Morphology and Characteristics of Starch Nanoparticles Self

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Morphology and characteristics of starch nanoparticles self-assembled via a rapid ultrasonication method for peppermint oil encapsulation Chengzhen Liu, Man Li, Na Ji, Jing Liu, Liu Xiong, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02938 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on September 5, 2017

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Journal of Agricultural and Food Chemistry

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Morphology and characteristics of starch nanoparticles self-assembled via a

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rapid ultrasonication method for peppermint oil encapsulation

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Chengzhen Liu†, Man Li†,Na Ji†, Jing Liu††, Liu Xiong†, Qingjie Sun*†

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†College of Food Science and Engineering, Qingdao Agricultural University (700

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Changcheng Road, Chengyang District, Qingdao, Shandong Province, 266109,

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China).

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† † Central laboratory, Qingdao Agricultural University (700 Changcheng Road,

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Chengyang District, Qingdao, Shandong Province, 266109, China).

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ABSTRACT

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Starch nanoparticles (SNPs) and peppermint oil (PO)-loaded SNPs were

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fabricated via an ultrasonic bottom-up approach using short linear glucan debranched

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from waxy maize starch. The effects of the glucan concentration, ultrasonic irradiation

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time, and chain length on the SNPs’ characteristics were investigated. Under the

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optimal conditions, i.e., short linear glucan concentration of 5% and ultrasonication

15

time of 8–10 min, SNPs were successfully prepared. The as-prepared SNPs showed

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good uniformity and an almost perfect spherical shape, with diameters of 150–200 nm.

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The PO-loaded SNPs also exhibited regular shapes, with sizes of approximately 200

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nm. The loading capacity, encapsulation efficiency, and yield of PO-loaded SNPs

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were ~25.5%, ~87.7%, and ~93.2%, respectively. After encapsulation, PO possessed

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enhanced stability against thermal treatment (80°C). The pseudo-first-order kinetics

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model accurately described the slow-release properties of PO from SNPs. This new

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approach of fabricating SNPs is rapid, high yield, and non-toxic, showing great

ACS Paragon Plus Environment


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potential in the encapsulation and sustained release of labile essential oils or other

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lipids.

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KEYWORDS:

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Encapsulation

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INTRODUCTION

Short

linear

glucan;

Ultrasonic

processing;

Essential

oil;

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Starch, as a naturally renewable and biodegradable biopolymer, is one of the most

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abundant reserve carbohydrates, and it constitutes a fundamental material for food and

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nonfood use.1 Native starch granules exhibit a size of 1–100 µm, making them

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microscale granules. Starch is predominantly composed of linear amylose and

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branched amylopectin. In recent years, nanoscale starch particles have drawn

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considerable attention as novel and biofunctional materials in diverse applications,

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including drug (or bioactive substance) delivery carriers,2 film fillers,3 emulsion

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stabilizers, and fat replacers.4 In the past few decades, various techniques have been

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developed for the preparation of starch nanoparticles (SNPs), including acid

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hydrolysis,

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treatments.2 Nevertheless, these methods are associated with environmental pollution,

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low yields, or high energy costs.4 Recently, Sun et al. (2014) proposed a simple,

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environmentally friendly technique for obtaining SNPs by pullulanase debranching of

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waxy maize starch followed by recrystallization.5 Moreover, Qiu et al. (2016)

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developed an easy method of preparing SNPs using nanoprecipitation of debranched

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waxy corn starch.6 Although these new methods are green, simple, and scalable, a

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time-saving technique is still lacking.

mini-emulsion

crosslinking,

enzymatic

treatment,


and

physical

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Ultrasonic technology has also been used to prepare SNPs within a short duration

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of time. Ultrasonication generates ultrasonic cavitation in the solution and causes

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microbubbles. When microbubbles collapse, high energy is released and converted to

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high pressure and high temperature.7 Sun et al. (2014) reported that waxy corn starch

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granules are converted from the micrometer to the nanometer scale after oxidation

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followed by ultrasonic treatment for 3 h, with the particle size of SNPs ranging from

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20 to 60 nm.8 Bel et al. (2013) reported on nanoparticles of 30–250 nm in size

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prepared from waxy maize starch granules using a high-intensity ultrasonication

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method for more than 75 min.9

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In the published literature using the ultrasonication method, the SNPs were all

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fabricated via a top-down process. In such a process, large starch granules can be

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gradually broken into nanoscale particles through a mechanical size-reduction

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process.10 However, this top-down ultrasonication method consumes a great deal of

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energy and still has a long duration, on the timescale of hours. Alternatively, the

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bottom-up approach to preparing nanoparticles mainly relies on eliciting specific

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interactions between molecules to drive an autonomous self-assembly process under

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appropriate conditions.11 This method makes it easy to quickly fabricate uniform

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nanoparticles compared with the top-down approach. To the best of our knowledge,

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there is no information on producing SNPs using a bottom-up approach combined

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with ultrasonication.

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The linear unbranched amylose fraction of starch is known to form inclusion

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complexes with low–molecular weight substances, such as iodine, alcohols, lipids,



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and aromatic compounds.12 Meng et al. (2014) prepared starch–palmitic acid

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complexes through heating combined with high pressure homogenization.13 Ocloo et

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al. (2016) reported that amylose–lipid complexes are formed during pasting of high

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amylose maize starch with stearic acid under pressure.14 Moreover, Qiu et al. (2016)

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reported that essential oils are encapsulated in SNPs prepared by short glucan

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chains.15 Natural essential oils, a concentrated aromatic hydrophobic liquid, are

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widely used in perfumes, cosmetics, food and drink, and medicines. However, low

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water solubility, high volatility, and strong odor limit their applications.16 To

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overcome these drawbacks, various encapsulation techniques have been studied. Lv et

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al. (2014) have prepared jasmine essential oil nanocapsules by gelatin and gum arabic

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based complex coacervation.17 de Oliveira et al. (2014) have successfully fabricated

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the nanoparticles of alginate/cashew gum for encapsulation of Lippia sidoides

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essential oil via spray-drying.18 Although some progress has been made, it is still

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urgent to develop facile and fast approaches for loading the fragile essential oils.

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The main objective of this work is to develop a facile, rapid approach for

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fabricating SNPs via an ultrasonic bottom-up method employing various chain lengths

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of short linear glucan (SLG). The encapsulation and release properties of peppermint

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oil (PO)-loaded SNPs are also investigated.

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MATERIAL AND METHODS

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Materials

Waxy maize starch (approximately 2% amylose and 98%

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amylopectin) was supplied by Tianjin Tingfung Starch Development Co., Ltd.

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(Tianjin, China). PO was obtained from Spectrum Chemicals & Laboratory Products


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(Gardena, CA). Pullulanase (E.C.3.2.1.41, 6000 ASPU/g, 1.15 g/mL) was purchased

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from Novozymes Investment Co., Ltd. (Beijing, China). All reagents used were of

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analytical grade.

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Preparation of short linear glucan from starch

Three types of SLGs with

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different chain lengths were prepared to investigate the formation of SNPs using the

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ultrasonication method. Primary SLG was prepared by debranching waxy maize

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starch according to Liu et al.’ (2016b) method, with some modifications.19 First, waxy

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maize starch was dispersed in disodium hydrogen phosphate and citric acid buffer

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solution (pH 4.6) for fully gelatinization and debranched with pullulanase at 58°C for

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8 h without stirring. Then, the obtained linear starch molecule solution was

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precipitated using 4× absolute alcohol (solution: absolute alcohol, v/v) at room

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temperature (25°C), washed three times with distilled water until a neutral pH was

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acheived, and freeze-dried at -86°C for 48 h to obtain primary SLG powder. To

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further fractionate the SLG into two components, absolute ethanol (solution:

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ethanol=1:3, v/v) was added to fully cooked SLG solutions at ambient temperature.

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The supernatant and precipitation were freeze-dried to obtain short SLG (S-SLG) and

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long SLG (L-SLG), respectively.

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Preparation of starch nanoparticles

SNPs were fabricated using the

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ultrasonication method as illustrated in Scheme 1. The primary SLG, S-SLG, or

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L-SLG powders were dispersed in ultrapure water with various concentrations (1, 5,

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and 10%, w/v, respectively) and autoclaved at 121°C. After cooling to room

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temperature, each aqueous solution was irradiated with a high-intensity ultrasonic



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horn (JY 92-IIN, 20–25 kHz, 990 W/cm2) operating at 10% efficiency for different

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lengths of time (5, 8, and 10 min). The formed nanoparticle suspension was allowed

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to cool to room temperature to determine the morphology and particle size. The

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suspension was centrifuged at 10,000 g for 10 min, and then the sediments were

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lyophilized for 48 h to obtain dry powders.

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For the preparation of PO-loaded SNPs, the cooked SLG solution (5%, w/v)

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containing PO (4.5% w/w, percentage of SLG) was irradiated with a high-intensity

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ultrasonic horn for 8 min (primary SLG and L-SLG) or 10 min (S-SLG). The

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nanoparticle suspensions were then centrifuged at 10,000 g for 20 min with

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ultrafiltration centrifuge tubes and a molecular weight cut-off of 5 kDa. The

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supernatant was removed and the sediments were washed with ethanol and then

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washed three times with water by centrifugation (10,000 g, 20 min). The supernatants

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were collected for calculations of the encapsulation efficiency (EE) and loading

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capacity (LC) of PO in the SNPs. The sediments were lyophilized for 48 h to obtain

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dry powders for further analyses.

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Encapsulation efficiency and loading capacity

The PO content was

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calculated using an ultraviolet-visible (UV-vis) spectrometer (290 nm) and a standard

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calibration curve that was plotted against the different concentrations of PO. The EE

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and LC of PO in SNPs were calculated using Eqs. 1 and 2, respectively (Liu et al.,

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2016c):20

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EE (%) = (total content of PO (mg) − content of PO in supernatant (mg)) / total content of PO (mg) × 100


(1)

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LC (%) = (total content of PO (mg) − content of PO in supernatant (mg)) / total

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weight of dry PO-loaded SNPs × 100

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Chain length distributions

(2)

Chain length distributions of SLG were analyzed

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using a high-performance size-exclusion chromatography (HPSEC) system. HPSEC

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was carried out according to Patindol et al.’ (2007) method, with some

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modifications.21 The SLG powder was dispersed in ultrapure water (1‰ w/v) and

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then autoclaved at 121°C for 30 min. An aliquot (100 µL) of solution was filtered

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through a filter membrane (0.22 µm pore size). The filtrate was used for chain length

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distributions analysis. Pullulan standards (Mw 342, 1,320, 6,200, 10,600, 21,700)

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were used as references for the determination of the SLG powder’s chain length.

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Transmission electron microscopy (TEM)

The morphology and size of the

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SNPs and PO-loaded SNPs were analyzed with a Hitachi 7700 transmission electron

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microscope (Tokyo, Japan) at an acceleration voltage of 80 kV. A droplet (about 10

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µL) of SNP or PO-loaded SNP suspension was diluted (5:1000) with ultrapure water

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and drop-cast onto a carbon-coated copper grid (400 meshes) and lyophilized for

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more than 6 h to obtain dry samples for further observation.

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Dynamic light scattering (DLS)

The particle size distributions of SNPs and

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PO-loaded SNPs were determined using the dynamic light scattering (DLS) technique

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with a Zetasizer Nano ZS90 (Malvern, UK). The intensity of light scattered was

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monitored at 90° angle. The SNP or PO-loaded SNP suspensions were diluted to a

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concentration of approximately 1 mg/mL with ultrapure water to avoid multiple

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scattering effects, and placed into the measurement chamber. Then, they were



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equilibrated at 25±1°C prior to analysis. Differential scanning calorimetry (DSC)

The thermal properties of SNPs and

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PO-loaded SNPs were measured using a differential scanning calorimeter (DSC 1,

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Mettler-Toledo International Inc., Switzerland). Three milligram samples (dry basis)

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with excess water (1:2) were sealed in an aluminum pan. DSC was performed from 25

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to 125°C at a heating rate of 10°C/min.22 The onset (To), peak (Tp), and conclusion

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temperatures (Tc), and enthalpy change of gelatinization (∆H) of the samples were

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recorded. The ∆H values were calculated using the dry weight.

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X-ray diffraction (XRD)

The crystalline structure of SNPs and PO-loaded

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SNPs were determined by an X-ray diffractometer (D8-ADVANCE, Bruker AXS

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Model, Germany). The scanning range and rate were 4–40° (2θ) and 1.0°/min,

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respectively. The relative crystallinity (RC) of each sample was quantitatively

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calculated following Kim’s (2013) method, as follows: RC = Ac / (Aa + Ac), where

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Ac is the crystalline area and Aa is the amorphous area.23

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Fourier transform infrared (FTIR) spectroscopy

The SNPs and PO-loaded

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SNPs were mixed with potassium bromide (KBr) powder at a ratio of 1:100 (sample:

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KBr, w/w). These admixtures were ground into fine powders and then compressed

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into thin disk-shaped pellets. The pellets were analyzed using a Fourier transform

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infrared (FTIR) spectrometer (Tensor 27, Bruker, Germany) with a mercury cadmium

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telluride detector. The FTIR spectra were obtained over the wavenumber range of 400

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to 4000 cm–1 at a resolution of 2 cm–1, and the total number of scans was 32.

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The release profile of peppermint oil

The release profile of PO-loaded SNPs


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in a hot water bath was determined according to Dong et al.’ (2011) method.24

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PO-loaded SNPs (10 mg) after lyophilized for 48 h were directly dispersed in 50 mL

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of distilled water. The suspensions were divided into five portions and then put in an

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80°C water bath at a stirring speed of 150 rpm. Portions were taken out at 30, 60, 90,

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120, and 150 min and filtered with a filter membrane (Mw = 3.5 kDa) with distilled

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water. The obtained residue was distilled using a Clevenger-type apparatus.

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Cumulative release (%) of the PO-loaded SNPs in the 80°C water bath was

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determined according to the following formula: Cumulative release (%)=(1−V2/ V1) ×100,

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(3)

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where V1 is the initial content of PO and V2 is the content of PO obtained from filter

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residue.

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Statistical analysis

All experiments were conducted in triplicate. The

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experimental data were subjected to statistical analysis with SPSS 17.0 software

190

(SPSS Inc., Chicago, USA). Duncan’s multiple range tests were also applied to

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determine the difference of means from the analysis of variance (ANOVA), using a

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significance test level of 5% (p < 0.05).

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RESULTS AND DISCUSSION

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Chain length distributions of short linear glucan The alcohol precipitation

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method is commonly used to separate polysaccharides from their aqueous solution

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due to its simplicity, rapidity, easy scalability, and cost effectiveness. The precipitation

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levels of polysaccharides at different concentrations of ethanol present different

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characteristics, including polymer recovery, chemical composition, molecular weight,



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and morphological appearance. In Liu et al.’s (2017) study, water-soluble

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polysaccharide was isolated and purified from Achatina fulica by papain enzymolysis

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and alcohol precipitation.25 In Wang et al.’s (2017) experiment, solvent extraction

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(water and alcohol) and organic solvent fractional extraction were used to extract

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crude polysaccharides from the dried pumpkin pulp.26

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In the present study, we added ethanol to the primary SLG aqueous solution to

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obtain various chain lengths of SLG; as a result, S-SLG was retained in the

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supernatant due to its higher solubility, and L-SLG was precipitated due to its lower

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solubility in the aqueous ethanol solution. Through a series of experiments, primary

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SLG was fractionated into S-SLG and L-SLG with the ethanol to water ratio of 3:1

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(v/v).

210

Chain length distributions of primary SLG, S-SLG, and L-SLG samples are

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shown in Figure 1, and the composition percentages are calculated in Table S1. The

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major population of SLG, with a low degree of polymerization (DP), was labeled F1;

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the rest, which had a high DP, was labeled F2. Compared with S-SLG, the content of

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minor components of F2 were somewhat higher in primary SLG and L-SLG, which

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were considered to represent amylopectin that was not fully debranched. The peak DP

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value of primary SLG was 13.5, with a shoulder peak at DP 30, and the relative

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content of F1 was 97.4±0.71% (Table S1). After fractionation with 3× absolute

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alcohol (solution: absolute alcohol=1:3, v/v), the S-SLG obtained from supernatant

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contained only one main peak (DP 9.5), which made up 78.1±0.58% (Table S1). In

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contrast, the L-SLG obtained from the precipitate had a main peak (DP 15.1) with a


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shoulder peak (DP 30). Hanashiro et al. (1996) categorized amylopectin branch chains

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into several types with their corresponding DP values, as follows: A chain (DP 6–12),

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B1 chain (DP 13–24), B2 chain (DP 25–36), and B3+ chain (DP 37-65).27 In our work,

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primary SLG was a mixture of debranched waxy maize starch with different DP

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values, mainly including A chains and B chains (B1 and B2).

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Effects of short linear glucan concentration

To investigate the effect of SLG

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concentration on SNP formation, we used primary SLG for self-assembly under

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ultrasonic treatment. The morphology and mean size of SNPs prepared at different

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SLG concentrations were determined by transmission electron microscopy (TEM) and

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DLS (Figure 2). When the concentration was 1%, SLG self-assembled to form small,

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irregular, spherical nanoparticles with a size ranging from 10 to 150 nm, and no

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agglomeration occurred. Moreover, the result of DLS showed two separated peaks,

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indicating that SNPs were non-uniform in size and distribution. When the SLG

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content was increased up to 5%, the obtained SNPs had a uniform, compact, smooth

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spherical shape, with diameters of 150–200 nm. The size distribution of SNPs

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determined by DLS showed only one peak, which was consistent with the TEM result.

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A further increment in the SLG concentration to 10% resulted in the formation of

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large and aggregated particles (200–300 nm). Furthermore, the DLS result showed

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two separated peaks, with the bigger one belonging to the micron level. Therefore, 5%

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SLG solution was the optimum concentration to produce uniform SNPs for

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subsequent studies. Similarly, Hebeish et al. (2013) found that increasing the

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concentration of native maize starch had a major adverse effect on the formation of



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monodispersity of the nanoparticles.28 Effect of ultrasonic irradiation time

Various ultrasonic irradiation times were

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employed to determine their effect on the morphology and particle size of SNPs

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prepared by different SLGs. Figures 3A–C, E–G, and I–K show the TEM images of

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primary SLG, S-SLG, and L-SLG nanoparticles, respectively, prepared at irradiation

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times of 5, 8, and 10 min. The morphology and size of primary SLG and L-SLG

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nanoparticles evolved with the same change tendency as a function of time. With

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increasing irradiation time, the diameter of the aggregates increased correspondingly.

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After ultrasonic processing for 5 min, the sizes of primary SLG (Figure 3A) and

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L-SLG (Figure 3I) nanoparticles were 100 and 150 nm, respectively. As

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self-assembling continued (Figure 3B and J), monodisperse nanoparticles with regular

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shapes (200 nm) were formed. It was noted that the SNP aggregates were formed after

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ultrasonication for 10 min (Figure 3C and K). As for S-SLG, only small portions of

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nanoparticles were formed after treatment for 5 min. Nanoparticles of irregular shape

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were formed following sonication for 8 min. Significantly, S-SLG took somewhat

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long period (10 min) to form compact nanoparticles with a spherical morphology; the

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size of the SNPs was determined to be 150 nm.

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Referring to the DLS measurement, the particles size was rather larger than that

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revealed by TEM observation. For the primary SLG sample, the particle size of

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formed SNPs increased from 100 to 2,000 nm (Figure 3D) as irradiation time

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increased from 5 to 10 min, which could have occurred because more energy provided

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by ultrasonication made the SNPs grow and then aggregate. The DLS results also


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showed that S-SLG and L-SLG nanoparticles exhibited increased size with increasing

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irradiation time. These results suggested that the shapes and sizes of SNPs produced

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via ultrasonication were highly influenced by irradiation time. Abbas et al. (2007) also

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reported an increase in the mean size of sodium chloride particles by applying

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sonication within 20 min.29

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The above results suggested that SLG could rapidly form nanoscale starch

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particles with a size of around 200 nm via the ultrasonic bottom-up method. To

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explore the application of these newly developed SNPs as a nanocarrier, PO was used

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as a model substance to be loaded into SNPs. The morphological characteristics,

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crystal structure, EE, LC, and release profile of the resulting SNPs were investigated.

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Encapsulation efficiency and loading capacity

The effects of various SLG

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chain lengths on the EE and LC of PO in SNPs were measured. As shown in Table 1,

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the EE of the three PO-loaded SNPs dropped from 87.7% to 74.5% with decreasing

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chain lengths from DP 15.1 to DP 9.5. The LC of the primary SLG (DP 13.5) and

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L-SLG (DP 15.1) nanoparticles was higher than that of the S-SLG (DP 9.5)

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nanoparticles. The yield of the PO-loaded SNPs increased from approximately 81.8%

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to 93.2% with the increase of the SLG chain lengths. The results suggested that the

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EE and LC values of the PO-loaded SNPs were enhanced as the SLG chain lengths

283

increased. With a high EE of 74.5-87.7% and high LC of 21.3-25.5%, the prepared

284

SNPs could be a potential carrier for essential oils. In a previous work, the Lippia

285

sidoides essential oil was encapsulated by a chitosan and “angico” gum matrix, with

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LC between 3% and 7% and EE in the range 16–77%.30



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In general, the PO-loaded SNPs fabricated using the ultrasonic bottom-up method

288

generated

higher

LC,

EE,

and

encapsulation

yields

compared

to

the

289

polycaprolactone-coated nanocapsules incorporated with the essential oil by the

290

emulsion-diffusion method.31 De Oliveira et al. (2014) reported that essential oil–

291

encapsulated alginate/cashew gum nanoparticles were successfully prepared via

292

spray-drying, and the encapsulated oil levels varied from 1.9% to 4.4%, with an EE of

293

up to 55%.18 The ultrasonic bottom-up approach seems to be the best among those

294

used to prepare nanocarriers in terms of the EE and LC of essential oil loading.

295

Characteristics of starch nanoparticles and peppermint oil-loaded starch

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nanoparticles The morphology and particle size of PO-loaded SNPs are shown in

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Figure 4. After encapsulation, the nanoparticles remained spherical, with a size of

298

around 200 nm for all three samples (Figure 4A, C, and E). PO incorporation led to

299

the formation of particles with slightly larger diameters from the DLS results (Figure

300

4B, D, and F). Bilenler et al. (2015) also reported that the dimensions of essential oil–

301

loaded zein particles are greater than that of blank zein particles.32

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The thermal properties

The thermal properties of SNPs obtained using the

303

ultrasonic process were determined by DSC. The SNPs prepared by primary SLG,

304

S-SLG, and L-SLG all showed one endotherm, with temperature ranges of 64.0–

305

94.3°C, 50.3–81.1°C, and 75.1–97.6°C, respectively, as depicted in Figure 5A.

306

However, primary SLG did not show an endothermic peak, reflecting its mainly

307

amorphous structure. Furthermore, the melting temperature of SNPs was positively

308

correlated with the DP of SLG. This behavior was probably due to the more perfect


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crystal structure formed by SLG with a higher DP value, which requires a higher

310

temperature to dissociate the crystallization. It has been reported that a minimum

311

chain length of DP 10 is required for double helix formation in a pure oligosaccharide

312

solution.33

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The thermal properties of PO-loaded SNPs are shown in Figure 5B. The melting

314

temperature ranges of primary SLG and L-SLG with PO were about 79.7–106.9°C

315

and 83.6–110.0°C, respectively. The addition of PO resulted in a further increase of

316

the Tp in SNPs. The shift to the higher temperature and wider range for melting

317

indicated that a more crystalline structure of the V-type complexes was formed by

318

primary SLG or L-SLG with PO. Similar results have been reported by Jane (2009),

319

who stated that the semicrystalline amylose inclusion complexes melt at 100–125°C.34

320

The same trend was noted by Maphalla et al. (2016) and Ai et al. (2013), who showed

321

that the addition of lipid to starch increased the melting temperature.35 However, the

322

SNPs fabricated by S-SLG and PO had two endothermic peaks, with temperature

323

ranges of about 48.0°C–79.8°C and 91.3–98.3°C. This may be attributed to the

324

formation of a double-helix structure by SLG self-assembly and a single-helix

325

structure between SLG and PO in the first and second peaks, respectively. Recently,

326

Le-Bail (2015) reported that the thermogram obtained from amylose complexes with

327

linoleic acid has a complex shape with two endotherms, the first at 87°C and the

328

second at 106°C.36

329 330

X-ray diffraction analysis (XRD)

The RCs of SNPs formed by primary SLG,

S-SLG, and L-SLG were also measured. The RC value of primary SLG was about 6%,



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indicating that it had an almost amorphous structure. There was no difference between

332

the X-ray diffraction (XRD) patterns of the three SNP samples. The XRD results

333

showed that the SNPs were characteristic of the B-type, with strong peaks at 2θ of 17°,

334

22°, and a weak peak at 24° (Figure 6A). This was consistent with a previous report

335

stating that recrystallized nanoscale starch particle samples prepared from proso millet

336

starch displayed a typical B-type crystalline structure with the main diffraction peaks

337

at 2θ = 5.6°, 17.1°, 22.5°, and 24.3°.37 The RCs of SNPs increased remarkably (from

338

23.5±0.9 to 31.6±1.5%) with the increasing SLG chain lengths.

339

In contrast to the B-type diffraction pattern of bare SNPs, the PO-loaded SNPs

340

exhibited a distinct V-type single-helix crystalline structure (Figure 6B). The

341

characteristic diffraction peaks of PO-loaded L-SLG nanoparticles were at 2θ = 7.5°,

342

13.1°, and 20.3°. Similarly, the main reflections of PO-loaded SNPs obtained by

343

primary SLG at 2θ were about 7.5°, 12.4°, and 19.5°. However, the PO-loaded S-SLG

344

nanoparticles had two weak peaks at 2θ of about 16.0° and 22.1°. The RCs of

345

PO-loaded S-SLG, primary SLG, and L-SLG nanoparticles were 24.3±1.7%,

346

31.4±1.4%, and 36.2±2.4%, respectively, which were higher (p < 0.05) than the

347

corresponding RCs of bare SNPs. These results proved that SLG had the capability to

348

form a V-type single-helical structure with essential oils under ultrasonic treatments.

349

Le-Bail et al. (2015) demonstrated that V-amylose forms helices with hydrophobic

350

helical cavities; they suggested that these helices could entrap the guest molecules to

351

various extents.36

352

Fourier transform infrared (FTIR) spectroscopic study


To investigate the

Page 17 of 36


17 353

interactions that occurred during the formation of SNPs, further studies were carried

354

out using FTIR. The spectra of the SNPs showed a characteristic peak around 3400

355

cm–1 that may be assigned to inter- and intramolecular hydrogen-bonded hydroxyl

356

groups (Figure 7A). A small peak at 2930 cm–1 was attributed to the C–H stretching

357

vibrations. The peak at 1640 cm–1 was a feature of tightly bound water present in the

358

nanoparticles. Similarly, the characteristic bands of FTIR spectra of primary SLG

359

were identical to those of the SNPs. However, the band of SNPs decreased in intensity

360

when compared with primary SLG due to inter- and intramolecularly bound hydroxyl

361

groups. These results were comparable to the findings of a recent report, which

362

showed that the OH stretching band at 3435.7 cm–1 in SNPs decreased in intensity

363

compared to that in the native maize starch.28

364

FTIR spectroscopy was further applied to study the interactions between PO and

365

SNPs in PO-loaded SNPs. Compared to bare SNPs, the characteristic peak of

366

PO-loaded SNPs shifted towards a shorter frequency (around 3937 cm–1), which

367

indicated that there were stronger intermolecular hydrogen bonds between PO and

368

SLG, as observed by Qiu et al. (2017) in a similar study. These authors reported that

369

characteristic bands at 3000–3700 cm–1 in the SNPs-menthone spectrum shift to a

370

shorter wavelength, indicating stronger hydrogen bonding between the hydroxyls of

371

SLG and menthone in the menthone-loaded SNPs.15 The PO characteristic bands,

372

particularly those of aromatic compounds (at 2960, 1440, 1580, and 1600 cm−1),

373

exhibited low intensity or disappeared. The changes in the FTIR spectra on PO-loaded

374

SNPs can be explained by the complex formation between SLG and PO.



Page 18 of 36

18 375

Release profile of peppermint oil–loaded starch particles at high temperature

376

The release behavior of PO-loaded SNPs under heating conditions was further

377

evaluated. Compared to pure PO (data not shown), the cumulative release amount of

378

PO in SNPs markedly decreased (Figure 8A). It was observed that PO released from

379

the three PO-loaded SNPs reached a level of maximal release (plateau) around 120

380

min. The decrease in cumulative release of PO can probably be attributed to the

381

compact SNPs formation between SLG and PO. Compared to the other two types of

382

PO-loaded SNPs, the PO-loaded S-SLG nanoparticles showed a slightly higher

383

release amount. Approximately 33% of the PO was released from the S-SLG

384

nanoparticles after incubation for 150 min. In contrast, the primary SLG and L-SLG

385

nanoparticles released about 28% and 27% of the PO, respectively, after 150 min of

386

incubation.

387

To understand the PO release process, the most common pseudo-first-order

388

kinetics model was used to analyze PO release in solution. The equation was

389

expressed as follows: Qt = Qe(1 – exp(–kt)),

390

(4)

391

where k is the rate constant of pseudo-first-order release (min–1), Qe is the maximal

392

release amount of PO at an infinite time, and Qt is the cumulative release amount of

393

PO at time t.

394

The fitting results of the release profiles and kinetic model parameters of PO from

395

the SNPs are shown Figure 8B and Table S3, respectively. The best correlation

396

coefficient (R2 > 0.99) was obtained for the kinetic model. Here, PO-loaded S-SLG


Page 19 of 36


19 397

nanoparticles exhibited a high maximal release amount. In contrast, low release of

398

SNPs and L-SLG nanoparticles was observed. In the whole release profile, it could be

399

observed that PO-loaded SNPs displayed a noticeably low release property, indicating

400

that they are stable against high temperature. This result suggested that SNPs may be

401

effective carriers for essential oil or other hydrophobic substances.

402

In conclusion, the current study presented the formation of SNPs via an ultrasonic

403

bottom-up approach using different SLG chain lengths. The new method was also

404

used to encapsulate PO into SNPs, which showed a rapid process at a minute

405

timescale (5 or 8 minutes), high LC of 25.5%, and high EE of 87.7%. The as-prepared

406

nanoparticles and PO-loaded SNPs exhibited spherical shapes with smooth surfaces.

407

Particle sizes were in the range 150–220 nm, with no aggregates. Compared with

408

SNPs, PO-loaded SNPs exhibited increased crystallinity, as determined via XRD

409

analysis. The thermal analysis and X-ray studies provided evidence that the

410

PO-loaded SNPs formed the V-type of crystallinity. Encapsulation of POs in the SNPs

411

greatly slowed their release in hot water. The present approach has the advantage of

412

being rapid, presenting a higher yield, and not requiring any chemical treatment. The

413

knowledge obtained from this study will be helpful in the design and development of

414

new strategies to encapsulate essential oils. Essential oil–loaded SNPs can used for

415

applications in medicine, functional foods, and the cosmetics field.



Page 20 of 36

20 416

ASSOCIATED CONTENT

417

Supporting Information

418

Data of chain length distributions, data of DSC, kinetic model parameters, Table

419

S1-S3.

420

AUTHOR INFORMATION

421

Corresponding Author

422

*Tel.: +86-0532-88030448. Fax: +86-0532-88030449. E-mail: [email protected] (Qingjie Sun).

423

NOTES

424

The authors declare no competing financial interest.

425

ACKNOWLEDGEMENTS The study was supported by National Natural Science Foundation, China (Grant No. 31671814).

426 427

REFERENCE

428

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thyme essential oil encapsulated in zein particles. Flavour and Fragrance Journal 2015, 30 (5),

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33. Andrew. D.; Abell, M. J.; Ratcliffe, J. G. Ascorbic acid-based inhibitors of α-amylases.

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Bioorganic & Medicinal Chemistry Letters, 1998, 8(13):1703-1706.

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with stearic acid and xanthan gum. Carbohydr Polym 2016, 136, 970-978; (b) Ai, Y.; Hasjim, J.; Jane, J.

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Page 24 of 36

24 Table captions Table 1 Encapsulation Efficiency (EE) and Loading capacity (LC) of Peppermint Oil (PO)-loaded Starch Nanoparticles (SNPs) Table S1 Chain Length Distributions of Primary Short Linear Glucan (SLG), Short SLG (S-SLG), and Long SLG (L-SLG) Table S2 Onset, Peak, and Melting Temperature (To, Tp, and Tc, Respectively) and Enthalpy Change (∆H) of Starch Nanoparticles (SNPs) and Peppermint Oil (PO)-loaded SNPs Table S3 Kinetic Model Parameters for Peppermint Oil (PO)-loaded Starch Nanoparticles (SNPs) Figure captions Scheme 1: Schematic diagram of the fabrication of starch nanoparticles (SNPs) and peppermint oil (PO)-loaded SNPs via an ultrasonic bottom-up method employing various chain lengths of short linear glucan (SLG). Figure 1 Chain length distributions of primary short linear glucan (SLG) (A), short SLG (S-SLG) (B), and long SLG (L-SLG) (C). SC: standard curve. DP: degree of polymerization. F1 and F2 represent small and large molecular weight fractions, respectively. Figure 2 Transmission electron microscopy (TEM) images of starch nanoparticles (SNPs) prepared by primary short linear glucan (SLG) at concentrations of 1% (w/v) (A), 5% (w/v) (B), and 10% (w/v) (C). Particle size distributions of SNPs were determined by dynamic light scattering (DLS) (D).


Page 25 of 36


25 Figure 3 Transmission electron microscopy (TEM) images of starch nanoparticles (SNPs) prepared at ultrasound irradiation times of 5 min (A, E, and I), 8 min (B, F, and J), and 10 min (C, G, and K). Particle size distributions of SNPs were measured using dynamic light scattering (DLS) (D, H, and I). Figure 4 Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) analyses of peppermint oil (PO)-loaded primary short linear glucan (SLG) (A, B), short SLG (S-SLG) (C, D), and long SLG (L-SLG) nanoparticles (NPs) (E, F), respectively. Figure 5 Differential scanning calorimetry (DSC) thermal profiles of starch nanoparticles (SNPs) (A) and peppermint oil (PO)-loaded SNPs (B). Figure 6 X-ray patterns of starch nanoparticles (SNPs) (A) and peppermint oil (PO)-loaded SNPs (B). RC: relative crystallinity. Figure 7 Fourier transform infrared (FTIR) spectra of starch nanoparticles (SNPs) (A) and peppermint oil (PO)-loaded SNPs (B). Figure 8 Release profile (A) of peppermint oil (PO) from primary short linear glucan (SLG), short SLG (S-SLG), and long SLG (L-SLG) nanoparticles (NPs) in hot water at 80°C and fitted by the pseudo-first-order kinetic model (B).



Ultrasonic

Starch

SNPs

S-SLG

SLG SLG

Cavitation

PO-loaded SNPs

L-SLG

Scheme 1: Schematic diagram of the fabrication of starch nanoparticles (SNPs) and peppermint oil (PO)-loaded SNPs via an ultrasonic bottom-up method employing various chain lengths of short linear glucan (SLG).


Page 26 of 36

Page 27 of 36


Table 1 Encapsulation Efficiency (EE) and Loading Capacity (LC) of Peppermint Oil (PO)-loaded Starch Nanoparticles (SNPs) SNPs

EE (%)

LC (%)

Yield (%)

SLG

82.90.54b

25.10.14b

86.20.24b

S-SLG

74.50.35c

21.30.17c

81.80.32c

L-SLG

87.70.38a

25.50.21a

93.20.36a

Values mean ± SD indicates the replicates of three experiments. Values with different letters (a, b, c, and d) are significantly different (p < 0.05). SLG, S-SLG, L-SLG: peppermint oil (PO)-loaded primary short linear glucan (SLG) nanoparticles, short SLG nanoparticles, and long SLG nanoparticles, respectively.



Page 28 of 36

B

A

6

6

1.0

1.0

DP=9.5

5

DP=13.5

5

0.8

0.8

4

SC

0.4

RI Signal

3

DP=30

0.6 3

0.4

SC 2

2

F1

0.2

Log Mw

0.6

Log Mw

RI Signal

4

0.2

1

1

F1 0.0

F2 0

2

4

6

8

10

F2

0.0

0

0

12

2

6

0

8

10

12

Elution time (min)

Elution time (min)

C

4

6 1.0 5

DP=15.1 0.8

0.6 DP=30

3

0.4

SC

Log Mw

RI Signal

4

2 0.2

1 F1 F2

0.0 0

2

4

6

0 8

10

12

Elution time (min)

Figure 1 Chain length distributions of primary short linear glucan (SLG) (A), short SLG (S-SLG) (B), and long SLG (L-SLG) (C). SC: standard curve. DP: degree of polymerization. F1 and F2 represent small and large molecular weight fractions, respectively.


Page 29 of 36


A

B

C

D 18 16 14

Intensity (%)

12 10 8 6 4 2 0 10

100

1000

10000

Size (nm)

Figure 2 Transmission electron microscopy (TEM) images of starch nanoparticles (SNPs) prepared by primary short linear glucan (SLG) at concentrations of 1% (w/v) (A), 5% (w/v) (B), and 10% (w/v) (C). Particle size distributions of SNPs were determined by dynamic light scattering (DLS) (D).



Primary SLG

Page 30 of 36

S-SLG

L-SLG

A

E

I

B

F

J

C

G

K

D

H

L

5 min

8 min

10 min

25

15

20

10 5

25

20

Intensity (%)

25

Intensity (%)

Intensity (%)

30 20

15 10

0

0 10

100

1000

Size (nm)

10000

10 5

5

0

15

10

100

1000

10000

Size (nm)

10

100

1000 Size (nm)

Figure 3 Transmission electron microscopy (TEM) images of starch nanoparticles (SNPs) prepared at ultrasound irradiation times of 5 min (A, E, and I), 8 min (B, F, and J), and 10 min (C, G, and K). Particle size distributions of SNPs were measured using dynamic light scattering (DLS) (D, H, and I).


10000

Page 31 of 36


A

B 25

Intensity (%)

20

15

10

5

0 10

100

1000

10000

1000

10000

1000

10000

Size (nm)

C

D

25

Intensity (%)

20

15

10

5

0 10

100

Size (nm)

E

F 20

Intensity (%)

15

10

5

0 10

100

Size (nm)

Figure 4 Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) analyses of peppermint oil (PO)-loaded primary short linear glucan (SLG) (A, B), short SLG (S-SLG) (C, D), and long SLG (L-SLG) nanoparticles (NPs) (E, F), respectively.



A

Page 32 of 36

B S-SLG-NPs

-4

Endo (mW)

Endo (mW)

-4

-6

-8

-6

-8 40

60

80

Temperature ( C)

100

120

40

60

80

100

Temperature (C)

Figure 5 Differential scanning calorimetry (DSC) thermal profiles of starch nanoparticles (SNPs) (A) and peppermint oil (PO)-loaded SNPs (B).


120

Page 33 of 36


A

B

Intesity (a. u.)

RC=31.6 1.5% RC=23.5 0.9%

RC=27.3 1.2%

SLG-PO S-SLG-PO L-SLG-PO

Intensity (a. u.)

SLG SLG-NPs S-SLG-NPs L-SLG-NPs

RC=36.2 2.4% RC=24.3 1.7%

RC=31.3 1.4%

RC=6.04 2.1%

5

10

15

20

25

30

35

40

5

10

15

20

25

30

35

2 

2 ()

Figure 6 X-ray patterns of starch nanoparticles (SNPs) (A) and peppermint oil (PO)loaded SNPs (B). RC: relative crystallinity.


40


SLG

A

Page 34 of 36

B

S-SLG-NPs

Intensity (a. u.)

Intensity (a. u.)

S-SLG-PO

4000

3500

3000

2500

2000 1500

1000

500

4000

3500

3000

2500

2000 1500

1000

-1

Wavenumber (cm )

-1

Wavenumber (cm )

Figure 7 Fourier transform infrared (FTIR) spectra of starch nanoparticles (SNPs) (A) and peppermint oil (PO)-loaded SNPs (B).


500

Page 35 of 36


A

B 35

30

Cumulative release (%)

30

Cumlative release (%)

35

SLG-PO S-SLG-PO L-SLG-PO

25 20 15 10

25 SLG-PO S-SLG-PO L-SLG-PO SLG-PO fitting S-SLG-PO fitting L-SLG-PO fitting

20 15 10 5

5

0

0 0

30

60

90

120

150

0

30

60

90

120

150

Time (min)

Time (min)

Figure 8 Release profile (A) of peppermint oil (PO) from primary short linear glucan (SLG), short SLG (S-SLG), and long SLG (L-SLG) nanoparticles (NPs) in hot water at 80°C and fitted by the pseudo-first-order kinetic model (B).



TOC Graphic Ultrasonic

Starch

SLG-NPs

S-SLG

SLG SLG

SLG-PO Cavitation

L-SLG


Page 36 of 36

Morphology and Characteristics of Starch Nanoparticles Self

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