Ionic Strength and pH Responsive Permeability of Soy Glycinin

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Ionic strength and pH responsive permeability of soy glycinin microcapsules Nannan Chen, Jinglin Zhang, Lei Mei, and Qin Wang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01559 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Ionic strength and pH responsive permeability of soy glycinin microcapsules Nannan Chen1, Jinglin Zhang1, Lei Mei1, Qin Wang1,2,* 1

Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, United States of America 2

College of Food and Bio-engineering, Xihua University, Chengdu 610039, China

Please send correspondence to: *Qin Wang, Associate Professor Department of Nutrition & Food Science University of Maryland College Park 0112 Skinner Building College Park, MD 20742 Phone: (301) 405-8421 E-mail:

[email protected]

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Abstract Recently, hollow protein microcapsules have been made simply by heating the microphase separated soy glycinin microdomains. However, the properties (e.g. mechanical properties and permeability) that relate to the application of these microcapsules are unknown. In this study, the permeability of the soy glycinin microcapsules was investigated by confocal laser scanning microscopy (CLSM) as influenced by ionic strength and pH using fluorescein isothiocyanatedextran (FITC-dextran). The glycinin microcapsules kept the integrity between pH 1 and 11.5, swelled when pH was below 3 or above pH 11. The glycinin microcapsules would dissociate at pH above 11.5 and de-swell slightly at pH 1. When pH increased above 11, the permeability of the microcapsule significantly increased. Remarkably, when pH was below the isoelectric point of glycinin (≈pH 5), FITC-dextran spontaneously accumulated inside the microcapsule with a significantly higher concentration than that in bulk solution as evidenced by the strong intensity increase of fluorescence. This unique feature significantly increased the loading amount of FITCdextran. Permeability of microcapsules was also increased by adding salt but less significant than by adjusting pH. The surface of the microcapsules became coarser when the permeability increased, which was revealed by scanning electron microscopy. These results show that soy glycinin has a great potential to be used as the wall material to fabricate hollow microcapsules that could find applications in biomedicine and food industry. Keywords: Soy glycinin, phase separation, microcapsules, permeability, encapsulation

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Introduction Microcapsules, usually defined as spherical particles with the size varying between 50 nm to 2 mm with core-shell structures, act as physical barriers between the encapsulated compounds and the external medium.1 They could protect and control the release of sensitive ingredients, which are widely used in biomedicine and food industry. Both the materials and fabrication methods have influences on the properties of the microcapsules so as to their ability to carry lipophilic or hydrophilic cargos. Proteins have been proved to be robust materials to make microcapsules owing to their functional groups that can be easily modified and self-assembly capacities under different environment conditions as well as their desirable biocompatibility.1-3 Layer-by-layer (LbL) method with protein built on decomposable colloid templates has been developed to fabricate hollow protein microcapsules.1, 4-6 Crosslinkers are applied to further stabilize the protein on the templates. Thereafter, the colloid templates are dissolved by adding appropriate chemicals.7-8 Permeability of the microcapsules can be adjusted by controlling the number of layers and environmental conditions such as pH, ionic strength and temperature, so that the ingredients can be encapsulated and released by permeating in or out of the shell. Microcapsules made by LbL method have been successfully used to encapsulate hydrophilic compounds. The main drawbacks of the LbL technique are the tedious and time consuming fabrication process and the waste of materials.4 Another widely used method to make protein microcapsules is based on oil-in-water emulsions with protein located at the oil-water interface followed by spry-drying, coacervation etc. This method has been extensively used in food industry due to its low cost and high efficiency and has been successfully used to encapsulate many kinds of lipophilic bioactives, such as omga-3, curcumin, vitamin D etc.2, 9-11 However, it has an inherent disadvantage that the oil droplets are not suitable to dissolve hydrophilic

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compounds. Very recently, Chen et al. have fabricated soy protein microcapsules based simply on the salt induced microphase separation of soy glycinin, followed by subsequent heating.12 Soy glycinin is one of the major components of soy protein which is abundant, in low-cost, and with high nutritional value. The molecular weight of the soy glycinin (11S) is around 350 kg/mol.13 It is composed of six subunits and each subunit contains a basic and an acid polypeptide which are crosslinked by the disulfide bond.14 It is reported that soy glycinin microphase separates when NaCl concentration increases to around 0.1 M, forming spherical protein microdomains which are unstable.12 However, these protein microdomains transfers into stable hollow microcapsules with the diameter between 1–40 µm depending on the protein concentration when the microdomains are heated above 60 oC for a short period of time. Compared to the two widely used methods to prepare protein microcapsules as mentioned above, the method reported by Chen et al. has several advantages. First, it is much simpler as neither a template nor an oil phase is needed. Second, the glycinin microcapsules are readily dispersed in water after fabrication with water filled cores, which are easy to be isolated and become superior candidates to encapsulate the hydrophilic components. Third, the stabilization of the protein on the microcapsule shell is dependent on the non-reversible aggregation of protein under heating due to the denaturation so no additional crosslinker is required. Further application and modification of these microcapsules necessitate the investigation of their properties in details which however have not been explored. Herein, we fabricated the soy glycinin microcapsules based on the NaCl induced microphase separation with subsequent heating as reported by Chen et al.12 We used fluorescein isothiocyanate (FITC) labeled dextran with different molecular weight (Mw) as representative 4


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hydrophilic biomacromolecules to investigate the permeability of the microcapsules. The microstructure was observed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results indicated that the permeability of soy glycinin microcapsules showed pH and ionic strength responsive property. Moreover, a spontaneous accumulation effect for FITC-dextran in acidic condition was found which however has not been reported with any other protein microcapsules. The possible mechanism of the permeability change under different conditions was also discussed. By tuning the permeability of the microcapsules, FITC-dextran was successfully encapsulated.

Experimental Section Materials. Defatted soy flour was purchased from Shandong Yuwang Group (P. R. China), defined as source A and Xinjiahua company (P. R. China) defined as source B. Defatted soy flour (Prolia®FLR-200/70) was a gift from Cargill (Cargill, Cedar Rapids, USA), defined as source C. Rhodamine B, FITC-dextran and sodium bisulfite were purchased from Sigma-Aldrich (St. Louis, MO. USA). According to the manufacturer, the Stokes’ radii of FITC-dextran with Mw of 20, 500, and 2000 kDa was 3.3, 15 and 27 nm, respectively. Isolation of soy glycinin. Preparation of glycinin followed the method of Nagno et al. with slight modification.15 Briefly, the defatted soy flour was dispersed in 15-fold water in weight and adjusted to pH 7.5 with 2 M NaOH. This slurry was then centrifuged (9000 g, 30 min) at 4 oC. Dry sodium bisulfite (SBS) was then added to the supernatant (0.98 g SBS/L), the pH of the solution was adjusted to 6.4 with 2 M HC1, and the obtained turbid dispersion was kept in 4 oC overnight. After that, the dispersion was centrifuged (6500 g, 30 min) at 4 oC. The precipitates which turned out to be glycinin were dispersed in 3 fold water and the pH was adjusted to pH

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7.2. The glycinin solution was then dialyzed again Milli-Q water for two days at 4 oC and then freeze-dried. Preparation of glycinin solution. Protein solutions of 20 g/L were prepared in salt-free Milli-Q water with 3 mM NaN3 to avoid bacterial growth. All solutions were centrifuged at 15000 g for 30 min to removed insoluble materials. The pH of the stock solution was adjusted by adding 0.1 M NaOH or HCl. The protein concentration was determined by measuring the UV absorption at 278 nm using an extinction coefficient of 0.89 L cm-1 g-1. Preparation of soy glycinin microcapsules. Soy glycinin microcapsules were made by inducing the microphase separation of glycinin solution with adding NaCl, followed by immediately heating (Figure 1).12 Specifically, at 20 oC and under vigorous stirring (700 rpm), soy glycinin solutions (20 g/L) with pH 7.2 were mixed with equal volume of 0.1 M NaCl to a final NaCl concentration of 0.05 M and protein concentration of 10 g/L. The samples were then immediately heated in a thermostat bath at 80 oC for 20 min with an accuracy of ± 0.2 °C. Isolation of the microcapsules was by centrifugation at 3000 g for 3 min. The isolated microcapsules were then dispersed in pure water. Heating

NaCl

Glycinin solution Microphase separation

Microcapsules

Figure 1. Schematic illustration of the procedure for preparation of soy glycinin microcapsules.

Confocal laser scanning microscopy (CLSM). Microcapsule dispersion was mixed with Rhodamine B to investigate the morphology of microcapsules or FITC-dextran to study the permeability by CLSM. The diffusion fraction of FITC-dextran was estimated by dividing the 6


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fluorescence intensity inside the microcapsule by that in the bulk solution. The final concentration of Rhodamine B was 5 ppm and that of FITC-dextran was 1 mg/mL. In order to investigate the morphology and permeability under different environmental conditions, the pH of the microcapsule dispersion was adjusted by adding 0.1 M NaOH or HCl and the ionic strength of the solution was adjusted by adding concentrated NaCl solution. Confocal images of microcapsule dispersion were obtained at 20 °C with a Zeiss LSM 710 Confocal Microscope (Zeiss, Germany). A water immersion objective lens (63×/1.2NA) was used. The incident light was emitted by a laser beam at 561 nm for Rhodamine B and at 488 nm for FITC. The fluorescence intensity was recorded between 568 and 650 nm for Rhodamine B and between 492 and 544 nm for FITC. Samples were inserted between a concave slide and a coverslip. The images were taken after 18 h, unless otherwise indicated. Scanning electron microscopy (SEM). SEM imaging of protein microcapsules was performed on a Hitachi SU-70 scanning electron microscope (Pleasanton, CA, USA) with an electric current of 5 kV in vacuum. A drop of microcapsules dispersion was air-dried at room temperature on silicon wafers and then sputter-coated with gold before imaging.

Results and Discussion Influence of protein source on the microcapsule formation. Soy glycinin was extracted from three sources of defatted soy flour obtained from three companies as indicated in the experimental section. The microcapsules fabricated under the same conditions were compared. It was showed that glycinin extracted from source C formed bigger microcapsules than those from sources A and B (Figure 2). For glycinin extracted from source A, more microgels (solid not hollow structure) were formed, accompanying by some small microcapsules. The difference on

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formation of microcapsules that varied in size between the three different sources of glycinin under the same condition might be caused by the various proportions of different groups of subunit of glycinin which had different chemical and physical characters in salt solution.16-18 Small amounts of free basic polypeptides were present in glycinin extracted from source B and C as observed by the non-reducing SDS-PAGE (Figure S1), which however was not present in glycinin from source A. The reducing SDS-PAGE patterns of all samples were similar (Figure S1). Obviously, more work is needed to reveal the key components that lead to the microcapsules formation, which is under investigation in our research group. In the rest of this study, we have used the glycinin extracted from source C.

Figure 2. CLSM images of microcapsule dispersion obtained from different sources in the presence of 5 ppm rhodamine B. The scale bar is 10 µm.

Influence of NaCl and pH on the morphology of microcapsules. After the microcapsules were isolated and re-dispersed in water, the pH of the solution was around 7 without adding salt. We then adjusted the ionic strength of the microcapsule dispersion by adding concentrated NaCl solution. The corresponding pH of the dispersion varied between 6.4 at highest ionic strength (0.4 M NaCl) and 7 at the lowest one without adding salt. CLSM images showed that the diameter and shell thickness were not influenced in the range of salt concentration investigated and no clustering of the microcapsules was observed either (Figure S2). The surface morphology 8


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was examined by SEM (Figure 3). After water evaporation, the microcapsules collapsed to a pancake like morphology without folds and creases. The surface of microcapsules became coarser and grainier after adding salt with visible pores. However, the differences between various ionic strengths were not significant (Figure S3). The change of the surface morphology was thought to be caused by the screening effect of salt which changed the electrostatic interaction between protein side chains. It has been found that at room temperature, the stability of soy glycinin was sensitive to the change of ionic strength with significant association when NaCl concentration was around 0.1 M, which resulted in the microphase separation of glycinin, forming spherical microdomains.12 By increasing or decreasing the ionic strength, the microdomains gradually disappeared, indicating this kind of aggregation was reversible. The soy glycinin microcapsules which were formed by heating the soy glycinin microdomains however, showed increased stability to ionic strength change compared to glycinin before heating (Figure S2), indicating that the aggregation induced by heating were irreversible. By dispersing the microcapsules in 1% sodium dodecyl sulfate (SDS), we observed a significant decrease in the turbidity (Figure S4a). SDS was reported to destroy the hydrophobic interaction.19 The decrease of the turbidity suggested that hydrophobic interaction was important in stabilizing the microcapsule structure. Reducing and non-reducing SDS-PAGE were further performed for the soy glycinin microcapsules (Figure S4b) to see if disulfide bonds were formed. The nonreducing SDS-PAGE of glycinin microcapsules showed some protein aggregates accumulated on top of the gel which however was not observed for glycinin before heating (Figure S1). These aggregates dissociated and entered the gel under reducing SDS-PAGE (Figure S4b). The sample buffer of the reducing SDS-PAGE contains dithiothreitol (DTT), which can break the disulfide bonds. These results indicated that apart from hydrophobic interaction, intermolecular disulfide

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bonds were formed during heating.

Figure 3. SEM images of soy glycinin microcapsule at 0 (a, b) and 0.0125 M NaCl (c, d) after drying. The scale bar is 2 µm in a and c, and 300 nm in b and d.

To study the effect of pH on the morphology, the pH of the microcapsule dispersion without adding salt was adjusted and the microstructure of the dispersion at each pH was observed with CLSM. Representative images were shown in Figure 4. When the pH was between 4 and 6, the clusters of microcapsules were observed. The microcapsules began to re-disperse when the pH was below 4 or above 6. This phenomenon was due to the change of net charge density of soy glycinin which was zero at the isoelectric point (≈pH 5).12, 20 When pH was away from the isoelectric point, the electrostatic repulsion between proteins gradually increased.20 Interestingly, we found that small amount of free glycinin that did not participate in constructing the shell was entrapped inside the core of the microcapsules. Between pH 6 and 4, the free glycinin aggregated into clusters as the pH was near the isoelectric point (Figure S5). This free glycinin may come from the destroying of the hydrogen bonding between proteins in the microdomain during 10


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heating. The free glycinin inside the microcapsule is supposed to induce a self-accumulation phenomenon which will be discussed later. The microcapsules were stable down to pH 1. However, when the pH increased to 12, the microcapsules dissociated and lost their integrity (Figure 4). The microcapsules at pH 11.5 also lost the integrity with time. Jiang et al. have investigated the structural change of soy glycinin at extreme pH levels.21 Under both extreme acidic and alkali conditions, soy glycinin unfolded but structural change of the soy glycinin was much more significant at the extreme basic pH than that at the extreme acidic pH. Significant increase of surface sulfhydryl groups with time were found at pH 12 which might be due to the breakdown of disulfide bonds.21 While at acidic conditions, the change of surface sulfhydryl group was less significant.21 Decomposition of protein disulfide bonds in dilute alkali solution was also reported earlier.22-24 The non-reducing and reducing SDS-PAGE for the microcapsules (Figure S4b) have shown that disulfide bonds between molecules were formed when glycinin transferred into the microcapsules. Therefore, the dissociation of the microcapsules at extreme alkali condition may be partially due to the breakdown of disulfide bonds. The swelling of the microcapsule was observed when pH > 11 or pH < 3 (Figure 4). The diameter increased from 4–14 µm at pH 7 to 6–24 µm at pH 2 and pH 11.5 while the shell sickness increased from an average of around 0.9 µm at pH 7 to around 1.4 µm at the extreme pH. When 3 < pH < 11, the diameter and shell thickness were not significantly changed (Figure S6). The swelling of the microcapsules were caused by the strong electrostatic repulsion between protein side chains when the pH was far away from the isoelectric point (≈pH 5).12, 20 However, at pH 1, the microcapsule de-swelled slightly compared to that at pH 2 (Figure 4) which was due to the increased ionic strength that screened the charge of the protein molecules caused by the

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larger amounts of HCl needed to the lower the pH to 1. It is also found for bovine serum albumin (BSA) and silk protein microcapsules that they only swelled at an extreme acidic or alkali condition while no shrinking or swelling was found in a wide range of pH. This might be that the repulsion force between protein molecules was not strong enough in the intermediate pH range.78

The SEM images in Figure 5 showed the surface morphology of the microcapsule as influenced by pH. At neutral pH, the surface of the microcapsule was relatively smooth. When the pH was adjusted from neutral to acidic or alkali conditions, the surface of the microcapsule became much coarser and grainier. At the acidic condition, the surface was rougher and with more visible pores than that at the alkali condition. Ye et al. found similar morphology change when changing the pH of silk ionomer microcapsule dispersion.8 They considered the changes in morphology of silk ionomer capsules were related to a reduction of ionic bonding at extreme alkali and acidic pH. The variation in surface morphology and swelling character by changing pH or ionic strength may suggest a tunable permeability of the soy glycinin microcapsules.

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Figure 4. CLSM images of soy glycinin microcapsule dispersion under different pH in the presence of 5 ppm rhodamine B. The scale bar is 10 µm.

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Figure 5. SEM images of soy glycinin microcapsules at different pH after drying. The scale bar is 500 nm.

Influence of ionic strength on the permeability. FITC-dextran was used as a representative hydrophilic biomacromolecule to investigate the permeability of the soy glycinin microcapsule. The microcapsule dispersion without adding salt at neutral pH was incubated with FITC-dextran with different molecular weight (Mw). If the pore size of the microcapsule was large enough, the FITC-dextran would permeate through the shell. As shown by Figure S7, the amounts of FITC-dextran in the interior of the microcapsules decreased with increased Mw of FITC-dextran. The diffusion fraction for FITC-dextran with Mw of 20 kDa was about 80% while that for 2000 kDa was about 20%. The soy glycinin carries net negative charge at neutral pH and FITC-dextran is slightly negatively charged so the shells of the microcapsules remained dark due 14


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to the electrostatic repulsion force.25-26 The FITC-dextran with Mw of 2000 kDa was chosen for the following study as the indicator for the permeability change of microcapsules. Figure 6a shows the microcapsule dispersion before adding salt. When 0.05 M NaCl was added, the fluorescence intensity of the FITC-dextran inside the microcapsules increased significantly which was about 58% of that in bulk solution (Figure 6b). However, further increasing the salt concentration did not lead to higher diffusion fraction (Figure S8). It has been suggested that the charge shielding effect caused by increased ionic strength could induce a transition of a dense polyelectrolyte complex to a loose structure, which triggers an increased permeability of the microcapsules.27-28 On the other hand, the pH of the dispersion was above the isoelectric point (≈ pH 5) of soy glycinin so the microcapsules carried net negative charges. Increase in the screening effect of salt ions on the negative charges in the microcapsule could reduce the repulsion between FITC-dextran with the negative charged groups of proteins so that increased the permeability.26, 29 The microcapsule dispersion as shown in Figure 6b was then centrifuged under 3000 g for 3 min and the precipitate was re-dispersed in pure water. It was shown that the FITC-dextran had been successfully encapsulated (Figure 6c). The concentration of FITC-dextran inside the microcapsules remained unchanged even after one month (Figure S9). However, when the ionic strength was increased back to 0.05 M, the FITC-dextran would permeate out of the microcapsules (Figure 6d). The releasing was quite fast. After 1.5 h, about 75% of the FITCdextran inside the microcapsule was released. (Figure S9). One week later, FITC-dextran was completely leaked out. The releasing speed among different samples with different ionic strength was not significantly different (Figure S9). These results indicated that the permeability of the soy glycinin microcapsules can be reversibly adjusted by changing ionic strength.

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Figure 6. CLSM images of the soy glycinin microcapsule dispersion exposed to FITC-dextran (2000 kDa) at neural pH without NaCl (a), with 0.05 M NaCl (b), removal of salt and dispersed in pure water (c) and again, adding 0.05 M NaCl (d). Scale bar is 10 µm.

Influence of pH on the permeability. The pH of the soy glycinin microcapsule dispersion in the presence of 2000 kDa FITC-dextran was adjusted without adding salt and the CLSM image was taken after 18 h (Figure 7). When pH was increased from pH 6.7 to 11.5, the concentration of FITC-dextran inside the microcapsule gradually increased. At pH 11.5, the fluorescent intensity inside the microcapsule was almost as the same as that in bulk solution indicating that most of the FITC-dextran permeated through the shell (Figure S10). The equilibrium at pH 11.5 could reach within 3 hours. Between pH 6 and 6.7, the permeability is relatively low. Further decreasing the pH, the concentration of FITC-dextran inside the microcapsule not only increased but also gradually became higher than that in bulk solution which was quite obvious down to pH 4.5 (Figure 7). At pH 4.5, the concentration of FITCdextran was about 5 times higher than that in the bulk solution as evidenced by the fluorescent 16


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intensity (Figure S10). This was intriguing as the FITC-dextran could spontaneously diffuse from the low concentration area to the high concentration microcapsule interior region. This indicated that decreasing the pH below the isoelectric point, the permeability of the microcapsules not only increased but there was a strong attraction force that has led FITCdextran to self-accumulate inside the microcapsules. This driving force made the concentration of FITC-dextran inside the microcapsules much higher than that in bulk dispersion. The intensity of the shell also increased slightly with reducing pH down to pH 4.5 but with further decreasing pH, it increased sharply and became higher than that of the interior of the microcapsule (Figure 7). The interior concentration of the FITC-dextran was still higher than that in the bulk solution when pH was lower than 4.5 which could be best seen later when the pH was adjusted back to 7 to avoid the influence of acidic pH on the fluorescence intensity (Figure 9).

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Figure 7. CLSM images of the soy glycinin microcapsule dispersion exposed to FITC-dextran (2000 kDa) under different pH conditions. The scale bar is 10 µm.

Many protein microcapsules have been reported to have the pH responsive permeability change which is caused by the change of charge upon pH variation that might induce reorganization of the wall structure.7-8, 30-31 Our results of SEM confirmed the change of wall structure when the microcapsules were exposed to alkali or acidic conditions as observed with increased roughness which was more significant in acidic conditions (Figure 5). The increased permeability was accompanied by an increased surface roughness which was also observed by Ye et al.8 However, none of these previous reports on protein microcapsules observed the accumulation effects as we are reporting here. This accumulation effect could be best seen when 18


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we looked into the process with time. For example, at pH 4.5, the fluorescent intensity of shell increased sharply just after sample preparation (Figure 8a). At the same time, the free glycinin that entrapped inside the microcapsules also attracted FITC-dextran as it showed significant fluorescent intensity of the protein clusters (Figure 8d). Interestingly, with time, the fluorescent intensity of the shell decreased while that of the interior increased (Figure 8b). Finally, the fluorescent intensity of the interior became higher than that of the shell and the bulk solution (Figure 8c). Similar phenomenon was observed between pH 4 and 5. However, decreasing pH slowed down the decrease of fluorescent intensity of the shell while speeding up the increase of fluorescent intensity inside the microcapsules. Below pH 3.5, the fluorescent intensity of the microcapsules did not change significantly over time. The isoelectric point of the glycinin is around pH 5, below which it carries net positive charge. Therefore, we proposed that the selfaccumulation phenomenon was due to the increasing positive charged groups of the remaining free soy glycinin inside the microcapsules with decreasing pH that enhanced the electrostatic attraction with the slightly negatively charged FITC-dextran. The self-accumulation effect has also been found in some colloidal template-based polymer microcapsules which is due to the remaining of the dissolved core materials in the microcapsules that have opposite charge with the encapsulated components.25, 32-34 When decreasing pH blow the isoelectric point, the fluorescent intensity of the shell increased sharply which was due to the electrostatic attraction between FITC-dextran and soy glycinin. However, near the isoelectric point (pH 4-5), this interaction was weakened with time as observed by the decreasing fluorescent intensity. Possibly, this is a pH domain where recharging and rearrangement processes take place which we have not yet understood. When pH was blow pH 4, there were more net positive charges on the shell so that it enhanced the absorption of the FITC-dextran.

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Figure 8. CLSM image of soy glycinin microcapsule dispersion at pH 4.5 exposed to FITC-dextran (2000 kDa) at 3 min (a, d), 3 h (b, e) and 16 h (c, f). The scale bar is 20 µm in a, b and c, and 5 µm at d, e and f.

The pH of microcapsule dispersion (pH ≤ 5) in the presence of 2000 kDa FITC-dextran was adjusted back to 7 to compare the loading amount under the same pH as the fluorescence of FITC was influenced by pH (Figure 9). The amount of entrapped FITC-dextran increased with decreasing initial pH till pH 3.5. This was due to the increasing positive charge of the free soy glycinin inside the microcapsule with reducing pH that contributed to the deposition of more FITC-dextran through electrostatic attraction. The concentration the FITC-dextran inside the microcapsule deposit at pH 3.5 could reach 8 times higher than that in bulk solution. However, further reducing the pH had no contribution to accumulate more FITC-dextran which might be due to the increased ionic strength at extreme low pH that screened the protein and FITC-dextran interaction.35 Even though increasing pH to 7 would destroy the electrostatic attraction between the soy glycinin and FITC-dextran that was formed at acidic conditions, the concentration of 20


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FITC-dextran was still higher inside the microcapsules. This was due to the decreased permeability at neutral pH so that the FITC-dextran deposit at acidic conditions was captured.

Figure 9. CLSM images of the soy glycinin microcapsule dispersion exposed to FITC-dextran (2000 kDa) at different pH of 5 (a) 4.5 (b) 4 (c) 3.5(d) 3 (e) and 2 (f) with subsequently adjusting the pH to 7. The scale bar is 10 µm.

Finally, the soy glycinin microcapsule dispersion in the presence of FITC-dextran was centrifuged, the pH of which was first adjusted to 3.5 and subsequently adjusted back to 7. After centrifugation, the precipitate of the microcapsules was re-dispersed in pure water. It was found that the concentration of FITC-dextran deposit inside the microcapsules did not change even after one month (Figure 10a,b). When 0.05 M salt was added, the FITC-dextran concentration in 21


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the microcapsules only slightly decreased after one month (Figure 10c,d).We observed similar phenomena at other ionic strength. When the pH of the dispersion without salt was increased to 11.5, swelling of the microcapsules were observed with slightly release of FITC-dextran after 1.5 h (Figure 10e) and after one day, almost all FITC-dextran was released (Figure S11). The microcapsules lost its integrity with time at this extreme alkali condition, accompanied by the complete release of FITC-dextran and no microcapsules were observed (Figure 10f). The release of FITC-dextran at other alkali pH below pH 11.5 was very slow (Figure S11). Compared to the microcapsules without initially adjusting pH to 3.5, the permeability of the microcapsules after the acid treatment was less sensitive to ionic strength and pH change. This indicated that the change of permeability induced by pH modification was only partially reversible. This may be due to some irreversible structure changes of soy glycinin at acidic condition as reported by Jiang et al.21, 36

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Figure 10. CLSM images of the soy glycinin microcapsules loaded with FITC-dextran (2000 kDa), dispersing in pure water without NaCl (a, b) with 0.05 M NaCl (c, d) and at pH 11.5 without NaCl (e, f). Images were taken after 1.5 h (a, c, e) and one month (b, d, f). The scale bar is 20 µm.

Conclusions Protein microcapsules fabricated by heating salt induced microphase separated soy glycinin dispersion showed good stability in a wide pH range. It swelled at pH > 11 and pH < 3, caused by the strong electrostatic repulsion when pH was far away from the isoelectric point. But it dissociated when pH > 11.5 which was thought to be caused by the dissociation of the disulfide bonds that stabilized the microcapsule structure. Both increasing the ionic strength and adjusting 23


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pH away from neutral led to an increased roughness of the microcapsule surface accompanied by an increased permeability. By adjusting ionic strength or pH, FITC-dextran was successfully encapsulated which did not leak out in the salt free neutral solution. Increasing the ionic strength or changing the pH, the encapsulated FITC-dextran was released. The encapsulation efficiency was significantly higher by adjusting pH below the isoelectric point where the microcapsules showed strong accumulation effect for FITC-dextran which was due to the electrostatic attraction between FITC-dextran and the free glycinin inside the microcapsules. By exploiting the spontaneous selfaccumulation effects, FITC-dextran can be successfully encapsulated in larger quantity than that by adjusting ionic strength.

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Associated Content Supporting Information Reducing and non-reducing SDS-PAGE of soy glycinin extracted from different soy flours and soy glycinin microcapsules. Effects of salt on the morphology of soy glycinin microcapsules. Permeability of soy glycinin microcapsule in the presence of FITC-dextran with different Mw and NaCl concentration. Influence of time, pH and ionic strength on the releasing of FITCdextran. Intensity analysis of CLSM images.

Acknowledgement This study is partially supported by Maryland Soybean Board. We are grateful to imaging core at the University of Maryland for providing support of CLSM measurement. Thanks also go to Dr. Yang Yuan and Dr. Qingzhu Zeng for their helps on the idea generation of this study.

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Ionic Strength and pH Responsive Permeability of Soy Glycinin

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