Nano-structure of octenyl succinic anhydride modified starch micelle

Food Hydrocolloids 32 (2013) 1e8

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Nano-structure of octenyl succinic anhydride modified starch micelle Jie Zhu, Lin Li, Ling Chen, Xiaoxi Li* Ministry of Education Engineering Research Center of Starch & Protein Processing, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 July 2012 Accepted 29 November 2012

The micelle nano-structures of octenyl succinic anhydride (OSA-starch) modified starches with different Mw (weight average molecular mass) in aqueous solution were studied. The micelle aggregates observation, maximum dimension (rmax) calculated from Pair Distance Distribution Function p(r) using General Indirect Fourier Transformation (GIFT) and radius of gyration (Rg) obtained from Guinier fitting of the small angle X-ray scattering data indicated the obvious geometric disparity of micelles originated from different OSA-starches. The micelle presented increased monodispersity in more concentrated aqueous solution. The OSA-starch molecules with the highest Mw formed the largest micelle. Hydrodynamic radius (Rh) via dynamic light scattering of the micelle with the highest Mw decreased in larger extent (from 80 nm to 46 nm), as well as more distinct change of Rg/Rh with increasing concentration, from which it could be deduced that the micelle conformation with higher Mw was more flexible and more sensitive to the interparticle electrostatic repulsion. Moreover, the most compact interior structure formed within the micelle with the highest Mw. The p(r) plots show typical profiles yielded from some oblate ellipsoid with a small subunit, long rod with a limited cross-section dimension and flattened prolate ellipsoid. It is anticipated that these nano-structures differences could be close related to stability and other properties of OSA-starch applied in oil-in-water system. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: OSA-starch Micelle Nano-structure Dimension Conformation

1. Introduction The chemical modification of starch with octenyl succinic anhydride (OSA) is achieved by a standard esterification reaction (Bhosale & Singhal, 2006; Liu et al., 2008). Hydrophobic octenyl succinic groups give the starch molecule amphiphilic nature and thus surface active properties, which have been applied in the formation and stabilization of dispersed food system (Nilsson & Bergenståhl, 2006). The hydrophobic part of OSA-starch molecule contains a carboxylic acid which can be negatively charged (Magnusson & Nilsson, 2011; Nilsson & Bergenståhl, 2007). The substitution with OSA can occur at carbons 2, 3, and 6 in the glucose molecule and the typical degree of substitution (DS) for food applications is between 0.01 and 0.03 (Bai & Shi, 2011; Bai, Shi, Herrera, & Prakash, 2011; Nilsson & Bergenståhl, 2006; Shogren, Viswanathan, Felker, & Gross, 2000). OSA-starch acquires the ability to stabilize oil-in-water emulsions by combining the hydrophobicity of the octenyl group with the hydrophilic carboxyl or sodium carboxylate groups (Segura-Campos, Chel-Guerrero, & Betancur-Ancona, 2008). Based on the amphiphilicity, OSA-starch is used as emulsifier and stabilizer in many food, cosmetics and * Corresponding author. Tel./fax: þ86 20 8711 3252. E-mail address: [email protected] (X. Li). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.11.033

pharmaceutical products (Liu et al., 2008; Ortega-Ojeda, Larsson, & Eliasson, 2005; Segura-Campos et al., 2008; Varona, Martín, & Cocero, 2009; Yusoff & Murray, 2011). Since widespread applications of OSA-starch in industrial products, the effects of reaction conditions on the synthesis of OSAstarch are of huge research interest. The production of OSA-starch was influenced by reaction condition factors, such as pH, temperature, starch concentration, and reaction time, and especially by the interaction of these factors (Liu et al., 2008; Ruan, Chen, Fu, Xu, & He, 2009; Segura-Campos et al., 2008; Shogren et al., 2000; Wang et al., 2011). During esterification, OSA groups first attack the surface and then some pores form with little change in the crystalline pattern (DS 0.045) (Ruan et al., 2009). The distribution of OSA within the modified starch granule and the effect of extent of modification on different physicochemical properties of starch also have been discussed (Bao, Xing, Phillips, & Corke, 2003; Ruan et al., 2009; SeguraCampos et al., 2008; Shogren et al., 2000; Thirathumthavorn & Charoenrein, 2006). OSA groups locate uniformly over the crosssection of modified waxy maize starch granules, which are probably distributed in the interior, amorphous domains of amylopectin molecules as well as on the outside of the granule (Shogren et al., 2000). Enzymatic pretreatment of native starch enhances the appearance of the OSA groups in the crystalline regions of the granules (Huang et al., 2010). It was found that the modification of

2

J. Zhu et al. / Food Hydrocolloids 32 (2013) 1e8

starch physical properties, including pasting viscosity, swelling volume, thermal temperatures and enthalpy, was dependent on not only the DS but also the plant origin of the starches (Bao et al., 2003). After enzyme treatment, the OSA-starches with DS ¼ 0.0208 showed an excellent emulsification capacity and stability (Liu et al., 2008). Stabilizing an emulsion results from the adsorption of OSAstarch molecules to the interface of water/oil (Nilsson & Bergenståhl, 2006; Ruan et al., 2009). The high surface loads, which closely depend on the DS, rms radius and molar mass (Nilsson & Bergenståhl, 2007), are mainly due to jamming at the interface caused by the polymer orientation (Nilsson & Bergenståhl, 2006). The existence of interactions between OSA-starch and some macromolecules in aqueous solution has been discussed (Krstonosi c, Dokic, & Milanovi c, 2011; Magnusson & Nilsson, 2011). The addition of xanthan gum, for instance, decreases the specific viscosity and increases the surface tension and the critical micellar concentration (CMC) values compared to the single OSA-starch solutions (Krstonosi c et al., 2011). The combination of xanthan gum with OSA-starch has been proved to present excellent stability properties (Ntawukulilyayo, De Smedt, Demeester, & Remon, 1996). Xanthan gum and OSA-starch interact through formation of inclusion complexes between OSA tails and xanthan gum helix. The interaction is affected by the concentrations of both components and facilitates the formation of the mixed micelles between them. Upon further increase in OSA-starch concentration, the complexes between formed mixed micelles occur. The presence of xanthan gum, as well as the structure of OSA-starch itself, influences the solubilization process of organic component in OSA-starch aqueous solution. The high molecular weight and branched polymer structure of OSA-starch may give rise to steric stabilization. The concentrations of OSA-starch and other macromolecules, as well as the mixing order, seem to affect the stability (Magnusson & Nilsson, 2011). However, in absence of other ingredients, the amphiphilic OSA-starch molecules above CMC (Krstonosi c et al., 2011; Varona et al., 2009) begin to form micelles via hydrophobic bonding, whereby the hydrophobic part forms the kernel of the micelle and hydrophilic part is oriented outwards to the water. Krstonosi c et al. (2011) previously reported that OSA-starch molecules curled and shrank at CMC in order to minimize contact between OSA groups and water molecules. The single molecules present smaller hydrodynamic size above CMC than that before CMC. The different micelle structure may relate to the properties as stabilizer in oil-inwater system. Consequently, the structure of micelles without other polymers should be of research interest. In aqueous solution the amphiphilic OSA-starch molecules form micelles via hydrophobic bonding. In this paper, we chose three different OSA-starches with different molecular weight and investigated the nano-structures of respective micelles. Knowledge of their behavior, when they are used in formulations, could be important to predict the stability and other properties in oil-in-water system. 2. Materials and methods 2.1. Materials Corn starch with different molecular weight was obtained from Huanglong Food Industry Co., Ltd. (Changchun, China). OSA was purchased from Aroma Chemical Co., Ltd. (Hangzhou, China). Three species of OSA-starch, respectively labeled with Sample 1, Sample 2 and Sample 3, were synthesized in the laboratory and all the species have a similar degree of substitution (DS z 0.02). The moisture contents of sample 1, 2 and 3, determined by Moisture Analyzer (MA35, Sartorius Stedim Biotech GmbH, Germany), were 7.88%, 8.17% and 7.45%.

2.2. Methods 2.2.1. Preparation of OSA-starch and OSA-starch aqueous solution The OSA-starch samples were synthesized in the laboratory according to the method previously reported by Bao et al. (2003) with some modifications. Corn starch with different molecular weight (50 g, dry weight) was suspended in water with agitation at 40% solid content, and the pH of the slurry was maintained between 8 and 9 by adding 3% (wt%) NaOH. Different amounts of OSA (3 or 5% based on the weight of starch) were added, and agitation was continued for 14 h at room temperature with pH maintained between 8 and 9 with dilute NaOH or HCl solution. Then pH was adjusted to 6.5 with dilute HCl; samples were collected by filtration, and then washed three times with distilled water and once with 95% ethanol, and oven-dried at 40  C for 48 h to about w8% moisture. A weighed quantity of OSA-starch powder (dry weight) was dispersed in 25 mL phosphate buffer solution with pH ¼ 4.0. The mixture with different OSA-starch concentration (0.1e4%) was placed in the orbital shaker (30  C, 200 rpm) for 4 h to obtain homogeneous solution for the characterization. 2.2.2. Gel permeation chromatography-multi angle light scattering (GPC-MALS) experiment A Waters GPC system, equipped with a pump (1515, Waters Co., USA) and a thermostated autosampler (717, Waters Co., USA) and followed by MALS (Wyatt Technology Co., USA), was used to measure the molecular weight for the samples. The GPC column (8.0 mm  300 mm length) was HEMA-Linear 10 mm (MZ-Analysentechnik GmbH, Mainz, Germany). MALS data were gathered with 18-angle light scattering detector (laser wavelength l ¼ 658.0 nm) (DAWN HELEOS, Wyatt Technology Co., USA) and differential refractive index (RI) detector (OptilabrEX). The mobile phase was 0.5 M NaCl and 0.02%(w/v) NaN3 filtered through a 0.22 mm filter and then degassed with ultrasound treatment and the flow rate was 0.7 mL/min. 10 mg OSA-starch samples were dissolved in 10 mL of mobile phase, shaking at 60  C to ensure full dissolution in the mobile phase. All solutions were filtered through a 0.45 mm filter before they were injected into the GPC column. The column temperature was controlled at 25  C. The OSA-starch molecular weight distribution was analyzed using the ASTRA V software. 2.2.3. Atomic force microscopy (AFM) observation AFM images were collected by NanoScope IIIA Multimode AFM (Veeco Metrology Group, Santa Barbara, CA, USA). For tapping mode, a Veeco silicon RTESP cantilever was used (k 20e80 N/m). Before silicon nitride tip engagement, the drive frequency of the silicon nitride tip was tuned with the aid of Nanoscope 5.30 software and fixed at 237e286 kHz for further scanning. The dilute OSA-starch aqueous solution (0.05%, w/v) was adsorbed onto the pretreated mica plates, which were cleansed by distilled water. Diluted solutions were allowed to sit for room temperature evaporation. All of the collected images were flattened for drifts and piezo creep before further analysis. Section analysis, which was incorporated in the Nanoscope 5.30 software, was utilized to obtain the quantitative and qualitative information of OSA-starch aggregates morphology. 2.2.4. Dynamic light scattering (DLS) experiment The hydrodynamic diameter (Dh) of OSA-starch micelle in distilled water was determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS instrument (measurement range of 0.6 nme6 mm) (Malvern Instruments, Worcestershire, UK). The Nano ZS instrument incorporates noninvasive backscattering (NIBS)

J. Zhu et al. / Food Hydrocolloids 32 (2013) 1e8

kT 3phDh

3. Results and discussion 3.1. Molecular weight comparison Fig. 1(a) shows broad peaks for all samples, resulting from the fragment formation in non-uniform size after random breakage of glycosidic bond under enzymolysis of native starch (Liu et al., 2008). As seen from Fig. 1(b), all samples presented wide molecular weight distribution. Comparatively, Mw (weight average molecular mass) of Sample 1 is higher than 10,000 g/mol, with the ratio of Mw > 100,000 g/mol accounting for over 50%. While Sample 3 presented distinctly discrepant Mw distribution with over 90% lower than 50,000 g/mol, especially the ratio of Mw < 6500 g/mol accounts for around 31%. Sample 2 comprises chiefly 10,000e 50,000 g/mol (>50%). Table 1 lists the molecular weight and polydispersities of all samples. The molecular weight ranks in the order Sample 3 < Sample 2 < Sample 1. The OSA-starch molecules (similar DS) with higher molecular weight contain more hydrophilic groups, and they have higher tendency to stay in the water (Krstonosic et al., 2011). Consequently, the micelles originated from different OSA-starch molecules should present structure diversities.

10

Sample 1

7

10

6

10

0.8

5

10

Sample 2

0.6

4

10

3

10

Sample 3

0.4

2

10

1

where k is the Boltzmann constant (1.38  1023 m2 kg s2 K1), T is the absolute temperature (K), h is the viscosity coefficient of the solvent (Pa s), and Dh the hydrodynamic diameter of the particles in solution. The sample solution was illuminated by a 633 nm laser at 25  C, and the intensity of light scattered at an angle of 173 was measured by an avalanche photodiode.

10 0.2

0

10

-1

10 0.0

-2

10 4

6

8

10

12

14

16

Time (min)

(b)

Distribution Ratio (%)

2.2.5. Small angle X-ray scattering (SAXS) experiment SAXS experiments were performed using a SAXSess camera (Anton-Paar, Graz, Austria). A PW3830 X-ray generator with a long fine focus sealed glass X-ray tube (PANalytical) was operated at 40 kV and 50 mA. A focusing multilayer optics and a block collimator provide an intense monochromatic primary beam (CuKa, l ¼ 0.1542 nm). A semi-transparent beam stop enables measurement of attenuated primary beam at zero scattering vector. The samples were filled into a capillary of 1 mm diameter and 0.01 mm wall thickness. The capillary was placed in a TCS 120 temperature-controlled sample holder unit (Anton Paar) along the line shaped X-ray beam in the evacuated camera housing. The sample-to-detector distance was 261.2 mm, and the temperature was kept at 26.0  C. The 2D scattered intensity distribution recorded by an imaging-plate (IP) detector was read out by a Cyclone storage phosphor system (Perkin Elmer, USA). The 2D data were integrated into the one-dimensional scattering function I(q) as a function of the magnitude of the scattering vector q (q ¼ 4psin q/l, where 2q is the scattering angle). Each measurement was collected for 30 min. All I(q) data were normalized so as to have the uniform primary intensity at q ¼ 0 for transmission calibration. The background scattering contributions from capillary and solvent were corrected. Desmearing is necessary because of the line collimation.

8

1.0

Rayleigh Ratio

D0 ¼

(a)

Molecular Weight (g/mol)

optics. This technique measures the time dependent fluctuations in the intensity of scattered light that occur because particles undergo Brownian motion. The analysis of these intensity fluctuations enables the determination of the diffusion coefficients (D0) of particles, which are converted into a size distribution according to StokeseEinstein equation (Hoo, Starostin, West, & Mecartney, 2008; Tominaga, Suda, Osa, Yoshizaki, & Yamakawa, 2002):

3

50

Sample 1 Sample 2 Sample 3

40

30

20

10

0 5E4-1E5 < 6500 6500-1E4 1E4-5E4 1E5-4E5 Mw Distribution (g/mol)

> 4E5

Fig. 1. Molecular weight comparison. (a) Apparent molecular weight and Rayleigh ratio, as a function of the elution time for all samples. (b) Distribution ratio vs. Mw distribution histogram of all samples.

3.2. OSA-starch micelle aggregates morphology The OSA-starch molecule is amphiphilic with hydrophobic octenyl groups and hydrophilic carboxyl groups. Based on Van der Waals force, in aqueous solution the hydrophobic part forms the kernel of the micelle and hydrophilic part is oriented outwards to the water. Fig. 2 exhibits the height (related to surface morphology) AFM images of OSA-starch samples. The self-assembly of OSAstarch molecules could be viewed as either approximately spherical or ellipsoidal shapes from the top view. The water evaporation affects the stability of OSA-starch molecules in aqueous solution, which could facilitate the micelle formation. However, according to the size information from the images, Sample 1 (with the highest Mw) formed aggregates of around 130e150 nm, whilst for samples 2 and 3 (having lower Mw) these were around 100 nm. The images actually displayed the micelle aggregates occurred by the

Table 1 Weight average molecular mass (Mw), number average molecular mass (Mn) and polydispersity (Mw/Mn) obtained from GPC-MALS. Sample (1 mg/mL)

Mw (g/mol)

Mn (g/mol)

Mw/Mn

Sample 1 Sample 2 Sample 3

1.695eþ5 (0.5%)a 4.628eþ4 (0.5%) 1.799eþ4 (0.5%)

8.568eþ4 (0.7%) 1.759eþ4 (2%) 8.899eþ3 (1%)

1.978 (0.8%) 2.630 (2%) 2.021 (2%)

a

Precision of global fit.

4

J. Zhu et al. / Food Hydrocolloids 32 (2013) 1e8

Fig. 2. Tapping mode (AFM) height images of OSA-starch with the scan sizes of 5 mm  5 mm and 500 nm  500 nm (top to bottom). (Left) Sample 1, (Middle) Sample 2, (Right) Sample 3.

3.3. Micelle conformation To elucidate the nano-structure of OSA-starch micelles in aqueous solution, SAXS experiments were performed to investigate the relationship between the samples conformation and Mw. The Guinier plots (Ln [I(q)] vs q2) of the measured SAXS data revealed various conformational forms of OSA-starch micelles. As seen in Fig. 3, all the curves in the region of q2 < 0.07e0.15 nm2 were well approximated by a straight line, while the upper q limit of the Guinier rule, qRg < 1 (Glatter & Kratky, 1982; pp: 119e196), or 1.3 (Haydyn & Dmitri, 2010), was q2 < 0.03e0.12 nm2, indicating that the micelles are of considerable monodispersity. Although the molecular weight presents wide distribution (GPC-MALS results), which means non-uniform OSA-starch molecule fragments, the monodisperse micelles formed above CMC. The better linear fitting to SAXS data (Fig. 3) indicated the micelle presents increased monodispersity in concentrated OSA-starch solution, from which we could deduce that the micelle conformation tends to be more uniform with increasing concentration. Micelle Rg values were estimated from the slope value of the regression line according to the Guinier equation (Glatter & Kratky, 1982), Ln [I(q)] ¼ Ln [I(0)]  R2gq2/3 within the Guinier region, which represents the micelle particles (not single molecule) comprised of a number of single OSA-starch molecules. The determined Rg value increases in the order Sample 3 < Sample 2 < Sample 1 (Table 2), which is consistent with the comparison in terms of Mw. The

different Rg values indicate the micelle particles characterized with different dimensions. Interestingly, there was no significant increase of Rg value for each sample when the concentration increased. Generally, the increasing concentration could give rise to the formation of larger particles upon micelles, i.e. the significant micelle aggregates, which would be indicated by an upward deviation from the straight line fit in the small-angle region. However, as seen from Fig. 3, we suggest that the lack of such a feature indicates there are no aggregates or interaction between micelle particles. The SAXS pattern (Fig. 4) also showed no intensity

4% Sample 3 4% Sample 2 4% Sample 1 2% Sample 3

Ln[I(q)] (a.u.)

association of neighboring micelles because of the evaporation of dilute solution (0.05%, w/v). The size of micelle aggregates originated from Sample 1, 2 and 3, presented obvious disparity. Sample 1 with the highest Mw generated the micelle with the largest dimension.

2% Sample 2 2% Sample 1 1% Sample 3

1% Sample 2 1% Sample 1

0.00

0.02

0.04

0.06

0.08 0.10 2 -2 q (nm )

0.12

0.14

Fig. 3. Guinier plots of the scattering data of all samples.

0.16

J. Zhu et al. / Food Hydrocolloids 32 (2013) 1e8

5

Table 2 Structural parameters of OSA-starch micelle obtained from SAXS and DLS. Sample

c (w/v)

Dah (nm)

sb

Sample 1

0.1% 0.2% 0.5% 1% 2% 4% 0.1% 0.2% 0.5% 1% 2% 4% 0.1% 0.2% 0.5% 1% 2% 4%

80.48 63.01 53.56 47.46 45.82 48.46 30.10 25.12 26.35 26.33 27.54 31.79 36.29 30.24 30.85 33.78 32.85 33.70

4.0570 1.7560 3.4330 3.3150 5.1780 0.2554 3.8830 1.4390 1.8710 0.5522 0.5237 0.2380 1.7690 1.4310 0.1457 2.6900 0.9791 1.6050

Sample 2

Sample 3

a

Rcg (nm)

I (0)d (a.u.)

Chi2

e

7.495  0.087 7.891  0.086 7.294  0.046

0.900  0.018 1.976  0.033 3.449  0.033

1.844 1.528 1.681

34.0 29.0 29.5

4.654  0.056 4.291  0.030 4.766  0.059

0.450  0.007 0.866  0.007 1.658  0.019

1.700 2.419 2.018

20.8 21.3 22.0

3.789  0.077 4.378  0.063 4.074  0.037

0.273  0.005 0.637  0.009 1.138  0.009

1.411 2.037 2.495

18.8 21.9 23.0

rfmax(nm)

(hydrodynamic diameter) and b (standard deviation) were obtained from Dispersion Technology Software (DTS) version 5.0. were calculated from Saxsquant 3.0. was calculated by fitting the experiment data in General Indirect Fourier Transform (GIFT).

c,d,e f

maximum, i.e. a downward deviation at low scattering angles, to suggest the absence of particle interaction. Jin et al. (2008) have reported the optimal concentration of 10 mg/mL for protein solution, which is suitable for data processing to avoid interaction between protein molecules. Although the minimal OSA-starch concentration of the series was 1% (i.e. 10 mg/mL) in SAXS experiment, the micelle number was much less compared with OSAstarch monomers in 1% solution. As for micelle Mw, since the I(0)/ c (c is the sample concentration) of the SAXS data depends linearly on the Mw of the biological macromolecules solutes in solution (Moitzi et al., 2011; Watanabea & Inoko, 2009, 2011), the results calculated from Table 2 also confirm the conclusion described above that OSA-starch molecules with higher Mw formed larger micelles and no micelle aggregation occurred. The increasing OSAstarch concentration increased the micelle number in solution. Although the micelle Rg values change slightly with increasing concentration for all three species, Dh values via. DLS decreased more distinctly (from 80 nm to 46 nm for Sample 1) (Fig. 5). Rh (1/2 Dh) corresponds to the radius of an effective sphere that moves as a Brownian particle, containing the excluded volume of the micelle

involving the hydrated water molecules. The dramatic decrease in Dh along with increasing concentration only arose for Sample 1. Comparatively, Dh for Sample 2 and 3 changed in a narrow range. Krstonosi c et al. (2011) have reported that the hydrophobic tails in dilute solutions associate intra-molecularly in order to minimize contact with water, and the OSA-starch chains probably curl and shrink above CMC with a smaller hydrodynamic size. The surface of OSA-starch micelles were negatively charged by virtue of hydrophilic carboxyl or sodium carboxylate groups. An increase of OSAstarch concentration led to the increase in micelle number, which could aggravate the electrostatic repulsion between micelles, and thus further compressed the micelle, including the hydrated water layer. These results demonstrated that for Sample 1 the micelles displayed more distinct conformation variation under different OSA-starch/H2O ratio than for Sample 2 and 3 solutions. Further, when comparing Rh obtained from DLS with Rg obtained from SAXS (Moitzi et al., 2011; Wu & Wang, 1998) (Table 2), Rg/Rh values are strangely small (0.2e0.3) for OSA-starch micelles. We assumed that Rg remained invariant in more dilute aqueous solution (0.1%, 0.2% and 0.5% are excessively diluted for SAXS test), which could be

90

10

Hydrodynamic Diameter (nm)

2% 1% 0.5%

I(q) (a.u.)

Sample 1 Sample 2 Sample 3

80

4% -1

-2

10

0.1% -3

10

-4

70 60 50 40 30 20

10

-1

0

1

2

3 -1 q (nm )

4

5

6

7

Fig. 4. SAXS plots of Sample 1 in aqueous solution with different concentration (Plots for Sample 2 and 3 are not shown).

0.1%

0.2%

0.5% 1% c (w/v)

2%

4%

Fig. 5. The hydrodynamic diameter (Dh) determined by dynamic light scattering for aqueous solutions. Some error bars are not visible within the symbols.

J. Zhu et al. / Food Hydrocolloids 32 (2013) 1e8

hand, for Sample 1, Rh value decreased and Rg/Rh value increased more dramatically with increasing concentration. This would indicate that the conformation of micelles from Sample 1 with more flexibility was more sensitive to the increasing interparticle electrostatic repulsion. Although the micelle number increased, the

(a) p(r) (a.u.)

0.05 0.04 0.03 0.02

1% Sample 3

0.01 0.00 0

1% Sample 2

0.1

10

15

25

30

35

Sample 1 (top) Sample 2 (middle) Sample 3 (bottom)

0.16

(b)

0.14

p(r) (a.u.)

0.12

2% Sample 3

0.10 0.08 0.06 0.04 0.02 0.00

2% Sample 2

0

5

10

15

20

25

30

r (nm)

2% Sample 1

1

-1

q (nm )

Sample 1 (top) Sample 2 (middle) Sample 3 (bottom)

0.25

4% Sample 3 p(r) (a.u.)

0.20

α = -1.86

I(q) (a.u.)

4% Sample 2

α = -2.08

20

r (nm)

1

-1

q (nm )

(c)

I(q) (a.u.)

5

1% Sample 1

0.1

4% Sample 1

Sample 1 (top) Sample 2 (middle) Sample 3 (bottom)

0.07 0.06

I(q) (a.u.)

deduced from the similarity of SAXS plots of Sample 1 in aqueous solution with different concentration (Fig. 4). As Rg/Rh of Sample 1 solution changed more dramatically with concentration increase (from approximately 0.15 for 0.1% to 0.3 for 4%) than that of Sample 2 and 3, this would indicate that the conformation of micelles from Sample 1 was more sensitive to the interparticle electrostatic repulsion, i.e. demonstrated more flexibility. It should be prominently recognized that Rg is model independent with no information about the shape or the conformation of the particle, which means that micelle particles with different shapes could have the similar Rg value. Consequently, the comparison of Rg/Rh variation with concentration further verified the conformational differences between micelles originated from OSA-starch sample 1, 2 and 3. At larger q-values, where the scattering experiment probes a reduced characteristic length scale (given by 2p/q), the contributions from the inner structure on these length scales are primarily observed. As the internal structure is proposed to be fractal, a power-law decay of the scattering curve should be observed. The slope or fractal dimension is directly related to the compactness of the scattering particles, where densely packed structures give rise to a scattering intensity which is decaying more steeply than more open structures with a smaller fractal dimension, i.e. the more compact structure corresponds to a larger mass fractal dimension (Schaefer, 1989; Schmitt et al., 2010). The internal structure of the micelle was mainly reflected in the decay of the scattering curve at large q-vectors. As seen from Fig. 6, the modulus of slopes of the decay in SAXS plots decreases in the order Sample 3 < Sample 2 < Sample 1, indicating the most compact structure within Sample 1 micelles. On the contrary, the interior structure of Sample 2 and 3 micelles was relatively loose. All the differences presented above were supposed to be relevant to the stability when applied to an oil-in-water system. Previous studies (Krstonosi c et al., 2011; Ntawukulilyayo et al., 1996) have investigated the stabilization and solubility of organic molecules in OSA-starch suspension. Krstonosic et al. (2011) have found that the solubility rapidly increased after OSA-starch micelle formation and further increased with higher OSA-starch concentration above CMC. Moreover, the solubilization was more obvious in OSA-starch solution with higher Mw. The OSA-starch with higher Mw formed micelles with larger dimension and more compact interior structure, which could provide “firmer” spatial structure for the encapsulated components in aqueous solution. On the other

I(q) (a.u.)

6

4% Sample 3

0.15 0.10 0.05 0.00

4% Sample 2

0

5

10

15

20

25

30

r (nm)

α = -2.14 4% Sample 1

0.1

-1

1

q (nm ) Fig. 6. Linear fitting in middle region of SAXS data (lg [I(q)] vs lg (q)) of 4% OSA-starch solution.

0.1

-1

q (nm )

1

Fig. 7. Pair distance distribution function (PDDF) p(r) of each OSA-starch micelle obtained from experimental SAXS data: (a) 1%, (b) 2%, (c) 4%.

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interparticle electrostatic repulsion prevented the micelles with smaller Rh from aggregating, which is supposed to enhance the stability of solution. This variation tendency should also occur in the mixed solution with the addition of organic component. 3.4. Geometric size and shape of micelle The structural findings were confirmed by a model-independent alternative way to analyze the SAXS curves using General Indirect Fourier Transformation (GIFT). This procedure results in a so-called Pair Distance Distribution Function (PDDF) p(r), an essential realspace function that containing information about size, shape, and internal structure of the scattering particles (Sato, Fukasawa, Aramaki, Glatter, & Buchner, 2011). For homogeneous particles, p(r) represents the histogram of distances between pairs of points within the particle. Obviously, its value is uniformly zero when r exceeds rmax, the maximum dimension of the particle (Koch, Vachette, & Svergun, 2003). As illustrated from the inset in Fig. 7, the rmax values of OSA-starch micelles were obtained (Table 2). Reduction of rmax was observed with concentration increase for Sample 1 micelles, while increase was noted for Sample 2 and 3. The real space representation is more intuitive and information about the particle shape can often be deduced by straightforward visual inspection of p(r) (Glatter & Kratky, 1982). According to the previous review (Svergun & Koch, 2003), globular particles display bell-shaped p(r) functions with a maximum at about rmax/2. Elongated particles have skewed distributions with a clear maximum at small distances corresponding to the radius of the cross-section. Flattened particles display a rather broad maximum, also shifted to distances smaller than rmax/2. A maximum shifted toward distances larger than rmax/2 is usually indicative of a hollow particle. Particles consisting of well-separated subunits may display multiple maxima, the first corresponding to the intra-subunit distances, the others yielding separation between the subunits (Mertens & Svergun, 2010; Svergun & Koch, 2003). The p(r) plots show typical profiles yielded from multi-domain particles with multiple shoulders and oscillations (see the arrows in Fig. 7) corresponding to intra and inter-subunit distances, which could be corresponding to some oblate ellipsoid with a small subunit. The micelles in 2% Sample 2 and 4% Sample 3 solution seem to be long rod with a limited cross-section dimension. Some flattened prolate ellipsoid formed in 4% Sample 1 solution. These conformation changes could be due to a compress of more random structure into more relatively symmetrical structure with increasing OSA-starch concentration. 4. Conclusions In this paper AFM/DLS/SAXS were utilized to characterize the micelle nano-structure of three different OSA-starch representatives with different Mw determined by GPC-MALS. Rg, Rh and rmax values of micelle particles were obtained. In absence of other ingredients, the amphiphilic OSA-starch molecules above CMC form micelles via hydrophobic bonding. The geometric size presented obvious disparity. No aggregation or interaction between micelle particles was observed in aqueous solution. The OSA-starch micelles presented increased monodispersity under higher solution concentration. Sample 1 molecule with the highest Mw facilitated the largest micelle formation, which displayed more distinct conformation variation under different OSA-starch/H2O ratio due to the aggravating electrostatic repulsion between micelles. Rg/Rh of Sample 1 solution changed more dramatically with concentration than that of Sample 2 and 3, it could be inferred that the conformation of Sample 1 micelles with more flexibility was more sensitive to the interparticle electrostatic repulsion. The p(r) plots

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show typical profiles yielded from some oblate ellipsoid with a small subunit, long rod with a limited cross-section dimension and flattened prolate ellipsoid. These conformation variations along with the concentration change could be due to compression of more random structure into more symmetrical structure. Moreover, the different OSA-starch micelles presented different compactness. The most compact structure formed within Sample 1 micelles compared to the relatively loose interior structure of Sample 2 and 3 micelles. It is anticipated that the dimension, flexibility and compactness of OSA-starch micelles would be closely related to the stability and other properties of OSA-starch. However, it should be recognized that when other polymers and organic molecules are added in real food system, the interaction and recombination between these components should generate complexes with different structures. Further work is expected to characterize the detailed structure and establish exactly what structures dominate these properties. Acknowledgments The authors would like to acknowledge the National Natural Science Funds of China (No. 31071503, 21076086, 31130042), the National Key Technology R&D Program (2012BAD33B04, 2012BAD34B07, 2012BAD37B01) and Guangdong Natural Science Foundation (S2011010001677). References Bai, Y., & Shi, Y.-C. (2011). Structure and preparation of octenyl succinic esters of granular starch, microporous starch and soluble maltodextrin. Carbohydrate Polymers, 83, 520e527. Bai, Y., Shi, Y.-C., Herrera, A., & Prakash, O. (2011). Study of octenyl succinic anhydride-modified waxy maize starch by nuclear magnetic resonance spectroscopy. Carbohydrate Polymers, 83, 407e413. Bao, J., Xing, J., Phillips, D. L., & Corke, H. (2003). Physical properties of octenyl succinic anhydride modified rice, wheat, and potato starches. Journal of Agricultural and Food Chemistry, 51, 2283e2287. Bhosale, R., & Singhal, R. (2006). Process optimization for the synthesis of octenyl succinyl derivative of waxy corn and amaranth starches. Carbohydrate Polymers, 66, 521e527. Glatter, O., & Kratky, O. (1982). Small angle X-ray scattering. London, UK: Academic Press. Haydyn, D. T. M., & Dmitri, I. S. (2010). Structural characterization of proteins and complexes using small-angle X-ray solution scattering. Journal of Structural Biology, 172, 128e141. Hoo, C. M., Starostin, N., West, P., & Mecartney, M. L. (2008). A comparison of atomic force microscopy (AFM) and dynamic light scattering (DLS) methods to characterize nanoparticle size distributions. Journal of Nanoparticles Research, 10, 89e96. Huang, Q., Fu, X., He, X., Luo, F., Yu, S., & Li, L. (2010). The effect of enzymatic pretreatments on subsequent octenyl succinic anhydride modifications of cornstarch. Food Hydrocolloids, 24, 60e65. Jin, K. S., Kim, D. Y., Rho, Y., Le, V. B., Kwon, E., Kim, K. K., et al. (2008). Solution structures of RseA and its complex with RseB. Journal of Synchrotron Radiation, 15, 219e222. Koch, M. H. J., Vachette, P., & Svergun, D. I. (2003). Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution. Quarterly Reviews of Biophysics, 36, 147e227. Krstonosi c, V., Doki c, L., & Milanovi c, J. (2011). Micellar properties of OSA starch and interaction with xanthan gum in aqueous solution. Food Hydrocolloids, 25, 361e367. Liu, Z., Li, Y., Cui, F., Ping, L., Song, J., Ravee, Y., et al. (2008). Production of octenyl succinic anhydride-modified waxy corn starch and its characterization. Journal of Agricultural and Food Chemistry, 56, 11499e11506. Magnusson, E., & Nilsson, L. (2011). Interactions between hydrophobically modified starch and egg yolk proteins in solution and emulsions. Food Hydrocolloids, 25, 764e772. Mertens, H. D. T., & Svergun, D. I. (2010). Structural characterization of proteins and complexes using small-angle X-ray solution scattering. Journal of Structural Biology, 172, 128e141. Moitzi, C., Donato, L., Schmitt, C., Bovetto, L., Gillies, G., & Stradner, A. (2011). Structure of b-lactoglobulin microgels formed during heating as revealed by small-angle X-ray scattering and light scattering. Food Hydrocolloids, 25, 1766e 1774. Nilsson, L., & Bergenståhl, B. (2006). Adsorption of hydrophobically modified starch at oil/water interfaces during emulsification. Langmuir, 22, 8770e8776.

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