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Exploiting salt induced micro phase separation to form soy protein microcapsules or microgels in aqueous solution Nannan Chen, Mouming Zhao, Taco Nicolai, and Christophe Chassenieux Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 19, 2017
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Biomacromolecules
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Exploiting salt induced micro phase separation to form soy protein
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microcapsules or microgels in aqueous solution
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Nannan Chen1,2, Mouming Zhao1, Taco Nicolai2*, Christophe Chassenieux2
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1
7
Guangzhou, China
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2
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72085 Le Mans cedex 9, France
School of Food Science and Engineering, South China University of Technology, 510640,
LUNAM Université du Maine, IMMM UMR-CNRS 6283, Polymères, Colloïdes et Interfaces,
10
Email :
[email protected] 11
Tel : (33)-243833139
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Abstract
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Self-assembly of native glycinin at room temperature was investigated as a function of the pH
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and the NaCl concentration. Microphase separation leading to the formation of dense protein
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microdomains was observed by confocal laser scanning microscopy. Depending on the
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conditions, the microdomains coalesced into a continuous protein rich phase or associated into
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large clusters. Addition of β-conglycinin inhibited phase separation and reduced the pH range
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in which it occurred. Microdomains of glycinin that were formed in the presence of 0.1 M NaCl
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transformed into hollow stable cross-linked microcapsules when heated above 60°C with
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diameters between 3 and 30 µm depending on the protein concentration and a shell thickness
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between 0.9 and 1.4 µm. The microcapsules were stable to dilution in salt free water, whereas
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microdomains formed at room temperature redispersed. Microdomains formed in mixtures
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with β-conglycinin did not transform into microcapsules, but became stable cross-linked
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homogeneous microgels.
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Keywords phase separation, glycinin, soy protein, microgels, microcapsules
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Introduction
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Soy protein isolate (SPI) has two main components: glycinin and β-conglycinin. The
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properties of soy proteins in aqueous solution and their aggregation or gelation behaviour
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during heating have been reviewed.1-3 Both components are complexes consisting of several
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different peptide chains4, 5 and have molar masses of about M≈3.6x105 g/mol for glycinin and
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M≈2.0x105 g/mol for β-conglycinin at neutral pH and high ionic strength.3, 6 However, it was 2 ACS Paragon Plus Environment
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found that depending on the pH and the ionic strength, the complexes may dissociate into
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smaller parts or associate into larger aggregates.7-10 In a pH range around the isoelectric point
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of the proteins (IEP≈5.0 for glycinin and IEP≈4.2 for β-conglycinin11, 12), native soy proteins
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assemble into large aggregates, which causes a strong increase of the turbidity.7, 8, 13-15 A
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fraction of the proteins precipitates under gravity or after centrifugation at moderate speeds.
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The pH-range where this occurs is narrower for β-conglycinin than for glycinin and is
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intermediate for mixtures. Remarkably, at pH 7-8, large scale aggregation of soy proteins is
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stronger at intermediate NaCl concentrations around 0.1 M than at either higher or lower
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concentrations.8, 13, 14
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When soy globulin is heated, the proteins denature, which leads to irreversible
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aggregation of the proteins and at higher protein concentrations to gelation.1-3 At neutral pH
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and without salt, the denaturation temperature was determined by DSC to be 65°C and 80°C for
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β-conglycinin and glycinin, respectively, but it decreases with increasing pH and increases
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with increasing ionic strength.13, 16-18 Nevertheless, Jiang et al.13 observed that the turbidity of
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SPI solutions at pH 7.0 increased more strongly during a heating ramp if 0.1 or 0.6 M NaCl was
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added than in deionized water, indicating that aggregation is favored by screening of
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electrostatic repulsion even if it leads to an increase of the denaturation temperature. The rate
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of thermal aggregation increases strongly with increasing temperature and protein
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concentration and has therefore been mostly investigated at elevated temperatures (T>80°C)
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and high protein concentration where it is rapid.19, 20 In the absence of salt, the aggregation rate
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of SPI was found to have an Arrhenius temperature dependence characterized by an activation
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energy (Ea) of 180 kJ/mol in the range 65–85°C.21,
22
Lakemond et al.23 studied thermal
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aggregation and gelation of glycinin solutions at pH 7.6 and 3.8, i.e. below and above the pH
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range where glycinin is not fully soluble. Gels were formed during heating for 30 min at 95°C
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if the protein concentration exceeded 7% at 0.03 M salt and above 5% at 0.2 or 0.5 M. At lower
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protein concentrations, precipitation of proteins was observed.
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The effect of self-assembly of native soy proteins in aqueous solution on the turbidity of
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the solution and the solubility of the proteins has been reported in some detail as a function of
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the pH and the ionic strength.7, 8, 13-15 However, the microscopic structure of the systems has not
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yet been investigated. Here, we present in the first part, a systematic investigation of the
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structure of self-assembling native glycinin in aqueous solutions at different pH and NaCl
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concentrations using confocal laser scanning microscopy. It will be shown that dense protein
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microdomains are formed that with time either coalesce into a continuous dense protein phase
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or agglomerate into large flocks. The effect of mixing β-conglycinin with glycinin has also
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been investigated. The kinetics was studied by measuring the turbidity as a function of time. In
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the second part, we discuss the effect of heating on micro phase separated soy protein solutions.
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We will show that when the microdomains of glycinin are heated above 60°C, hollow
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cross-linked stable microcapsules are formed spontaneously, whereas in mixtures with
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β-conglycinin, microgels are formed.
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As far as we are aware, spontaneous formation of protein microcapsules in aqueous
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protein solutions has not yet been reported in the literature. The currently used methods to form
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protein microcapsules have been reviewed by Jaganathan et al.24 and Nesterenko et al.
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reviewed the literature on microcapsules formed by plant proteins.25 Most often, microcapsule
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formation involves emulsification of an incompatible liquid in water, usually an oil, and 4 ACS Paragon Plus Environment
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formation of a crosslinked protein layer around the dispersed droplets via coacervation or spray
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drying. Soy protein capsules have been formed with a variety of oils via coacervation26, 27 or
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spray drying.28-30 For example, Lazko et al.27 formed capsules with a diameter of about 100 µm
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by emulsifying hexadecane in an aqueous glycinin solutions at pH 2, followed by increasing
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the pH to 5.0 while stirring, which induced coacervation of glycinin at the surface of the oil
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droplet, and covalently cross-linking the highly irregular glycinin layer with glutaraldehyde.
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Of course, in order to form hollow capsules, the core material needs to be removed. Formation
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of soy protein microgels with diameters of 15-25 µm has been reported by Chen and
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Subirade.31 It involved dispersing a preheated aqueous SPI solution in soy oil and gelling the
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dispersed droplets of SPI aggregates at room temperature by adding CaCl2 to the water phase.
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Here we demonstrate that stable hollow protein capsules or microgels can be produced more
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simply by exploiting salt induced micro phase separation of soy proteins in aqueous solution.
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Microcapsules and microgels can be used to protect sensitive ingredients by encapsulation
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with a protein layer or embedding them within the microgel and to control their release for
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applications in food, cosmetics and pharmaceutics.32-36 Further research will be needed to
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determine if soy protein capsules and microgels formed by heating micro phase separated soy
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protein solutions have properties that make them attractive for applications, but this was
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outside the scope of the present investigation.
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Experimental Section
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Materials. Purified fractions of glycinin and β-conglycinin were isolated from defatted
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soybean flakes following the method reported by Wu et al.37 The total protein concentration
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in the purified glycinin and β-conglycinin fractions was determined by measuring the
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nitrogen content and was found to be 87 wt% and 81 wt%, respectively using the conversion
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factor Nx5.7.38, 39 The protein composition was determined by reducing SDS-PAGE and is
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shown in Figure S1 of the supporting information. Quantitative analysis of the absorbance of
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Coomassie brilliant blue assuming equal binding of the colorant to the two components
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showed that the purified glycinin sample contained 16% β-conglycinin and the purified
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β-conglycinin sample contained 39% glycinin. The assumption of equal binding was verified
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by analysing a 50/50 mixture of the purified samples. In the following, when we write
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glycinin or β-conglycinin solutions we refer to solutions prepared with purified glycinin or
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purified β-conglycinin powders, unless otherwise specified.
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Protein Solution Preparation. Protein solutions were prepared in salt-free Milli-Q
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water with 3 mM NaN3 to avoid bacterial growth. All solutions were filtered through 0.45 µm
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pore size Anatope filters before use without significant loss of protein. The pH was adjusted
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to within ±0.02 units by adding 0.1 M HCl or 0.1 M NaOH and the ionic strength was varied
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by adding aliquots of a concentrated NaCl solution (2 or 4 M). The samples were heated in a
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thermostat bath at different temperatures with an accuracy of ±0.2°C
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Confocal Laser Scanning Microscopy (CLSM). CLSM was used in the fluorescence
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mode. Observations were made at 20°C with a Leica TCS-SP2 (Leica Microsystems
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Heidelberg, Germany). A water immersion objective lens was used (HCxPL APO 63×NA =
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1.2). Soy protein was labeled by adding 5 ppm rhodamine B which binds spontaneously to the
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proteins.
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Electrophoresis. In order to reduce the disulphide bonds, 0.2 mL of the protein solution
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at C=2.5 g/L was mixed with an equal amount of buffer containing 30 mM DTT in an
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Eppendorf tube which was then put in the 95oC water bath for 10 min and centrifuged at 104 g
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for 10 min. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
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was done with a Mini-PROTEAN Tetra Cell (Bio-Rad, USA) using a discontinuous buffered
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system and combined 12% resolving gel and 5% stack gel. The electrophoresis was run at 15
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mA until all samples entered the resolving gel and thereafter at 30 mA. After the
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electrophoresis, the gels were stained with a Coomassie brilliant blue R250 solution for 1 h and
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excess coloring was removed by soaking in Milli-Q water overnight.
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Turbidity. The turbidity was measured in rectangular airtight cells at a wavelength of 500
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nm using a UV-visible spectrometer Varian Cary-50 Bio (Les Ulis, France). The temperature
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was controlled at 20±0.2oC using a thermostat bath.
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Determination of Solubility. The solubility of the proteins was determined by measuring
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the UV absorption at 278 nm of the supernatant after standing overnight. The extinction
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coefficient of the purified glycinin and β-conglycinin was determined by measuring the
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absorption of a series of solutions with known concentrations and was found to be 0.89 g/L/cm
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and 0.95 g/L/cm, respectively. In the case of mixtures, intermediate extinction coefficients
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were used.
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Titration. Potentiometric titration of soy globulin solutions was done using an automatic
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titrator (TIM 856, Radiometer Analytical) using a probe for low ionic strength protein 7 ACS Paragon Plus Environment
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solutions. The pH electrode was calibrated by a three-point calibration. The pH of all samples
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was first set to 8.0 with a standard solution of NaOH (0.1 M) and then titrated to pH 3 with a
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standard solution of HCl (0.1 M). Titrations were done at room temperature a rate of 0.08 ml
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per min while stirring. The variation of the charge density of the soy globulins (∆α) expressed
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in mol/kg was calculated as a function of the pH using the following equation:
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∆ () =
×
(7)
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where V is the volume of the HCl solution with molar concentration [HCl] that was added, m is
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the mass of soy globulin in the solution. The real charge densities (α) were subsequently
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calculated assuming that α=0 at the iso-ionic point, see below.
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Effect of the pH and the Ionic Strength on the Charge Density. The glycinin and the
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β-conglycinin samples were titrated (Figure 1) with HCl at C=10 g/L and the charge density (α)
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expressed in mol per kg protein was calculated as explained in the experimental section. Salt
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screens electrostatic interactions and therefore renders it easier to charge the proteins. As a
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consequence, the proteins are more strongly charged (positively or negatively) at a given pH
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when salt is added. A similar effect is obtained when the protein concentration is increased due
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to the increase of the counterion concentration. We have shown this elsewhere in more detail
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for SPI.7 The titration curves obtained at different NaCl concentrations cross at the so-called
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isoionic point (IIP) where the net charge of the proteins is zero.40, 41 Here we find IIP=5.2 for
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glycinin and IIP= 4.7 for β-conglycinin. Expressed in mol/kg, β-conglycinin is much more
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strongly negatively charged than glycinin for pH>5 (notice the difference in scale).
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As was mentioned in the introduction, the isoelectric point where the zeta potential is zero
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was found to be 5.0 and 4.2 for purified glycinin and purified β-conglycinin, respectively.3, 6
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The IIP differs from the IEP if there is binding of ions to the proteins, which changes the zeta
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potential.41 Another explication for the difference between the IIP values found here and the
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IEP values reported in the literature could be the difference in composition of the purified
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glycinin and β-conglycinin.
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8 no salt 0.03 M 0.3 M
7
7
6
6
pH
pH
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5
5
4
4
β-conglycinin
glycinin 3 -0.4 -0.2
0.0
0.2
0.4
0.6
0.8
3 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0
1.0
−3
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α (10 mol/kg)
α (10 mol/kg)
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Figure 1. Titration curves of glycinin and β-conglycinin at C=10 g/L and different NaCl
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concentrations indicated in the figure.
174 175
Effect of pH on the Self-assembly of Native Glycinin in Salt Free Solutions. Figure 2
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shows the evolution of the turbidity of glycinin solutions at 10 g/L as a function of time after
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reducing the pH to different values down to 6.3 starting from pH 7.4. The turbidity increased
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significantly after setting the pH below 6.8 due to self-assembly of the proteins and it slowly
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continued to increase further with time. At pH5.9 is curious in the sense that high NaCl concentrations
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inhibit phase separation even though electrostatic repulsion is more strongly screened. This
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behaviour has already been reported in the literature 8, 13, 14 and Lakemond et al. suggested that
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it was caused by the influence of salt on the structure of glycinin.8 At neutral pH and [NaCl] >
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0.5 M, glycinin is a hexamer of six subunits, each of which consists of an acid and a basic
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polypeptide linked by disulfide bonds. Lakemond et al. showed that the acidic polypeptides are
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more exposed at high ionic strength.8 Acidic polypeptides are more hydrophilic and soluble
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than the basic polypeptides.45, 46 As a consequence of the rearrangement of the basic and acid
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polypeptides, the short range attractive interaction decreases with increasing NaCl
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concentration. It appears that the effect of screening electrostatic repulsion dominates up to
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about 0.1 M NaCl and the effect on the short attraction is more important at higher NaCl
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concentrations causing a maximum rate and extent of phase separation at about 0.1 M NaCl.
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A further complication at all pH is that glycinin does not phase separate in the form of
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individual proteins, but in the form of small aggregates. The size of these aggregates increases
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when the electrostatic repulsions are decreased and the protein concentration is increased.
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Elsewhere we showed for SPI that the aggregates have a self similar structure which is
396
characteristic for randomly aggregating particles.7 Aggregation causes a reduction of the
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mixing entropy and therefore favors phase separation. It explains why native globular proteins
398
such as β-lactoglobulin that are soluble at the IIP in the native form become insoluble when
399
they have formed aggregates.
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Remarkably, heating glycinin solutions at pH≈7 above 60°C just after addition of 0.1 M
401
NaCl caused transformation of homogeneous microdomains into microcapsules with diameter 24 ACS Paragon Plus Environment
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radius of 3-30 µm and a wall thickness of about 1 µm. Microcapsules were also sporadically
403
observed at 20°C, but only between pH 4-6.4 and 0.4-0.5 M NaCl. As was mentioned in the
404
introduction, formation of protein microcapsules has been reported before, but it involves
405
emulsification with an incompatible liquid followed by coacervation or spray drying.
406
Spontaneous formation of microcapsules by pure proteins solutions has not been reported
407
before.
408
The microcapsules formed at T≥60°C were stable after dilution in salt free water whereas
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the micro domains formed at 20°C completely dispersed, implying that strong crosslinks were
410
formed by heating. Most micro domains transformed into microcapsules when they were
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heated above 70°C that was when the denaturation temperature of the glycinin is approached.
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We may speculate that microcapsule formation is induced by heat induced configurational
413
changes of the glycinin structure, that increase attractive interaction and drive densification of
414
glycinin within the micro domains. The process is fast as little difference was observed
415
between heating during 5 min and during an hour. However, it is not clear why the glycinin
416
moves from the center to the periphery of the micro domains nor why the thickness of the shell
417
depends very little on the size of the microcapsules.
418
The diameter of the microcapsules is determined by the size of the micro domains.
419
Therefore, larger microcapsules are formed at higher protein concentrations, because micro
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phase separation is faster at higher concentrations so that the micro domains are larger. We
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speculate that increasingly indistinct agglomerates were formed above 15 g/L because the
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micro domains started to fuse during the heating. Clearly, more work is needed to elucidate the
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molecular mechanism. 25 ACS Paragon Plus Environment
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Addition of β-conglycinin reduced the pH range in which phase separation occurred. This
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can be explained by co-aggregation of β-conglycinin with glycinin, which changes the balance
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of attractive and repulsive interactions between the aggregates. It was already reported that the
427
presence of β-conglycinin reduces the aggregate size during thermal aggregation of soy
428
proteins.17, 47 Interestingly, β-conglycinin inhibited formation of microcapsules and instead
429
stable homogeneous microgels were formed during heating, perhaps because it reduced the
430
attractive interaction between glycinin that may be the driving force for densification of the
431
latter at the periphery.
432 433
Conclusion
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Setting the pH of salt free glycinin solutions between 5.9 and 4.5 leads to micro phase
435
separation of a fraction of the proteins into dense protein microdomains that spontaneously
436
cluster and sediment. The pH range where this phenomenon occurs widens when NaCl is added
437
up to [NaCl]=0.1M where it occurs in the pH range 7.5-4.0. The upper pH limit reduces again
438
when more NaCl is added. Clustering of protein microdomains is much reduced close to
439
neutral pH. Addition of β-conglycinin reduces the pH range in which microphase separation
440
occurs. The microdomains redisperse easily when the pH is increased or the ionic strength is
441
decreased, but this does not occur when the solutions are heated implying that strong bonds are
442
formed between the proteins during heating.
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The present investigation shows that microcapsules with diameters between 3 and 30 µm
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can be formed simply by heating micro phase separated glycinin solution above 60°C at pH
445
close to neutral and NaCl concentration close to 0.1 M. The protein concentrations should be 26 ACS Paragon Plus Environment
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between 4 and 15 g/L depending on whether larger or smaller microcapsules are desired, but
447
we stress that in all cases, a polydisperse size distribution is obtained. There is no need to use
448
glycinin with high purity, but if soy protein sample contains more than about 20%
449
β-conglycinin, microgels are formed instead, which may be desirable for some applications.
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The soy protein microparticles discussed here were stable to dilution in 0.1 M NaCl or in salt
451
free water and to heating up to 90°C. Protein microcapsules and microgels are of interest for
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encapsulation, delivery and controlled release of sensitive ingredients. A more detailed
453
investigation of the properties of these microparticles regarding their capacity to encapsulate
454
ingredients, the permeability of the shell, etc was outside the scope of the present investigation.
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Further research will be needed to assess the potential for application of the soy protein
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microparticles produced by the relatively easy method reported here.
457 458
Acknowledgement Anna Kharlamova is thanked for performing the titration measurements.
459 460
Associated Content
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Supporting Information
462
The Supporting Information is available free of charge on the ACS Publications website.
463
Reducing SDS- PAGE patterns of purified glycinin and β-conglycinin and the dilute top phase
464
and dense bottom phase of glycinin with [NaCl]=0.1M.
465
CLSM images of glycinin solutions and glycinin/ β-conglycin mixtures at various
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concentrations with [NaCl]=0.1M
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CLSM images of microcapsules obtained by heating at 70oC or 90°C for different durations. 27 ACS Paragon Plus Environment
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References
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E.; Nakagawa, S.; Mikami, B.; Utsumi, S. Crystal structures of recombinant and native
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soybean β‐conglycinin β homotrimers. Eur. J. Biochem. 2001, 268, 3595-3604.
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(7) Chen, N.; Zhao, M.; Chassenieux, C.; Nicolai, T. Structure of self-assembled native soy
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