2 s in the test

296

conditions. T2p is the intrinsic relaxation time of labile protons on the β-glucan chain, which

297

cannot be measured directly, and usually deduced from relaxation time of inert protons on the

298

polymer. The value of T2p can be estimated at about 30 µs as in the barley β-glucan film.52 And

299

kp is the rate constant of chemical exchange between water protons and labile protons on the

300

polysaccharide. Fp is the molar fraction of labile protons on β-glucan. In a 4% (w/w) β-glucan

301

solution, the calculated molar fraction of labile hydroxyl protons is 6.90 × 10-3. The value of T21

302

is about 10 ms in all the β-glucan samples. The exchange rate, kp, can be estimated from

303

Equation 5. The calculated value of kp is 25.4 × 103 s-1, which is twice of the proton exchange 15



Page 16 of 53

304

rate of water saturated starch42 (11.5 × 103 s-1) and chitosan-cyclodextrin hydrogel53 (about 10 ×

305

103 s-1). It indicates a fast proton exchange between oat β-glucan and water in the cross-links and

306

stable entanglements of the cryostructure.

307

T22 corresponds to water trapped in the cryogel microphase and T23 to bulk water in the

308

pores created by ice crystals. T22 is modified by labile protons on β-glucan chains in the cryogel

309

microphase. T23 is modified by labile protons on the polysaccharide both remains in bulk water

310

and constitutes the surface layer of cryogel microphase. Component 2 and 3 are in diffusive

311

exchange through the interface between bulk water microphase and cryogel microphase. Because

312

component 2 and 3 present two different spin-spin relaxation time, T22 and T23, instead of an

313

averaged spin-spin relaxation time, the diffusive exchange rate D/a2 is slow comparing to the

314

difference in effective relaxation rates (1/T22-1/T23), that is the ratio54 1 1 2√2 # − ( " ≫ 1 6 45 6

315

where D is the self-diffusion coefficient of water in the microphase, which is approaching the

316

value of bulk water, so D = 2.74 × 10-9 m2 s-1 is used (bulk water at 32 °C); and a is characteristic

317

distance scale of heterogeneity, it could be regarded as the diameter of the pores left by ice

318

crystals. T22 and T23 of OBG112 treated with 16 freeze-thaw cycles are 64.5 and 676.3 ms

319

respectively, the result from Equation 6 is that a ≫ 26.1 µm. That means the averaged diameter

320

of those pores should be much larger than 26.1 µm.

321

Morphological Study by Scanning Electron Microscopy. Oat β-glucan solutions and

322

cryostructures were lyophilized and observed with a scanning electron microscope. The

323

microstructure of the sample is exhibited in Figure 5. In general, the solution samples have a 16


Page 17 of 53


324

lamellar structure and the cryogel samples have a porous structure. In fact, the solution samples

325

were frozen in a refrigerator before lyophilization, and the solution structure had been modified

326

by the growth of ice crystals, so the SEM images were not a real reflection of the microstructure

327

in the solution samples. The lamellar structures in solution samples originate from the

328

concentrated oat β-glucan solution, and the space between the lamellar structures is created by

329

the growth of ice crystals in the slow freezing of samples in the refrigerator before freezing dry.

330

The layers of β-glucan are damaged by ice crystals, giving rise to the formation of holes on the

331

layers. It seems that the samples with lower molecular weight produce larger lamellar structure

332

with higher integrity, probably due to the higher mobility of β-glucan with lower molecular

333

weight. Interconnected pores formed in oat β-glucan cryogel samples after repeated freeze-thaw

334

cycles (Figure 5e-5h). The thin walls, with the thickness of several microns, constitute the

335

cryogel microphase. Component 1 and 2 are water molecules in the cryogel microphase. As

336

mentioned before, T21 is the spin-spin relaxation time of water protons within the cross-links and

337

stable physical entanglements. The similar values of T21 probably indicate the structures of the

338

cross-links and entanglements in different oat β-glucan cryogels are alike in spite of the

339

difference in molecular weight. Water entrapped in the interstitial between aggregates of oat

340

β-glucan chains is component 2. The interstitial structure is shown in Figure 5i. The highly

341

heterogeneous interstitial structure in the cryogel microphase is probably the reason for the

342

irregular variation of T22 in the cryogelation process (Figure 3c). The heterogeneous structure

343

also leads to multiexponential relaxation, T21 and T22, in the cryogel microphase. The bulk water

344

in the pores left by ice crystals has a relaxation time T23 between 450 and 680 ms, and a spin

345

density A23 from 85% to 95% when the samples are treated with 16 freeze-thaw cycles. It means

17



346

the pores occupy most volume of the cryogel, which is clearly shown in Figure 5e to 5h. The

347

pores and the thin walls around them indicate the microphase separation in the cryogel.

Page 18 of 53

348

The measured diameters at the cross-section of the pores from the SEM images of OBG112

349

with 16 freeze-thaw treatments have an average value of 68.5 ± 28.3 µm. It is in accordance with

350

the result from Equation 6 that the average diameter of those pores is larger than 26.1 µm.

351

Considering that the pores are interconnected, the effective size of these pores should be larger

352

than 68.5 µm, so the real value is much larger than calculated size 26.1 µm.

353

The macroscopic porosity was observed in barley β-glucan cryogel with naked eyes and

354

optical microscope.29 It was considered that the porosity increased with the reduction in

355

molecular weight. But the change with the molecular weight is not significant in SEM images

356

(Figure 5e-5h). The pore size increased with the number of freeze-thaw cycles in the cryogel of

357

starch due to the growth of ice crystals.55 The increase of pore size is speculated to contribute to

358

the rise of T23. Lamellar structures were observed in the oat β-glucan hydrogel prepared at 4 °C,

359

some pores in the hydrogel were believed to be created by the ice crystals during the freezing of

360

sample before freezing dry.56

361

Rheology Analysis of Cryogelation. The cryogelation process of oat β-glucan was also

362

investigated with rheological method. The storage modulus, G', at an oscillatory frequency of 1

363

Hz was plotted against the number of freeze-thaw cycles in Figure 6a. The variation of tan δ in

364

cryogelation is shown in Figure 6b. It should be pointed out that the cryostructures could not be

365

separated from bulk water through repeated freeze-thaw cycles, probably due to the high

366

concentration and viscosity of the samples, and a monolithic cryogel formed at last. So both the

367

cryogel microphase and water in the connected holes were subjected to rheological test as a

368

whole, which was different from the method adopted by Lazaridou and Biliaderis,27 the clear 18


Page 19 of 53


369

liquid phase was well separated from the cryogel in their experiments. The G' of high molecular

370

weight β-glucan is greater than that of β-glucan with low molecular weight, the result is also

371

different from that of Lazaridou and Biliaderis.27 It is supposed that the value of G' reflects not

372

only the cross-link density in the cryogel, but also the amount of stable physical entanglement

373

produced by oat β-glucan molecules. The higher G' of OBG494 means more entanglement in the

374

high molecular weight β-glucan sample. But the increment of G' of OBG494 with the number of

375

freeze-thaw cycles is not significant, indicating there is little increase in the density of cross-links.

376

Although the other low molecular weight β-glucan samples have lower initial values of G', they

377

exhibit significant increase in G' with more freeze-thaw cycles, and the final values of G' are

378

approaching that of OBG494. The increase of G' is considered to be derived from cross-links

379

produced by freeze-thaw treatments. The increment of G' can be defined as 9:

=

;′16 7 ;′0

380

where the G' (16) and G' (0) are the storage modulus after 16 and 0 freeze-thaw cycles. The plot

381

of log (IG') versus log (Mw) is shown in Figure 7. The negative relationship between log (IG') and

382

log (Mw) is consistent with the result that the density of cross-links is higher in the cryogel of oat

383

β-glucan with smaller size.27 The value of tan δ is considered as a better index of elasticity and

384

connectivity of a cryogel network.27 The initial value of tan δ increases remarkably with the

385

decreasing of molecular weight (Figure 6b). The tan δ of 4% OBG494 is below 1, indicating a

386

typical gel behavior, while the other samples exhibit typical solution behavior with tan δ well

387

above 1. With the increase of the number of freeze-thaw cycles, tan δ of OBG494 shows slight

388

decrease, but the values of the other samples decrease significantly and approach a constant at

389

the end of the test. The change of tan δ demonstrates a significant increment of cross-links in the 19



390

low molecular weight β-glucan samples, but a fairly low increment of cross-links in OBG494.

391

The final values of tan δ are still above 0.1, indicating that the resulted cryostructure is not a

392

highly elastic cryogel.

Page 20 of 53

393

LF-NMR provides useful information on the water distribution in oat β-glucan cryogel and

394

the evolution of water components during cryogelation process. Three components are

395

determined as water within the cross-links and stable physical entanglements, water confined in

396

the interstitial between aggregates of β-glucan chains, and water in the interconnected pores in

397

the cryogel. The cryogel structure and the water components in the microstructure are illustrated

398

in Scheme 1. The variations of spin-spin relaxation time and spin density of those water

399

components indicate that the amount of cross-links and stable physical entanglements increased

400

during cryogelation. The increment of cryogel microphase leads to the rise of water entrapped in

401

the interstitial between aggregates of β-glucan chains and the decrease of bulk water in the pores

402

left by ice crystals. The transfer of water from the pores to the walls around them results in the

403

decrease of β-glucan concentration in those pores. The spin-spin relaxation is analyzed with

404

chemical and diffusive exchange. The pore size calculated from relaxation is in accordance with

405

that measured from SEM image of the cryogel. It is found that the cryogelation rate can be

406

described with the maximum increment of T23. The rheological study confirmed the increased

407

cross-link density during cryogelation.

408 409

ABBREVIATIONS USED

410

DP3 3-O-β-cellobiosyl-D-glucose; DP4 3-O-β-cellotriosyl-D-glucose; LF-NMR low field

411

nuclear magnetic resonance; HPSEC-MALLS high performance size exclusion chromatography

20


Page 21 of 53


412

– multi angle laser light scattering; CPMG Carr-Purcell-Meiboom-Gill; SEM scanning electron

413

microscopy

414

ACKNOWLEDGMENTS

415

We thank Zhiyu Li, College of Food Science, Fujian Agriculture and Forestry University, for his

416

help in HPSEC-MALLS analysis.

417

Funding

418

This work was financially supported by the National Natural Science Foundation of China

419

(31101224) and Natural Science Foundation of Fujian Province (2013J05049).

420

Notes

421

The authors declare no competing financial interest.

422

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423

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Figure captions Figure 1. Double logarithmic plot of [η] or Rg against Mw of oat β-glucan samples. Figure 2. Spin-spin relaxation time distribution curve of OBG494 cryogel treated with 16 freeze-thaw cycles. Figure 3. Effect of number of freeze-thaw cycles and molecular weight on the relaxation time T2i and spin density A2i of protons of water component 1 (a, b), component 2 (c, d), and component 3 (e, f) during cryogelation of oat β-glucan. Figure 4. Dependence of cryogelation rate on the molecular weight of oat β-glucan. Figure 5. SEM images of lyophilized aqueous solutions of OBG494 (a), OBG250 (b), OBG155 (c), and OBG112 (d) as well as cryostructures of OBG494 (e), OBG250 (f), OBG155 (g), and OBG112 (h, i) treated with 16 freeze-thaw cycles. Figure 6. Variation of G' (a) and tan δ (b) with the number of freeze-thaw cycles recorded at 25 °C, 1 Hz, and 0.1% strain of cryostructures. Figure 7. log (IG') as a function of molecular weight. Scheme 1. The oat β-glucan cryogel microstructure and the corresponding water components.

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Tables Table 1. Composition of Oat β-Glucan Samples hydrolysis time

β-glucan

moisture

protein

starch

others

(min)

(%)

(%)

(%)

(%)

(%)

0

88.41 ± 1.26

5.86 ± 0.16

2.80 ± 0.13

1.89 ± 0.08

1.05 ± 0.04

15

90.13± 1.18

5.32± 0.10

2.12± 0.17

1.64± 0.05

0.78± 0.03

30

89.73 ± 1.04

5.35 ± 0.18

2.39 ± 0.10

1.47 ± 0.07

1.09 ± 0.05

45

89.64 ± 1.23

5.21 ± 0.14

2.26 ± 0.11

1.35 ± 0.04

1.53 ± 0.07

60

90.72 ± 1.58

5.28 ± 0.15

2.33 ± 0.12

0.86 ± 0.05

0.83 ± 0.06

90

91.80 ± 0.97

4.64 ± 0.17

2.33 ± 0.14

0.30 ± 0.06

0.93 ± 0.05

a

Results are presented as the mean ± standard deviation.

Table 2. Molecular Characteristics of Oat β-Glucan Samples

a

d

Rg c

[η]d

(nm)

(dL/g)

1.13

48.8

5.14

2.46

332

1.08

38.6

3.48

2.51

30

250

1.25

33.6

2.94

2.42

45

178

1.13

28.1

2.18

2.39

60

155

1.40

25.6

1.97

2.56

90

112

1.32

20.6

1.58

2.49

hydrolysis time

Mwa

(min)

(×103 g/mol)

0

494

15

Mw/Mnb

DP3/DP4e

Weight average molecular weight. bPolydispersity. cRoot mean square radius of gyration.

Intrinsic viscosity, dL = 100 mL. eMolar ratio, calculated from peak area of DP3/DP4 × 1.321. 30


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Figures

Figure 1. Double logarithmic plot of [η] or Rg against Mw of oat β-glucan samples.

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Figure 2. Spin-spin relaxation time distribution curve of OBG494 cryogel treated with 16 freeze-thaw cycles.

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Figure 3a. Effect of number of freeze-thaw cycles and molecular weight on the relaxation time T2i of protons of water component 1.

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Figure 3b. Effect of number of freeze-thaw cycles and molecular weight on the spin density A2i of protons of water component 1.

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Figure 3c. Effect of number of freeze-thaw cycles and molecular weight on the relaxation time T2i of protons of water component 2.

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Figure 3d. Effect of number of freeze-thaw cycles and molecular weight on the spin density A2i of protons of water component 2.

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Figure 3e. Effect of number of freeze-thaw cycles and molecular weight on the relaxation time T2i of protons of water component 3.

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Figure 3f. Effect of number of freeze-thaw cycles and molecular weight on the spin density A2i of protons of water component 3.

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Figure 4. Dependence of cryogelation rate on the molecular weight of oat β-glucan.

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Figure 5a. SEM images of lyophilized aqueous solutions of OBG494.

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Figure 5b. SEM images of lyophilized aqueous solutions of OBG250.

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Figure 5c. SEM images of lyophilized aqueous solutions of OBG155.

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Figure 5d. SEM images of lyophilized aqueous solutions of OBG112.

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Figure 5e. SEM images of lyophilized cryostructures of OBG494 treated with 16 freeze-thaw cycles.

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Figure 5f. SEM images of lyophilized cryostructures of OBG250 treated with 16 freeze-thaw cycles.

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Figure 5g. SEM images of lyophilized cryostructures of OBG155 treated with 16 freeze-thaw cycles.

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Figure 5h. SEM images of lyophilized cryostructures of OBG112 treated with 16 freeze-thaw cycles.

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Figure 5i. SEM images of lyophilized cryostructures of OBG112 treated with 16 freeze-thaw cycles.

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Figure 6a. Variation of G' with the number of freeze-thaw cycles recorded at 25 °C, 1 Hz, and 0.1% strain of cryostructures.

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Figure 6b. Variation of tan δ with the number of freeze-thaw cycles recorded at 25 °C, 1 Hz, and 0.1% strain of cryostructures.

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Figure 7. log (IG') as a function of molecular weight.

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Scheme 1. The oat β-glucan cryogel microstructure and the corresponding water components.

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TOC Graphic

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Avena nuda L. - American Chemical Society

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