Article pubs.acs.org/JPCB
Effect of Heating on Chain Conformation of Branched β‑Glucan in Water Shuqin Xu, Xiaojuan Xu, and Lina Zhang* Department of Chemistry, Wuhan University, Wuhan 430072, China ABSTRACT: The thermal stability of polysaccharides under heat treatment is an important factor to their functionality in food and pharmaceutical fields. The stiff branched β-glucan coded as AF1-1 isolated from Auricularia auricula-judae was investigated with viscometry, dynamic light scattering (DLS), and size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) in water at 25 to 170 °C. The chain conformation of AF1-1 in the aqueous solution exhibited a sharp decrease in viscosity, hydrodynamic radius (Rh), and weightaverage molecular weight (Mw) at elevated temperature in a narrow range of 140 to 160 °C. It was confirmed that the conformation transitions of the AF1-1 chains from rod-like chains to the flexible occurred during heating to 140−160 °C for 30 min, leading to the coexistence of the flexible chains and stiff chains at 155 °C as a result of the breaking of the intra- and intermolecular hydrogen bonds of the AF1-1 macromolecules. The results from scanning electron microscopy and atomic force microscopy further directly proved that the AF1-1 nanofibers in water were destructed into flexible coils consisting of individual chain at the elevated temperature higher than 155 °C, supporting the conformation transition. The conformational transition from stiff to flexible chains at 140−160 °C was irreversible. However, the chain shape and stiffness of AF1-1 was stable below 140 °C and hardly changed with an increase in the temperature. This was important for the application in the fields of food and pharmaceutical.
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INTRODUCTION Polysaccharides have attracted much attention because of their bioactivities and many functions.1 As an essential constituent of organisms, the polysaccharides possess an abundance of hydroxyl groups, leading to the water solubility, the conformational transitions, and the formation of aggregates and so on, which are relative to various functional roles in the life processes.2−4 It is noted that the 6-OH group of side glucoses along the main-chain can combine with water molecules through hydrogen bonds to form an associating water layer, leading to the increasing water solubility.5,6 The breaking of the associating structure with relatively lower energy occurs under slightly heating, resulting in the rotation of side chains in triple helix.7,8 There are β-(1→3)-glucans with triple helical structures, such as curdlan,9 schizophyllan (SPG),3 and lentinan,7 which have linear backbones with regular substitution at 6-OH on the main chain by β-(1→6)-D-glucose residues. The triple helix is stabilized by a triangular interhydrogen bonding, in which each backbone 2-OH protruded toward the inside serves as donor and acceptor to link with each other.6 The stiff triple helix can dissociates into single-strand coils by increasing NaOH concentration, adding DMSO, or elevating temperature as a result of the destruction of the intra- and intermolecular hydrogen bonds, which sustain the triple helical structure.3,7 Hydrogen bonding of the polysaccharides plays an important role not only in physical aggregates but also in the selfassembly.5,10,11 It is worth noting that the β-(1→3)-glucans can interact with certain polynucleotide to form a new triplestranded structure, in which the supramolecular helices consist © 2013 American Chemical Society
of two glucan strands and one polynucleotide strand by hydrogen bonding and hydrophobic interactions.3 More recently, we have successfully isolated a β-(1→3)-Dglucan (AF1) with comb-branched structure by using an improved extraction and purification method, showing high stability in water for long storage time.12 The stiff comb-like polysaccharide chain with short branches of AF1 can easily align in order and self-assemble into well-defined nanofibers with hydrophobic hollow cavity, driven by hydrogen bonding and hydrophobic interactions. The side glucose residues of the branched β-glucan contribute to the water solubility, whereas the backbone shows relative hydrophobicity.5 The nanofibers further organized sideby-side into lamella and finally curled up to a hollow fiber with high tensile strength. Usually, the heating of the polysaccharides and polymers in aqueous solutions can induce the transition of the chain conformation.7,13 Many water-soluble polysaccharides have been applied in food and pharmaceutical fields as thickening, stabilizing, and water-binding agents, which are often sterilized by autoclaving.14−16 The stiff triple helices can be dissociated into single random coils by heating to 135 °C and subsequently renatured as the linear and circular helical components in the samples.17,18 A “phase diagram” for schizophyllan in mixed dimethyl sulphoxide (DMSO)/water solvent solution at different temperature was mapped by using scanning calorimetry, indicating the multiple conformational transitions Received: April 1, 2013 Revised: May 30, 2013 Published: June 24, 2013 8370
dx.doi.org/10.1021/jp403202u | J. Phys. Chem. B 2013, 117, 8370−8377
The Journal of Physical Chemistry B
Article
where A is the measured baseline and β is a constant related to the coherence of the detected optics. For a polydisperse system, g(1)(q, τ) is related to the distribution of the characteristic line width G(Γ) by20,21
including a reversible transition at low temperature and an irreversible transition at high temperature.8 The influence of temperature on the chain structure of polysaccharides is very important for their fundamental research and clinical application in the medical and biological fields.19 In the present work, we attempted to study the effect of heating treatment on the chain conformation of the polysaccharide fraction AF1-1 in dilute aqueous solution with dynamic light scattering (DLS), size-exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS), and viscometry. Moreover, the chain shape and size of AF1-1 at different temperature were further clarified by scanning electronic microscopy (SEM) and atomic force microscopy (AFM). The aim was to provide meaningful information on the changing of the chain structure of AF1-1 in water at different temperatures as well as the destruction of intra- and intermolecular hydrogen bonding at elevated temperature.
|g(1)(q , τ )| =
∞
G(Γ)e−ΓdΓ
(2)
(1)
Thus, g (q,τ) can be converted to a line-width distribution G(Γ) by the CONTIN Laplace inversion algorithm in the correlator according to eqs 1 and 2. For a pure diffusive relaxation, Γ is related to the translational diffusion coefficient (D), and G(Γ) can be converted to a translation diffusion coefficient distribution G(D) by
Γ = Dq2
(3)
Thus, a hydrodynamic radius (Rh) can be calculated by using the Stokes−Einstein equation
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METHODS Sample Preparation. Crushed dried fruit bodies of A. auricula-judae are a commercial product cultivated in Fangxian (Hubei, China) and were purchased from a market in China. The polysaccharide sample was isolated and purified according to our previous method.12 AF1 was dissolved in ultrapure water with a concentration of 3 × 10−4 g/mL and exposed to 20−25 kHz ultrasonic irradiation by an ultrasonic cell disruptor (JY92-IID, Ningbo Scientz Biotechnology, China) for 5 min to obtain the sample with moderate molecular mass. The sonicated solution was further purified by using the reprecipitation method at 25 °C, and the second fraction was collected, which was dissolved in water again and then dialyzed against ultrapure water for 5 days. The resultant polysaccharide solution was finally lyophilized to give the white sample, coded as AF1-1, which was used in this work. AF1-1 consists of a β-(1→3)-D-glucan backbone with two β-(1→6)-D-glucosyl residues for every three main chain glucose residues, showing a comb-branched structure.12 All of the fresh solutions were carefully prepared by completely dissolving the proper amount of polysaccharide AF1-1 in water for over 24 h with stirring. To investigate the thermally induced transformation process at temperature higher than 80 °C, the solutions were vacuum-sealed in the 25 mL glass tubes and heated in the thermostatted oil bath at the desired temperature in the range from 25 to 180 °C for 30 min. After being quenched to 25 °C in an ice bath and kept for 2 h, then the solutions were used for all measurements.17 The above aqueous samples were filtered with 0.45 μm pore size filters (NYL, 13 mm syringe filter, Whatman, USA) before the viscosity and light scattering measurements. Dynamic Light Scattering, SEC-LLS, and Viscometry Measurements. DLS measurements were used to characterize the hydrodynamic radii (Rh) of the polysaccharide solution in water at 25 °C. The measurements were carried out on the modified commercial light scattering spectrometer (ALV/SP125, ALV, Germany) equipped with an ALV-5000/E multi-τ digital time correlator and a He−Ne laser (at λ = 632.8 nm). The precisely measured intensity−intensity time correlation function G(2)(q,τ) in the self-beating mode can be related to the normalized field−field autocorrelation function g(1)(q,τ) via the Siegert relation as20 G(2)(q , τ ) = A[1 + β|g(1)(q , τ )2 |]
∫0
Rh =
kBT 6πη0D
(4)
where kB is Boltzmann’s constant and η0 is the solvent viscosity. The dependence of weight-average molecular weight (Mw), radius of gyration (z1/2), and intrinsic viscosity ([η]) on temperature for polysaccharides in water was determined by using size -exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) equipped with a He−Ne laser at λ = 663 nm (Dawn-Heleos-II, Wyatt Technology, USA) and a viscometer (ViscoStar-II, Wyatt Technology). SEC-MALLS measurements were carried out on the multiangle laser photometer mentioned above at 18 angles from 14 to 163° with a Waters 515 HPLC pump equipped with a degasser (Flom Gastorr TG-14, USA). A column of Shodex− OHpak SB-806MHQ (8.0 mm × 300 mm) at a flow rate of 0.6 mL/min was kept at 40 °C by a CBL model 200 column heater. The chromatogram was measured with a differential refractive index detector (Optilab T-rEX, Wyatt Technology) at 25 °C. The Astra software (V. 6.1.1) was utilized for data acquisition and analysis. The specific refractive index increment (dn/dc) of AF1-1 was 0.136 mL/g in aqueous solution.22 The whole polysaccharide solutions were prepared at a concentration of 5 × 10−4 g/mL in water, then purified by a 0.45 μm Millipore filter, and the injection volumes were 200 μL. The eluents were 0.9% aqueous NaCl solution, which were purified by a 0.2 μm membrane and degassed before use. All of the solutions for viscosity measurement had the same original concentration of 5 × 10−4 g/mL. The reduced viscosities (ηsp/c) of the polysaccharide AF1-1 in water were measured at different temperatures by Ubbelohde capillary viscometer, and the ηsp/c value of AF1-1 in dry DMSO was measured at 25 °C. Kinetic energy correction was always negligible. Characterizations. The molecular architecture of the AF1-1 in solution was observed on a field-emission scanning electron microscope (FE-SEM, Hitachi, S-4800, Japan) by using an accelerating voltage of 5 kV. The unheated AF1-1 aqueous solution with a concentration of 5 × 10−4 g/mL was prepared for 24 h at room temperature. Part of the resulting AF1-1 solution was first heated to 160 °C for 30 min in a sealed vial to prepare the heated sample; then, both the heated and unheated samples were frozen in liquid nitrogen immediately and finally lyophilized for the observation. The wide-angle X-ray diffraction (WXRD) patterns were obtained on an XRD diffractometer (D8-Advance, Bruker, USA)
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dx.doi.org/10.1021/jp403202u | J. Phys. Chem. B 2013, 117, 8370−8377
The Journal of Physical Chemistry B
Article
elevated temperature (
