Accepted Manuscript

Title: Hydrophobic starch nanocrystals preparations through

crosslinking modification using citric acid

Author: Jiang Zhou Jin Tong Xingguang Su Lili Ren

PII: S0141-8130(16)30637-7
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.082
Reference: BIOMAC 6255

To appear in: International Journal of Biological Macromolecules

Received date: 1-4-2016

Revised date: 14-6-2016
Accepted date: 26-6-2016

Please cite this article as: Jiang Zhou, Jin Tong, Xingguang Su, Lili Ren,
Hydrophobic starch nanocrystals preparations through crosslinking modification
using citric acid, International Journal of Biological Macromolecules
http://dx.doi.org/10.1016/j.ijbiomac.2016.06.082

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Hydrophobic starch nanocrystals preparations through crosslinking modification

using citric acid

Jiang Zhoua, Jin Tonga, Xingguang Sub, Lili Rena,b,

a
Key Laboratory of Bionic Engineering (Ministry of Education), College of Biological

and Agricultural Engineering, Jilin University, Changchun 130022, China

b
College of Chemistry, Jilin University, Changchun 130022, China

*
Corresponding author. Tel.: +86 431 85095760 ext.8608; fax: +86 431 85095760 ext.888.

E-mail liliren@jlu.edu.cn (L.L. Ren)

1
Graphical abstract

Highlights

 Starch nanocrystals were hydrophobically modified with citric acid by a dry reaction

method.

 The modified starch nanocrystals possessed lower polarity and showed stronger

hydrophobicity.

 The hydrophobicity of the modified starch nanocrystals could be adjusted by

controlling the heating time.

 The crystalline structure of starch nanocrystals was partially preserved after the

modifications.

2
Abstract

Biodegradable starch nanocrystals prepared by an acid treatment process were modified

through crosslinking modification using citric acid as reactant by a dry reaction method.

The occurrence of crosslinking modification was evaluated by Fourier transform

infrared spectroscopy and swelling degree. X-ray diffraction, wettability tests and

contact angle measurements were used to characterize the modified starch nanocrystals.

It was found that the crosslinked starch nanocrystals displayed a higher affinity for low

polar solvents such as dichloromethane. The surface of starch nanocrystals became

more roughness after crosslinking modification with citric acid and the size decreased as

revealed by scanning electron microscopy and dynamic light scattering results. XRD

analysis showed that the crystalline structure of starch nanocrystals was basically not

changed after the crosslinking modification with shorter heating time. The resulting

hydrophobic starch nanocrystals are versatile precursors to the development of

nanocomposites.

Keywords: hydrophobicity; crosslinking; starch nanocrystals

1. Introduction

Fossil energy depletion and growing environmental degradation have been attracting

increasing interest in biodegradable materials from renewable and sustainable

non-petroleum based resources. Starch is one of the abundant biopolymers produced by

many plants as a source of stored energy and probably the most promising renewable

3
naturally biodegradable polymer [1]. Starch granules consist of amorphous and

crystalline domains. Through a mild hydrolysis using acids and/or enzymes at

temperatures below the gelatinization temperature of starch, the amorphous regions

could be dissolved leaving the rigid crystalline regions intact [1-4]. The residue after

acid hydrolysis contains the starch nanocrystals which have high crystallinity,

platelet-like, square-like or round-like morphology and specific surface area. Starch

nanocrystals were suggested to be used as an emulsion stabilizer in food industries [5].

Nontoxicity, biodegradability and biocompatibility of starch nanocrystals make them

excellent carriers for drug delivery [6,7]. In addition, starch nanocrystals have been used

as reinforcements in natural polymers and synthetic polymers matrices to prepare

nanocomposite materials by virtue of their advantages of biodegradability, low cost,

renewability, intrinsic rigidity, high crystallinity and low permeability [3,8]. Even when

small amounts of starch nanocrystals were added to a matrix, improvement in

mechanical and barrier properties was found [9-12]. However, starch nanocrystals have

a strong tendency to aggregate and settle in water, and the inherent polar and

hydrophilic nature of starch nanocrystals result in their poor dispersibility in organic

solvents and poor compatibility with hydrophobic polymers, which inhibited the

utilization of starch nanocrystals in foods, cosmetics, medicines as well as various

composites. Hence, it is necessary to modify starch nanocrystals to lower their polarity,

improve their dispersion in organic solvents and broaden the type of possible polymer

matrices by allowing the processing of nanocomposites from an organic solvent instead

of aqueous medium.

4
 Nanoscale dimensions and ample surface hydroxyl groups enable chemical

modification of starch nanocrystals to introduce virtually any desired functionality.

Common chemical modification of starch nanocrystals have been reviewed in detailed

by Lin et al. [13], including mainly modification by chemical reaction with small

molecules [4,14], grafting onto polymer chains with coupling agents [15-17], and

grafting from polymer chains with polymerization of a monomer [18-21]. Le Corre and

Angellier-Coussy [3] reported that crosslinking reactions were the other route of

chemical modification of starch nanocrystals in their recent review article. A novel type

of reduction-sensitive starch nanoparticles with disulfide crosslinker was prepared via

the reversed-phase micro-emulsion method by using disulfide-functionalized crosslinker

of N,N-bisacryloylcystamine [22]. Author’s previous works showed that the

crosslinking modification of starch nanocrystals with sodium hexametaphosphate

significantly improved dispersion of starch nanocrystals in water and the crystalline

structure of starch nanocrystals was preserved after the crosslinking [23]. Jivan et al. [24]

proposed to cast light on water redispersible starch nanocrystals developed by Ren et al.

[23] and found that crosslinked starch nanocrystals formed a significantly stronger

lattice than their unmodified counterpart and thus showed high thermal stability (no

phase transition up to 150 ℃). Ma et al. [25] prepared starch nanoparticles by

precipitating a starch paste solution within ethanol as the precipitant followed by

crosslinking with citric acid for 1.5 h at 130 ℃. The addition of crosslinked starch

nanoparticles improved mechanical and moisture barrier properties of thermoplastic

starch film due to the good interaction between filler and matrix. When being

5
incorporated as fillers in glycerol plasticized soy protein plastics, the citric acid

modified starch nanoparticles exhibited a homogeneous distribution in the matrix and

a good compatibility with soy protein matrix, which was the source of the

enhancements of mechanical performance and water resistance [26]. Starch nanocrystals,

starch crystallites, microcrystalline starch and hydrolyzed starches refer to the

crystalline part of starch obtained by hydrolysis. They have to be distinguished from

starch nanoparticles, of which they are just one kind, and which can be amorphous [3].

However, the effect of crosslinking modification with citric acid on the

structure/properties of starch nanocrystals has not been studied.

Citric acid is a trifunctional carboxylic acid, which can be used as crosslinking agents

to react with hydroxyl groups in starch to create intermolecular linkages so that

macromolecular networks were formed [27-29]. Compared to starch nanoparticles, the

crystalline structure of starch nanocrystals was preserved after acid hydrolysis. The

preserved crystalline structure may keep its integrity in the crosslinking modification

with citric acid and hydrophobic character can be imparted to starch nanocrystals so that

the application range of the crosslinked starch nanocrystals could be broadened.

Therefore our interest was focused on crosslinking modification of starch nanocrystals

with citric acid by a dry reaction method. The aim of this investigation was to

characterize the modified starch nanocrystals in terms of their novel physicochemical

properties by ensuring that the morphology and the crystallinity of nanocrystals were

remained during the crosslinking modification. Some other properties were

characterized by Fourier transform infrared (FT-IR) spectroscopy, wettability tests and

6
contact angle measurements to investigate the effect of the modifications.

2. Experimental Section

2.1 Materials

Waxy maize starch was supplied by Changchun Jincheng Corn Development Co. Ltd.,

Da Cheng Group (China). Sulfuric acid (≥95%), citric acid, ethanol and

dichloromethane were obtained from Beijing Beihua Fine Chemicals Co. Ltd. (Beijing,

China). All these materials were used as received without further purification.

2.2 Preparation and modification of starch nanocrystals

Starch nanocrystals were prepared by acid hydrolysis of waxy maize starch granules

according to the commonly used method described in literature with minor modification

[30]. 25 g of native waxy maize starch was mixed with 250 mL of 3.16 M H2SO4

solution in a 500 mL Erlenmeyer flask. The suspensions were then put on a platform in

a thermo-stated atmosphere and continuously stirred at 100 rpm for 5 d at 40 ℃. The

resultant suspensions were washed by successive centrifugations in distilled water until

neutrality. Finally, the loose starch nanocrystals powder was obtained by lyophilization.

3 g citric acid was dissolved in 30 mL of ethanol to prepare citric acid solution. 2 g

starch nanocrystals were mixed with 20 mL of citric acid solution in a Petri dish and

conditioned for 12 h at room temperature to allow the absorption of citric acid solution

by starch nanocrystals. The Petri dish was dried in ovens at 50℃ to weight equilibrium

for removing ethanol and then heated in oven at 130℃ for 2, 4, 6 h and 8 h to finish the

reaction. The modified starch nanocrystals were washed three times with water to

remove unreacted citric acid, and then with ethanol to remove water and dried at room

7
temperature and finally ground.

2.3 Characterization

2.3.1 Fourier transform infrared (FT-IR) spectroscopy

The intact and modified starch nanocrystals were characterized with FT-IR

(IRAffinity-1 spectrophotometer, Shimadzu, Japan). The specimens were prepared by

grinding starch nanocrystals together with KBr and then pressing the samples into a disc.

The resolution was 4 cm-1 and the total scans were 32.

2.3.2 X-ray diffraction (XRD)

The structures of the intact and modified starch nanocrystals were characterized by

using a Rigaku D/max-2500 X-ray diffractometer (Rigaku Corporation, Tokyo, Japan)

with Cu-Ka radiation (λ=1.542 Å) at 40 kV and 250 mA. The XRD patterns were

recorded at room temperature over the 2θ range of 3-35° at a speed of 1°/min. Relative

crystallinity was determined from the ratio of the areas of the diffraction peaks to the

area of the whole diffraction pattern after subtracting amorphous background patterns.

2.3.3 Particle size and zeta potential analysis

The mean size of the intact and modified starch nanocrystals and zeta potential

measurements of starch nanocrystals suspension were measured by dynamic light

scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK).

The intensity of the scattered light was detected at 90° to the incident beam. The

measurements were performed using the samples prepared by dispersing starch

nanocrystals in 1 mM NaCl at 25 at a ratio of 0.01%, w/v. The mean size and

polydispersity index (PDI) were reported as the average of six measurements. The

8
refractive index of fluid and particle used were 1.33 and 1.53, respectively.

2.3.4 Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) was carried out on a Zeiss EVO 18 SEM (Carl

Zeiss SMT Ltd, Cambridge, UK). The intact and modified starch nanocrystals were

sputtered with gold, and then observed and photographed. An accelerating potential of

20 kV was used.

2.3.5 Swelling degree (SD) and solubility (S)

Swelling degree (SD) and solubility (S) of the intact and modified starch nanocrystals

were measured. Accurately weighed sample (0.2 g) was put into a 100 mL beaker, and

then 20 mL deionized water was mixed using ultrasound treatment (KQ-600KDE NC

Ultrasonic Cleaner, Kunshan, China) at 100% power with continuous stirring for 30 min

in a water bath at 40 ℃. The obtained suspension was centrifuged at 4250 ×g for 10 min.

To measure the SD and S, supernatant and sediment were carefully transferred into Petri

dishes respectively and dried overnight in a hot air oven at 105 ℃. The SD and S were

calculated as:

W2  W1 W3
SD  , S 100%
W0 W3  W0

where W0 is the weight of sediment on dry basis, W1 is the weight of the centrifugal

tube, W2 is the weight of the centrifugal tube with swollen sediment, and W3 is the

weight of supernatant on dry basis. The tests were performed in triplicate for each

sample.

2.3.6 Contact angle measurements

9
 The dynamic contact angles of sessile drops of water (2μL) on discs of starch

nanocrystals were measured with an OCA20 automated and software-controlled video

based contact angle meter (DataPhysics Instruments GmbH, Filderstadt, Germany).

Discs of starch nanocrystals with smooth surface and a diameter of 13 mm were

obtained by compressing the dried powder of starch nanocrystals under a pressure of 8

MPa using an IR specimen die. The discs were dried again under vacuum at 40 ℃ for

12 h to remove residual water. All measurements were conducted at room temperature

(25 °C). The contact angles were measured on both sides of the drop and averaged.

Each reported contact angle was the mean value of at least five measurements taken at

different positions on the discs.

2.3.7 Wettability tests

Wettability tests were used to assess surface polarity of the modified starch

nanocrystals according to the method described by Namazi and Dadkhah [4]. Starch

nanocrystals were mixed with two immiscible solvents having different polarities and

densities in a vial at a concentration of 2.5 mg/mL at room temperature to observe the

wettability of the sample with the solvents.

2.3.8 Statistical analysis

The difference between factors and levels was evaluated by the analysis of variance

(ANOVA). Duncan’s multiple range tests were used to compare the means to identify

which groups were significantly different from other groups (p < 0.05). All data are

presented as mean ± standard deviation.

3. Results and Discussion

10
3.1 FT-IR analysis

When citric acid was heated, it dehydrated to yield an anhydride, which could react

with starch to form a starch-citrate derivative. When a terminal carboxyl esterifies, a

citric acid molecule form an effective crosslinking between two starch molecules

[31-33]. The schematic representation of the crosslinking reaction between starch and

citric acid was given in Fig. 1. Comparative FT-IR spectra of intact starch nanocrystals

and citric acid modified ones showed clear signs of modification. In the spectrum of the

intact starch nanocrystals (Fig. 2a), the characteristic peak occurred at 1651 cm-1 was

due to the presence of bound water and a broad band appeared at 3379 cm-1 was due to

hydrogen-bonded hydroxyl groups [34]. The peaks at 1155 cm-1 was attributed to the

stretching vibration of C-O in C-O-H groups and the peak at 1022 cm-1 was attributed to

the stretching vibration of C-O in C-O-C groups [4]. In the FT-IR spectrum of pure

citric acid (Fig. 2e), the peak occurred at 1709 cm-1 was due to the C=O stretching

vibration in carboxyl groups and the peak occurred at 1743 cm-1 was an interference

peak which could be found when the carboxyl groups were very close to each other in

diacid [35]. However, after washing the modified starch nanocrystals thoroughly with

water, the characteristic peaks occurred at 1709 cm-1 and 1743 cm-1 disappeared (Fig. 2d,

2f). For citric acid modified starch nanocrystals, there was a clear new peak which was

located at 1732 cm-1 in the spectra. Since this peak is associated with C=O ester stretch

and the modified starch nanocrystals were thoroughly washed to remove the unbound

citric acid, the presence of the ester carbonyl bands peak confirms the chemical linkages

between starch and citric acid. The change of the peak’s position from 1709 cm-1 in pure

11
citric acid to 1732 cm-1 in the modified starch nanocrystals demonstrated that the

crosslinking modification between starch and citric acid happened in the course of high

temperature heating [35]. In intact starch nanocrystals, the oxygen of the C-O-C group

could form the hydrogen-bond interaction with the hydrogen of hydroxyl groups, while

in the modified starch nanocrystals, the ester bonds as the crosslinking of starch

sterically hindered this hydrogen-bond interaction [25] and the weakening of this

interaction made C-O bond stretching of the C-O-C group shift to lower wave numbers

(from 1022 cm-1 to 1016 cm-1) as shown in Fig. 2b-d [36]. The results of FT-IR

suggested that new interactions formed between starch and citric acid after modification.

The area ratio of C=O peak at 1732 cm-1 to OH peak at 3379 cm-1 in FT-IR spectra was

determined as degree of substitution (DS, i.e. the average number of hydroxyl groups

substituted per glucose unit) of starch [37]. Fig. 2 listed DS values of the starch

nanocrystals modified with citric acid at 130 ℃ for various heating time. When the

heating time was increased from 2 h to 6 h, DS of the modified starch nanocrystals

increased from 0.1089 to 0.3249, suggesting that the increase of heating time gave rise

to a significant increase in DS.

3.2 XRD and relative crystallinity

XRD has been widely used to reveal the characteristics of the crystalline structure of

starch nanocrystals and to verify if any alteration of their crystallinity occurred upon

chemical modification. The XRD patterns and relative crystallinity data of waxy maize

starch, intact starch nanocrystals and the modified starch nanocrystals were shown in

Fig. 3A. Both waxy maize starch and intact starch nanocrystals showed the A-type

12
pattern with the clear diffraction peaks, at which 2θ values equal to 15.1°, 17.1°, 17.9°,

23.0° [38-40], which indicated that the acid hydrolysis did not destroyed or transform

the starch crystalline structure. The crystalline structure of the modified starch

nanocrystals with 2 h, 4 h and 6 h heating time at 130 ℃ kept intact although the

crystalline peaks became much smaller in comparison with intact starch nanocrystals,

and the modified starch nanocrystals with higher DS possessed lower intensity of

diffraction peaks. Before the test, all the samples were washed with adequate water. It

was found that the crystalline peaks of pure citric acid (Fig. 3g) disappeared on XRD

curves of the modified starch nanocrystals and a peak around 31.5° caused by the

residual citric acid did not appear [41], which illustrated that the unreacted citric acid

was removed in the process of washing with water. The crystallinity calculated from

X-ray diffraction patterns of the modified starch nanocrystals with 2 h, 4 h and 6 h

heating time at 130 ℃ ranged from 53.1% to 11.7%, which was contrary to the change

trend of DS (from 0.1089 to 0.3249). When the heating time increased to 8 h, the

modified starch nanocrystals lost most crystalline peaks except a peak at 2θ = 18.6° (Fig.

3e) and the color of sample became black (not shown), which might be because that

starch nanocrystals was gelatinized under the reaction with citric acid at 130 ℃ for a

longer heating time [31]. The new XRD pattern at 2θ = 18.6° may reflect a new

structure of amorphous regions in the modified starch nanocrystals with 8 h heating

time. These phenomena can be explained that the crosslinking reaction should occur

both in the amorphous phase and crystalline phase of starch, and when citric acid

penetrates the starch granules through channels and cavities, it could disrupt the

13
crystalline structure of granules due to a concentrated solution of citric acid, especially

at longer heating time [25,42].

3.3 SD and S analysis

Crosslinking modification has been reported to inhibit swelling and solubility of

starch, with the inhibition being linearly related to the degree of crosslinking [43],

therefore, SD and S were used to characterize crosslinking modification of starch

nanocrystals. Fig. 4 showed the SD and S for the intact and modified starch

nanocrystals. It can be seen that both SD and S increased with the increasing of heating

time, reached a maximum at 2 h, and then decline, when heating time is 6 h, both SD

and S of the modified starch nanocrystals were lower than those of intact starch

nanocrystals. Traditionally, substitution of citric acid groups on starch chains may alter

the associations between amylose and amylopectin and each component with itself in

the starch granules and thus preventing the starch granules from swelling and solubility

[42]. However, in this study the crystalline structure of starch nanocrystals was partially

destroyed after crosslinking modification (proven by XRD), which can not thoroughly

hinder the crosslinker from entering the inner of starch nanocrystals and so that the

crosslinking reaction could take place on the surface and in the inner of starch

nanocrystals. When the heating time was short, the crosslinking degree of the modified

starch nanocrystals was low and the crosslinked network structure was not sufficiently

to penetrate water into the inner of starch and inhibited swelling and solubility of

nanocrystals. Whereas, the increase of heating time led to a significant increase in DS

and higher DS means more crosslinked network structure have been formed, which

14
resulted in lower SD and S of the modified starch nanocrystals. The modified starch

nanocrystals with lower relative crystallinity contained lower SD and S, suggesting that

the decreased in swelling and solubility may be attributed to the formation of

crosslinked network structure, rather than the crystalline structuture.

3.4 Size and zeta potential analysis

Table 1 shows the mean size, PDI and zeta potential of the intact and modified starch

nanocrystals. Analysis of particle size showed that the intact starch nanocrystals were

micro-sized due to aggregation. The crosslinking modification significantly reduced the

particles size and narrowed the range of size distribution and the mean size of the

modified starch nanocrystals decreased with increasing of reaction time. Polydispersity

index (PDI), a parameter defining nanoparticle size distribution obtained from DLS,

ranges from a value 0.1-0.5 for suitable measurements and good quality of the colloidal

suspensions to values close to 0.7 for poor quality samples which have a very broad size

distribution and may contain large particles or aggregates. The PDI of the intact starch

nanocrystals was very close to 0.7, suggesting there were big particles or aggregates in

the suspension, which has been further confirmed by the wettability tests. For the starch

nanocrystals modified with citric acid at 130 ℃ for 2 h, 4 h and 6 h, PDI values

indicated that these samples were colloidal suspensions with good quality and suitable

for dynamic light scattering measurements. It was known that the effective surface

charge of particles primarily determines their dispersion and aggregation. Thus, the

effects of crosslinking modification with citric acid on zeta potentials of starch

nanocrystals suspensions (0.01%, w/v) were studied at pH 7.0. As shown in Table 1,

15
zeta potential of H2SO4-hydrolyzed starch nanocrystals suspension was negative (–6.19

mV), indicating that starch nanocrystals have negative surface charges. It was due to the

presence of carboxyl and sulfate esters on the surface of starch nanocrystals obtained

from H2SO4 hydrolysis [44]. While after crosslinking modification with citric acid, the

zeta potentials of the modified starch nanocrystals suspension significantly decreased.

This result demonstrated that the introduction of carboxyl and carbonyl groups

significantly increased the surface charge of starch nanocrystals, which was in

agreement with previous findings [45]. In addition, with increase of heating time, the

absolute zeta potentials of the modified starch nanocrystals increased. This is because

that longer heating time lead to a higher degree of modification and more carboxyl and

carbonyl groups have been introduced, resulting in higher surface charge of the

modified starch nanocrystals.

3.5 SEM analysis

SEM was used to investigate the morphological changes of the starch nanocrystals

associated with modification. Fig. 5a showed a SEM micrograph of the intact starch

nanocrystals, illustrating the formation of laminar aggregates with large specific area,

similar to the structures reported by Garcia, et al. [9] and Wei, et al. [45]. The existence

of nanocrysals aggregates suggested the presence of a high number of OH-groups per

individual nanocrystal, which became strongly associated by hydrogen bonding.

Crosslinking modification of starch nanocrystals with citric acid seemed to decrease the

aggregation of starch nanocrystals and the large particles of starch nanocrystals reduced

as shown in Fig. 5b-d. It was noted that the longer reaction time gave rise to the smaller

16
particle size. The surface of intact starch nanocrystals looked smooth, whereas the

surface of the modified starch nanocrystals exhibited roughness and there were some

connections between particles, suggesting that the crosslinking modification of starch

nanocrystals with citric acid did occur.

3.6 Wettability tests

Wettability tests are valuable and simple methods to qualitatively assess surface

polarity of starch nanocrystals. It was observed that, in the solvent system of water (d=1

g/cm3) and dichloromethane (d=1.335 g/cm3) (water/dichloromethane), the intact starch

nanocrystals can only stay in the aqueous medium of the mixed solvent systems because

of the strong hydrophilic character, and were not able to migrate into dichloromethane

even after shaking (Fig. 6a). But, the crosslinked starch nanocrystals could disperse in

both water and dichloromethane (Fig. 6b-d). It can be explained that in water medium

the hydrophobic C=O groups of citric acid will be rolled up, and the hydrophilic starch

backbone is in extended form. On the contrary, in dichloromethane the hydrophilic

hydroxyl groups of starch tend towards inside of starch nanocrystals, while the

hydrophobic groups of citric acid will be in stretched form. With increase of the heating

time, more and more crosslinked starch nanocrystals migrated toward the

dichloromethane, suggesting longer heating time results in higher hydrophobicity. This

is because longer heating time would increase the reaction extent and more hydroxyl

groups have been substituted in the crosslinking modification.

In addition, the intact starch nanocrystals started settling in several minutes due to

aggregation and almost completely settled to the bottom of the aqueous medium of the

17
mixed solvent systems in 20 min, while the crosslinked starch nanocrystals were still

well dispersed after 60 min (not shown). This could be ascribed to, compared to the

intact starch nanocrystals suspension, the higher absolute zeta potential and the relative

smaller particle distribution of the crosslinked starch nanocrystals generated the more

stable suspensions. These observations indicated that the crosslinking modifications of

starch nanocrystals using citric acid changed the surface nature of starch nanocrystals

and prevented starch nanocrystals from aggregation.

3.7 Contact angle measurements

Fig. 7 shows the dynamic behavior of the contact angle for a drop of distilled water

deposited onto the surface of both the intact and crosslinked starch nanocrystals. The

initial value of water contact angle presented in Fig. 7 for intact starch nanocrystals is

very close to that reported for starch nanocrystals obtained by acid hydrolysis of waxy

maize starch for 5 days at 40 ℃ in a 3.16 M aqueous H2SO4 solution [16,17,46] and the

low value can be attributed to the high hydroxyl group content of the constituting

glucose unit (3 hydroxyl groups per glucose moiety). It was found that a clear and

significant increase in initial contact angle value was observed for the crosslinked starch

nanocrystals compared to intact one, clearly showing the more hydrophobic nature of

the crosslinked starch nanocrystals and this is in agreement with the results obtained

from wettability tests. As shown in Fig. 7, the initial contact angle values of the

crosslinked starch nanocrystals increased with increasing of heating time. This is

because the increase of heating time gave rise to a significant increase in DS and higher

DS means more hydroxyl groups in starch molecules have been substituted by

18
hydrophobic carbonyl group, which result in higher hydrophobicity of the crosslinked

starch nanocrystals. In addition, it is also clear that the decrease of contact angle values

with time can be attributed exclusively to the spreading of the water drop rather than a

penetration by capillarity because of the porous character of the discs used in the

measurements [14,16]. The water droplet was rapidly spread on the surface of the discs

of intact starch nanocrystals, whereas it remained on the surface of the crosslinked ones

for a much longer time. These observations also suggested that crosslinking

modification gave rise to lower polarity of starch nanocrystals.

Conclusion

Starch nanocrystals were hydrophobically modified through mild crosslinking

modification with citric acid by a dry reaction method. The modification has changed

the surface character of starch nanocrystals and decreased the size of starch nanocrystals.

The modified starch nanocrystals exhibited enhanced hydrophobicity and dispersibility

compared to unmodified starch nanocrystals, and the hydrophobicity could be adjusted

by controlling the heating time. The crystalline structure of starch nanocrystals was

preserved after the modification with shorter heating time. These advantages clearly

pave the way to potential applications in foods, cosmetics, medicines as well as various

composites, for example, as can be used as emulsion stabilizer, rheology modifier, drug

delivery vehicle and reinforcements for the nanocomposites with hydrophobic polymer

matrix.

19
Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (No.

51273083 & No. 51405188) and China Postdoctoral Science Foundation (No.

2013M540253 & No. 2015T80307).

20
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Figure captions

Fig. 1 Schematic representation of the crosslinking reaction between starch (St-OH) and

citric acid.

Fig. 2 FTIR spectra for the intact starch nanocrystals (a), starch nanocrystals modified

with citric acid at 130 ℃ for 2 h (b), 4 h (c) and 6 h (d), pure citric acid (e) and starch

nanocrystals without washing after being modified with citric acid at 130 ℃ for 6 h (f).

Fig. 3 X-ray diffraction patterns of waxy maize starch (a), intact starch nanocrystals (b),

starch nanocrystals modified with citric acid at 130 ℃ for 2 h (c), 4 h (d), 6 h (e) and 8

h (f) and pure citric acid (g).

Fig. 4 Comparison of the SD and S for intact starch nanocrystals and starch nanocrystals

modified with citric acid at 130 ℃ for different heating time.

Fig. 5 SEM micrograph of intact starch nanocrystals (a) and starch nanocrystals

modified with citric acid at 130 ℃ for 2 h (b), 4 h (c) and 6 h (d).

Fig. 6 Dispersion of intact starch nanocrystals (a) and starch nanocrystals modified with

citric acid at 130 ℃ for 2 h (b), 4 h (c) and 6 h (d) in water/dichloromethane.

Fig. 7 Water contact angle with time for (a) intact starch nanocrystals and starch

nanocrystals modified with citric acid at 130 ℃ for 2 h (b), 4 h (c) and 6 h (d).

27
Fig 1

28
Fig 2

29
Fig 3

30
Fig 4

31
32
Fig 5

33
Fig 6

34
Fig 7

35
Table 1. Mean size, PDI and zeta potential of the intact and crosslinked starch
Sample Mean size (nm) PDI Zeta potential (mV)

Intact starch 1801±43.9d 0.63±0.22c -6.2±0.3d

nanocrystals
Crosslinked starch 503.1±35.2c 0.45±0.01ab -14.1±0.7c
nanocrystals-2 h
Crosslinked starch 479.3±23.9b 0.46±0.07b -15.5±0.5b
nanocrystals-4 h
Crosslinked starch 290.5±10.5a 0.42±0.04a -18.0±0.5a
nanocrystals-6 h
Values mean ± SD indicates that the replicates of six experiments. Means with the same letter in
each column are not significantly different (p<0.05).
Starch nanocrystals modified with citric acid at 130 ℃ for 2 h (Crosslinked starch
nanocrystals-2 h), 4 h (Crosslinked starch nanocrystals-4 h) and 6 h (Crosslinked starch
nanocrystals-6 h).

36