Self-Assembled Micelles Based on OSA-Modified Starches for

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Self-assembled Micelles Based on OSA-modified Starches for Enhancing Solubility of #-Carotene: Effect of Starch Macromolecular Architecture Quanquan Lin, Rong Liang, Fang Zhong, Aiqian M. Ye, Yacine Hemar, Zhi Yang, and Harjinder Singh J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 26, 2019

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Journal of Agricultural and Food Chemistry

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Self-assembled Micelles Based on OSA-modified Starches for Enhancing Solubility of

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β-Carotene: Effect of Starch Macromolecular Architecture

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Quanquan Lin,§, ‡, †,‖ Rong Liang, §, ⊥ Fang Zhong,§, †, ⊥, * Aiqian Ye,‡, ‖, * Yacine Hemar, ‡ Zhi Yang,#

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and Harjinder Singh‡

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§Key

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Wuxi 214122, China

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‡

Riddet Institute, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand

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†

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

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‖

School of Food Science and Bioengineering, Zhejiang Gongshang University, Hangzhou, Zhejiang

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310018, China

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⊥ School

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#

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Oak Ridge, TN 37831, United States

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*Corresponding authors:

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Tel.: +86 510 85197876 (F. Zhong); +64 6 350 5072 (A. Ye).

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E-mail addresses: [email protected] (F. Zhong), [email protected] (A. Ye).

Laboratory of Synthetic and Biological Colloids, Ministry of Education, Jiangnan University,

of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China

Biology and Soft Matter Division, Neutron Sciences Directorate, Oak Ridge National Laboratory,

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1

ACS Paragon Plus Environment


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ABSTRACT

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Self-assembled micelles based on octenyl succinic anhydride (OSA)-modified starch were prepared to

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enhance the solubility of β-carotene. The critical micelle concentration (CMC) was lower for

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OSA-modified starch with lower molecular weight (Mw) or higher degree of substitution (DS). Above

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the CMC, OSA-modified starch assembled into spherical micelles with an average hydrodynamic

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diameter of 0.05), suggesting that the Mw had no influence on the esterification of starches with the same

187

amount of added OSA. When the added OSA was increased from 5% to 9%, the DS of the starch

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increased from 2.54 ± 0.04% to 4.16 ± 0.01% at an acid hydrolysis time of 12 h, consistent with

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previous studies.28,30,33,34

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CMC of OSA-modified Starches. The CMC is also called the “critical aggregation

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concentration”. Amphiphilic polymers can form self-assembled micelles in aqueous solution above

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the CMC.22 The presence of hydrophilic hydroxyl groups and hydrophobic octenyl side chains

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imparts the amphiphilic property to OSA-modified starch, providing the ability to form micelles in

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water. In this study, the CMCs of the OSA-modified starches in water were measured by

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fluorescence spectroscopy using pyrene, which is a hydrophobic fluorescence probe that 9



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preferentially partitions into the hydrophobic core of the aggregate. The excitation peak of pyrene is

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at 334 nm in a hydrophilic environment and shifts to about 337 nm in a hydrophobic environment,

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such as in the core of polymer micelles.23 The CMC can be obtained by plotting the ratio of the

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peak intensities at 337 and 334 nm (I337/I334) against the polymer concentration.

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The I337/I334 ratio increased with increasing starch concentration (Figure 2). The concentration

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of OSA-modified starch at the turning point in the plot was determined to be the CMC. As shown in

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Table 1, the CMC values of the OSA-modified starches with the same DS decreased in the

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following order: H1-OSA5% > H6-OSA5% > H12-OSA5%, which suggested that OSA-modified

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starches with larger Mw formed micelles at higher concentration. This result was in agreement with

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a study conducted by Lei et al.25 At a given DS, increase in the Mw of OSA-modified starch may

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imply a reduction in the relative hydrophobicity, which led to lower the interfacial tension and

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further made it difficult to form micelles in the case of OSA-modified starches with larger Mw.

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However, Tizzotti et al.27 found the opposite, i.e., OSA-modified starches with larger

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macromolecular size formed micelles at lower concentrations for a given DS. These authors

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considered that an increase in the average number of OSA groups per starch chain, induced by

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increasing the Mw while keeping the DS constant, contributed to micelles forming more readily for

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OSA-modified starches with larger Mw. These different results can be attributed to the different

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structures of the polymers used. The effect of the Mw on the self-assembly of polymers is probably

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a result of the competition between the two opposing effects: enhanced by a greater average number

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of OSA groups per starch chain and decreased by a reduction in relative hydrophobicity. For

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OSA-modified starches with similar Mw, an increase in the DS (from 2.54 ± 0.04% to 4.16 ±

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0.01%) significantly decreased the CMC from 0.084% to 0.058% (Table 1), indicating that the

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number of OSA groups is a primary factor in the micellization process. It has been reported that the 10


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driving force in the formation of micelles is the hydrophobic interactions among the OSA groups.25

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The results in this current study are consistent with previous studies,21,25,27,35,36 which also showed

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that a higher DS of hydrophobic groups in the polymers leads to micelle formation at lower

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concentration.

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Particle Size of OSA-modified Starch Self-Assembly. The size of the self-assembled micelles

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is important to the appearance (e.g., transparency), stability, and encapsulation capacity of an

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OSA-modified starch solution. As an example, the particle size distribution of micelles formed by

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H12-OSA5%, as measured by DLS, was represented as both the intensity-weighted particle

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concentration and the volume-weighted particle concentration versus the particle diameter (Figure

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3). The intensity distribution is very sensitive to the presence of large particles, whereas the volume

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distribution is more sensitive to the presence of small particles (such as micelles). Peaks found in

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the intensity distribution (Figure 3) indicated the presence of large particles, which might be the

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aggregates of starch micelles. However, they could not be observed in the volume distribution due

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to their small volume number. In this study, we used the volume distribution to better characterize

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the more representative population of OSA-modified starch micelles.

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The influence of concentration, Mw, and DS of the starch on the particle size of the

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OSA-modified starch self-assembly are shown in Figure 4. The particle size distributions of each

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OSA-modified starch in water at concentrations above CMC, upon self-aggregation, were relatively

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monodisperse (Figure 4C-F). As seen in Figure 4A, the volume mean diameter of micelles formed

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from H1-OSA5% decreased markedly from 416.6 ± 268.8 to 15.8 ± 9.4 nm when the starch

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concentration was increased from 0.05% to 1%. Further increases in the starch concentration (
H6-OSA5% > H12-OSA5%, indicating that the Rg of the

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β-carotene-free micelles was significantly positively (p < 0.05) correlated with the Mw of the

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starch. The trend in the Rg of the β-carotene-free micelles was consistent with that in the Dh of these

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micelles. With similar Mw, the Rg of the β-carotene-free micelles was slightly smaller for

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H12-OSA9% than for H12-OSA5% at all starch concentrations. Although there was no significant

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change in the Dh with increasing DS, the Rg of the β-carotene-free micelles formed from

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OSA-modified starch with higher DS was smaller because of the formation of more compact

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micelles with enhanced intermolecular hydrophobic interactions among the starch molecules.

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In addition, there was a slight but significant increase (p < 0.05) in the Rg of all micelles after

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the incorporation of β-carotene (Table 2), which could be attributed to the enlargement of the

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hydrophobic core. Interestingly, Yu and Huang,23 reported that the incorporation of curcumin did

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not induce any changes in the Rg of OSA-modified starch micelles, as determined by SAXS. This

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may reflect different sizes of the lipophilic bioactive components; the Mw of β-carotene is 1.45

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times greater than that of curcumin. Besides, the trends in the Rg of all the β-carotene-loaded

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micelles were the same as those in the β-carotene-free micelles when the concentration, Mw, or DS

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of the OSA-modified starch was changed (Table 2). In detail, the Rg decreased when the

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concentration or DS of the starch was increased, whereas it increased when the Mw of the starch

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was increased. Finally, it is worth noting that the aggregates of starch micelles or higher-order

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assemblies with large particle size (> 100 nm) could not be measured by SAXS due to the limit of

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the high q range.

401 402

In summary, OSA-modified starch formed micelles at above the CMC, which correlated positively with the Mw but negatively with the DS of the starch. Both DLS and SAXS showed that 18


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the particle size of the starch micelles decreased with increasing concentration or decreasing Mw. A

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greater DS led to a smaller Rg of the starch micelles. The OSA-modified starch micelles markedly

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increased the apparent solubility of β-carotene in the aqueous phase through hydrophobic

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interactions between the core of the micelles and β-carotene. The incorporation of β-carotene

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enlarged the hydrophobic core and induced a significant increase in the Rg of the micelles

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determined by SAXS; and it may also promote the aggregation of micelles or the formation of

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higher-order assemblies resulting in a marked increase in the Dh determined by DLS. The

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encapsulation capacity of the OSA-modified starch micelles showed a positive relationship with the

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concentration, Mw, and DS of the starch. From a practical viewpoint, this work provides

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fundamental support for the design of innovative delivery systems for lipophilic bioactive

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compounds in the food industry.

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ABBREVIATIONS USED

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CMC, critical micelle concentration; Dh, hydrodynamic diameter; DLS, dynamic light scattering;

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DS, degree of substitution; FTIR, Fourier transform infrared; I337/I334, excitation fluorescence

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intensity ratio of pyrene; Mw, molecular weight; OSA, octenyl succinic anhydride; Rg, radius of

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gyration; Rh, hydrodynamic radius; SAXS, small-angle X-ray scattering; TEM, transmission

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electron microscopy; XRD, X-ray diffraction.

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Funding

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This research was supported by the National Key R&D Program of China (2016YFD0400801) and

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the National Natural Science Foundation of China (No. 31571891, 31801589). It was also

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supported by a program of the "Collaborative Innovation Center of Food Safety and Quality Control

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in Jiangsu Province", China, a national first-class discipline program of Food Science and 19



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Technology (JUFSTR20180204), and the Riddet Institute, a New Zealand Centre of Research

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Excellence, funded by the Tertiary Education Commission.

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Notes

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The authors declare no competing financial interest.

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SUPPORTING INFORMATION

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Supplementary Figure S1: XRD spectra of -carotene powder, powder of -carotene-free

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micelles, and powder of -carotene-loaded micelles formed by 5% of OSA-modified starch

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(H12-OSA9%).

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Table 1. Weight-average Mw, DS, and CMC of OSA-modified Starches.a hydrolysis time

addition of OSA

Mw

DS

CMC

(h)

(g/starch)

(×104 g/mol)

(%)

(%)

Native starch

‒

‒

5550.0 ± 277.5a

‒

H1-OSA5%

1

5

1243.0 ± 10.0b

2.60 ± 0.02b

0.140

H6-OSA5%

6

5

60.27 ± 0.06c

2.57 ± 0.01b

0.107

H12-OSA5%

12

5

19.24 ± 0.07d

2.54 ± 0.04b

0.084

H12-OSA9%

12

9

19.60 ± 0.05d

4.16 ± 0.01a

0.058

starch

a

Different superscript letters indicate significant differences (p < 0.05) in the same column.

27



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Table 2. Effect of Concentration, Mw, and DS of OSA-modified Starches on the Radius of Gyration (Rg, nm) of Micelles with and without Loaded β-Carotene.

β-carotenea starch concentration (%) H1-OSA5% H6-OSA5% H12-OSA5% H12-OSA9%

‒

+ a

1

8.45 ± 0.19

6.65 ± 0.14

5.54 ± 0.15

5.57 ± 0.06

2

8.11 ± 0.16

5.90 ± 0.02

5.01 ± 0.01

4.69 ± 0.00

5

5.68 ± 0.06

4.40 ± 0.02

3.86 ± 0.01

3.53 ± 0.01

1

8.78 ± 0.09

6.42 ± 0.08

5.66 ± 0.06

5.66 ± 0.03

2

8.17 ± 0.08

6.05 ± 0.02

5.01 ± 0.01

4.94 ± 0.01

5.74 ± 0.04

4.48 ± 0.02

3.93 ± 0.01

3.67 ± 0.01

5 ‒ indicates without β-carotene; + indicates with β-carotene.

28


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Figure captions Figure 1. Weight-average Mw and concentration signal versus elution volume of OSA-modified starches: solid line, concentration signal; dashed line, Mw distribution. Figure 2. Excitation fluorescence intensity ratio of pyrene (I337/I334) versus the concentration of OSA-modified starches: (A) the effect of Mw; (B) the effect of DS. Figure 3. Particle size distribution of starch micelles formed by 5% of H12-OSA5%. Figure 4. Volume mean diameter (A, B) and particle size distribution (C‒F) of starch micelles formed by OSA-modified starch: (C) H1-OSA5%; (D) H6-OSA5%; (E) H12-OSA5%; (F) H12-OSA9%. Figure 5. β-Carotene powder in water (left) and β-carotene-loaded starch micelle formed by 5% of H12-OSA5% (right). Figure 6. Effect of structural properties of OSA-modified starches on the concentration of β-carotene in starch micelles: (A) the effect of Mw; (B) the effect of DS. Figure 7. Particle size (A, B) and particle size distribution (C‒F) of β-carotene-loaded starch micelles formed by OSA-modified starch: (C) H1-OSA5%; (D) H6-OSA5%; (E) H12-OSA5%; (F) H12-OSA9%. Figure 8. FTIR spectra of β-carotene, powder of β-carotene-free micelles, and powder of β-carotene-loaded micelles formed by 5% of OSA-modified starch. Figure 9. TEM micrographs of starch micelles formed by 1% of H12-OSA5%: (A, B) β-carotene-free micelles; (C, D) β-carotene-loaded micelles. Figure 10. Guinier plots of the scattering data of starch micelles: (A) β-carotene-free micelles; (B) β-carotene-loaded micelles. The Guinier fit curves are shown as a solid red line in each plot.

29



Page 30 of 40

-5

10

10

a1, a2: H1-OSA5% b1, b2: H6-OSA5% c1, c2: H12-OSA5% d1, d2: H12-OSA9%

9

10

8

10

7

10

a2 -5

6

1.0x10

10 b2

c2 d2

4

c1 b1

5

10 10

d1

3

10

a1

0.0

2

8

10

12

14 Volume (mL)

Figure 1

30


16

18

10 20

Mw (g/mol)

Concentration (g/mL)

2.0x10

Page 31 of 40


A 1.2 H1-OSA5% H6-OSA5% H12-OSA5%

I337/I334

1.1 1.0 0.9 0.8 -4

10

-3

10

-2

10

-1

10

0

10

0

10

10

1

Concentration of starch (%) B 1.2

H12-OSA5% H12-OSA9%

I337/I334

1.1 1.0 0.9 0.8 -4

10

-3

10

-2

10

-1

10

Concentration of starch (%) Figure 2

31


10

1


Page 32 of 40

25

4.0 3.5

Intensity (%)

3.0 2.5

15

2.0 10

1.5 1.0

5

0.5 0.0

1

10

100 Size (nm)

Figure 3

32


1000

0 10000

Volume (%)

20

Page 33 of 40


B

A 800 H1-OSA5% H6-OSA5% H12-OSA5%

400 200 40 30 20 10 0

0

1

2

3

4

18

H12-OSA5% H12-OSA9%

16 Volume mean diameter (nm)

Volume mean diameter (nm)

600

14 12 10 8 6 4 2 0

5

0

1

C

3

D

Volume (%)

20 Volume (%)

10

0.05% 0.2% 1.0% 5.0%

5

1

10

100

1000

E 30

5 0

0.05% 0.2% 1.0% 5.0% 1

10

100

F 25

Volume (%)

Volume (%)

15 10

Volume (%)

Volume (%)

10000

20

1

10

10

100

0.05% 0.2% 1.0% 5.0%

5

10

1000

25

20

Size (nm)

1

100

Size (nm)

5

15

10

10

0

10000

20

0

1

Size (nm)

25

20

10

5

30

25

15

15

Size (nm)

0

5

20

15

0

4

25

25

20

Volume (%)

2

Concentration of starch (%)


100

1000

15

15 10 5 0

1

10

0.05% 0.2% 1.0% 5.0%

5 0

10000

100

Size (nm)

10

1

Size (nm)

10

100 Size (nm)

Figure 4

33


1000

10000


(a)

β-Carotene powder in water

β-Carotene-loaded starch micelle

Figure 5

34


Page 34 of 40

Page 35 of 40


β-Carotene concentration (μg/mL)

A 60 H1-OSA5% H6-OSA5% H12-OSA5%

50 40 30 20 10 0

0

1

2

3

4

5

4

5

Starch concentration (%)

β-Carotene concentration (μg/mL)

B 50 H12-OSA5% H12-OSA9%

40 30 20 10 0

0

1

2

3

Starch concentration (%) Figure 6

35



A 4000

B



900

H1-OSA5% H6-OSA5% H12-OSA5%

3000 2000

1000 800 600 400

H12-OSA5% H12-OSA9%

800 700 600 500 400 300

200

1

2

3

4

1

5

2

3

4

5



C

D 1% 2%

1% 2% 5%

15

Volume (%)

20 Volume (%)

Page 36 of 40

10

10

5

0

1

10

100

1000

0

10000

1

10

F 15

E 15 1% 2% 5%

Volume (%)

Volume (%)

10

5

0

10

100

1000

10000

1000

10000

1000

10000

1% 2% 5%

10

5

0 1

100 Size (nm)

Size (nm)

1

10

100 Size (nm)

Size (nm)

Figure 7

36


Page 37 of 40


A

B

H1-OSA5% containing -carotene

1719

1569 1644

2925

Transmittance (%)

3350

Transmittance (%)

H1-OSA5%

3350


1719

1569 1644

2925

-Carotene

1620

2917

3350

1719

1569 1643

2925

H6-OSA5%

1719 3350

1569 1643

2925

1368 965

4000

3500

3000

2500

2000

1500

4000

1000

3500

3000

2500

C

D H12-OSA5% containing -carotene

Transmittance (%)

Transmittance (%)

1720

1569 1644

H12-OSA5%

3350

4000

3500

1719

1569 1644

2925

3000

2500

2000

1000


1719 3350

1500

Wavenumbers (cm )

Wavenumbers (cm )

2925

2000 -1

-1

1500

3350

H12-OSA9%

1720 3350

4000

1000

1569 1644

2925

3500

1569 1644

2925

3000

2500

2000 -1

-1

Wavenumbers (cm )

Wavenumbers (cm )

Figure 8

37


1500

1000


(A)

(B)

(C)

(D)

Figure 9

38


Page 38 of 40

Page 39 of 40


A

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

1% H1-OSA5% 1% H6-OSA5% 1% H12-OSA5% 1% H12-OSA9% 2% H1-OSA5% 2% H6-OSA5% 2% H12-OSA5% 2% H12-OSA9% 5% H1-OSA5% 5% H6-OSA5% 5% H12-OSA5% 5% H12-OSA9% 0.00

0.04

0.08

0.12 2

0.16

0.20

0.24

-2

q (nm ) B

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

1% H1-OSA5% 1% H6-OSA5% 1% H12-OSA5% 1% H12-OSA9% 2% H1-OSA5% 2% H6-OSA5% 2% H12-OSA5% 2% H12-OSA9% 5% H1-OSA5% 5% H6-OSA5% 5% H12-OSA5% 5% H12-OSA9% 0.00

0.04

0.08

0.12 2

0.16

0.20

-2

q (nm ) Figure 10

39


0.24


β-Carotene-loaded starch micelle

β-Carotene-free starch micelle

The Graphic for the Table of Contents

Mw increase

DS increase

Mw increase

DS increase

OSA-modified starch molecules

40


β-carotene

Page 40 of 40

Self-Assembled Micelles Based on OSA-Modified Starches for

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