Characterization of Cationic Modified Debranched Starch and

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Characterization of cationic modified debranched starch and formation of complex nanoparticles with #-carrageenan and low methoxyl pectin Qing Liu, Man Li, Liu Xiong, Lizhong Qiu, Xiliang Bian, Chunrui Sun, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05045 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 24, 2019

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Journal of Agricultural and Food Chemistry

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Characterization of cationic modified debranched starch and formation of

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complex nanoparticles with κ-carrageenan and low methoxyl pectin

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Qing Liua, Man Lia, Liu Xionga, Lizhong Qiub, Xiliang Bianb, Chunrui Sunb, Qingjie Suna,*

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a College of Food Science and Engineering, Qingdao Agricultural University (Qingdao, Shandong

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Province, 266109, China)

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b Zhucheng Xingmao Corn Developing Co., Ltd (Weifang, Shandong Province, 262200, China)

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*Correspondence authors (Tel: 86-532-88030448, Fax: 86-532-88030449, e-mail: [email protected])

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ABSTRACT: The functional modifications of debranched starch (DBS) has been

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attracting the interest of researchers. This study marks the first time that DBS was

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modified by cationization through the use of (3-Chloro-2-hydroxypropyl) trimethyl

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ammonium chloride with the introduction of cationic functional groups. The

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physicochemical properties and structural characteristics of cationized debranched

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starch (CDBS) were systematically assessed. The results demonstrate that the

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maximum degree of substitution (DS) value obtained was as high as 1.14, and the

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corresponding CDBS exhibited significantly higher zeta potential values:

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approximately +35 mV. The minimal inhibitory concentration values of the CDBS of

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DS 1.14 against Escherichia coli and Staphylococcus aureus were 6 mg mL-1 and 8

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mg mL-1, respectively. In addition, nanoparticles were successfully prepared with a

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combination of CDBS and low methoxyl pectin (LMP) and a combination of CDBS

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and κ-carrageenan (CRG). The maximum encapsulation efficiency of nanoparticles

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for (–)-epigallocatechingallate can reach 87.8%.

ACS Paragon Plus Environment


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KEYWORDS: short chain amylose; functionality; antibacterial activity; electrostatic

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interaction


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INTRODUCTION

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Starch, one of the most abundant polysaccharides in nature, has been applied in

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various fields. It has been modified to increase its beneficial attributes and to reduce

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its disadvantages. Chemical, physical, enzymatical, and genetical methods have been

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widely investigated for their potential to modify starch.1, 2 Debranched starch (DBS),

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an enzymatically modified starch, has increasingly been the focus of research. DBS is

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composed of linear short glucan chain molecules3 and exhibits great potential for a

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variety of fields. For example, it has been a fat or protein substitute in food products,3

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and it has been used as a good tablet matrix to extend the duration of drug release.4

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Recently, Ji et al.5used DBS and proanthocyanidins to prepare nanocomposites for the

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oral delivery of insulin. In addition, debranched starch nanoparticles might possibly

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serve as particulate emulsifiers for achieving stability in Pickering emulsions.6 More

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recently, DBS was used to form nanoparticles by in situ self-assembly in gelatin

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matrices, and this significantly enhanced the mechanical strength of gelatin

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nanocomposite hydrogels.7

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Like native starch, DBS has been modified by a variety of methods to achieve

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functionality for industrial applications. Acetylation has been reported to improve the

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freeze-thaw stability and swelling power of debranched rice starch.8 DBS

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hydrophobically modified with octenyl succinic anhydride could self-assemble into

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micelles and vesicles for the delivery of hydrophobic functional ingredients or drugs.9

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DBS nanoparticles functionally modified with sodium hypochlorite exhibit enhanced

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the colloidal stability and heavy metal ion adsorption capacity of Pb2+ and Cu2+.10



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Recently, TEMPO-oxidized DBS was used to prepare nanoparticles of 30–50 nm with

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calcium ions by ionic gelation.11

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An important modification method, cation modification is increasingly being used

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to assess the functionality of polysaccharides. Li et al.12 successfully used quaternized

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alkali lignin and sodium dodecyl benzenesulfonate to prepare a new type of

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pH-responsive micelle for the encapsulation of hydrophobic drugs. Cationized starch

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was used to remove sulfate ions from an aqueous solution through an ultrafiltration

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technique.13 In addition, the antioxidant activity of chitosan was shown to be

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improved after quaternization because of the high number of quaternized groups

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created.14

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In this study, DBS was modified through the introduction of amino groups to

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obtain cationized debranched starch (CDBS) with various degrees of substitution (DS)

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and to investigate the structural and antibacterial properties of CDBS. A combination

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of positively charged CDBS and negatively charged κ-carrageenan (CRG) and a

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combination of CDBS and negatively charged low methoxyl pectin (LMP) were used

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to prepare novel polysaccharide nanoparticles. Besides, the nanoparticles obtained

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were used as carriers to load (–)-epigallocatechin-3-gallate (EGCG) as a model active

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ingredient. The encapsulation and release characteristics of EGCG in nanoparticles

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under simulated gastrointestinal fluids conditions were investigated.

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MATERIALS AND METHODS

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Materials. Waxy corn starch (approximately 98% amylopectin) was obtained

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from ingredion China Ltd. (Shanghai, China), and (3-Chloro-2-hydroxypropyl)


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trimethyl ammonium chloride (CHPTAC) (60 wt. % in H2O) was supplied by Aladdin

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industrial Corporation (Shanghai, China). Pullulanase (E.C.3.2.1.41, 6000 ASPU/g)

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was provided by Novozymes Investment Co. Ltd. (Beijing, China). The

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Gram-negative bacterium Escherichia coli (E. coli) (ATCC 25922) and the

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Gram-positive bacterium Staphylococcus aureus (S. aureus) (ATCC 25923) were

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offered by Nanjing Bianzhen Biological Technology Co. Ltd. κ-carrageenan (CRG)

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was provided by Beijing Solarbio Science & Technology Co. Ltd. Low methoxyl

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pectin (LMP) (degree of esterification 29%) was purchased from Yantai Andre Pectin

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Co., Ltd. (Yantai, China). Pepsin (≥400 units per mg protein) from porcine gastric

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mucosa and pancreatin (8×USP specifications) from porcine pancreas were purchased

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from Sigma-Aldrich (USA). All the reagents used were analytical grade.

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Preparation of Cationized Debranched Starch. DBS was obtained according to

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a previously described method.15 Briefly, waxy corn starch was fully gelatinized and

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debranched with pullulanase at 58 °C for 6 h and then centrifuged to obtain

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supernatant, which was precipitated with four times volumes of absolute ethanol.

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After centrifugation, the pellets were lyophilized to obtain DBS. The CDBS was

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prepared according to Zhou et al.16 with some modifications. The DBS (1 g) was

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dispersed in 50 mL distilled water and sonicated to achieve a homogeneous

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suspension. The suspension was then adjusted to pH 12 with 2 M NaOH and stirred at

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40 °C. Thereafter, 1 mL CHPTAC was added dropwise into the suspension while the

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pH value of the suspension was maintained at 12. The etherification reaction was

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carried out for 4 h; then the dispersions were titrated to pH 7.0. The mixture was



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centrifuged at 8000 g for 10 min to obtain the precipitate, which was washed

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successively with 95% ethanol until no chloride could be detected and then

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lyophilized to get the CDBS. The recovery of CDBS was about 75%. The same

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procedure was applied to other doses of CHPTAC (2 mL and 3 mL).

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Determination of Degree of Substitution. Elemental analysis was identified by

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a scanning electron microscopy with energy dispersive X-ray spectroscopy

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(SEM-EDS) (JEOL 7500F, Hitachi Instruments Ltd., Tokyo, Japan) according to

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Antunes et al.17 The DS was calculated according to the nitrogen analysis as following

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equation:

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162 × 𝑁

(1)

𝐷𝑆 = 1400 ― 151.5 × 𝑁

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where N was the amount of nitrogen determined by the SEM-EDS (%), 162 was the

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average molecular weight of an anhydroglucose unit, 1400 was 100 times the atomic

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weight of nitrogen, and 151.5 represents the molecular weight of CHPTAC without

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chloride group.

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Zeta Potentials. Debranched starch and CDBS were dispersed in ultrapure water

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at 0.05% (w/v). The zeta potentials were measured via the dynamic light scattering

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(DLS) technique using a Zetasizer Nano ZS90 (Malvern Instruments, U.K.).18

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Proton Nuclear Magnetic Resonance. The structure analysis of the samples was

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performed with a proton nuclear magnetic resonance (1H NMR) spectrometer

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(AVANCE 500 MHz, Bruker, Switzerland). Approximately 20 mg of the samples

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were dissolved in 0.6 mL dimethyl sulfoxide-d6 (DMSO-d6) using tetramethylsilane

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as the internal standard. The chemical shifts were expressed in ppm.9


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Fourier Transform Infrared. The structural characteristics of the samples were

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determined by using a Fourier transform infrared (FTIR) spectrometer (NEXUS-870;

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Thermo Nicolet Corp., USA). The samples were collected by the KBr pellet method.

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The wavenumber range was 4000–400 cm−1, and the resolution and the total number

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of scans were 4 cm−1 and 32, respectively.19

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Differential Scanning Calorimetry. A differential scanning calorimeter (DSC 1)

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(Mettler-Toledo International Inc., Switzerland) was used to evaluate the thermal

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properties of the DBS and CDBS. The samples (approximately 4 mg) with 8 L water

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were placed in hermetic aluminum pans, balanced for at least 4 h, and then heated at

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10 °C min−1 from 25 °C to 125 °C.20 The thermal parameter onset (To), peak (Tp),

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and endset (Te) temperatures and the enthalpy change (ΔH) were obtained. The

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samples were then cooled from 125 °C to 25 °C at 50 °C min−1, and rescanned from

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25 °C to 125 °C, and the parameters for the rescanning were determined.

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X-ray diffraction (XRD). The X-ray patterns of the samples were determined

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through an X-ray diffractometer (XRD) (D8-ADVANCE, Bruker AXS Model,

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Germany) equipped with Cu Kα1 radiation at 40 kV and 25 mA.21

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Iodine Staining Index. An iodine staining index (ISI) was used for quantifying

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and determining the iodine binding abilities of the samples. The procedure was

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performed according to the recommendations of Lu et al.22 with minor modifications.

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In brief, an aqueous solution of I2–KI was prepared with 0.2 g I2 and 2 g KI in 100

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mL distilled water and diluted 50 times before use. The sample (0.010 g) was

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dispersed in 10 mL deionized water and completely gelatinized. Then the sample



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solution was added to an equal volume of diluted iodine solution. The solution was

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mixed and incubated for 10 min. The absorbance of the CDBS–iodine complex was

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measured with a UV-visible spectrophotometer (Shimadzu-2600, Kyoto, Japan) at

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wavelengths of 400–800 nm. An absorbance of 570 nm was used to express the ISI

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value for the CDBS-iodine complex.23

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Antibacterial Activity. The test microorganism strains selected to determine the

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antibacterial activity of CDBS were the Gram-negative E. coli and Gram-positive S.

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aureus species. The growth of bacteria was determined by taking optical density (OD)

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at 600 nm, as was previously described, but with some modifications.24-26 Bacteria

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suspensions were cultivated to achieve approximately 1 × 108 CFU mL-1 bacterial

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concentration in each tube, and they were aerobically grown in broth medium

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supplemented with different concentrations of DBS or CDBS at 37 °C for 24 h. The

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culture with DBS served as the control. Finally, the mixtures were left untouched and

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allowed to settle for 30 min. The bacterial growth was monitored by measuring the

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OD at 600 nm. The IC50 was defined as the concentration of sample that inhibited

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50% of bacteria. The MIC was determined as the lowest concentration of sample with

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an OD600 close to 0. The inhibition efficiency of the CDBS was calculated as

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following equation:

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Inhibition efficiency =

𝑂𝐷𝐶600 ― 𝑂𝐷𝑆600 𝑂𝐷𝐶600

× 100%

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where 𝑂𝐷𝐶600 and 𝑂𝐷𝑆600 were the OD600 values of the culture medium for the

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control and the sample, respectively.


(2)

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Preparation of Cationized Debranched Starch Nanoparticles. The ionotropic

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gelation method was used to prepare nanoparticles with either CDBS and LMP or

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CDBS and CRG. The stock solutions (0.5 mg mL−1) of LMP and CRG were dissolved

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by heating at 60 °C and 80 °C, respectively. For the formation of nanoparticles, the

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CDBS suspension (0.5, 1, 2, and 3 mg mL−1, respectively, 20 mL) was dissolved in a

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boiling water bath. The CDBS solution was then added dropwise to an equal volume

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of LMP solution at 25 °C with constant stirring at 500 rpm. The same was done with

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an equal volume of CGR solution. After incubation for 2 h, each mixture was

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centrifuged at 12000 g for 30 min, and the precipitate was washed three times with

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deionized water. Finally, CDBS-LMP and CDBS-CRG nanoparticles were collected

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after lyophilization.

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Morphology and Size of Nanoparticles. The morphology and particle size of

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the nanoparticles were assessed with the method previously described.10 The

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morphologies were observed with transmission electron spectroscopy (TEM)

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(HT7700 TEM, Tokyo, Japan), and the size distributions were evaluated with a

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Zetasizer Nano ZS90 (Malvern Instruments, U.K.).

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Nanoparticle Stability in Simulated Gastrointestinal Fluids. Gastrointestinal

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conditions were simulated in accordance with previously reported protocols.27

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Nanoparticles of 5 mg mL−1 in distilled water were diluted 1:9 in simulated gastric

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fluid (SGF) solutions containing 32 mM HCl, 34 mM NaCl, and 0.32% (w/v) pepsin

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(pH 1.5), and in simulated intestinal fluid (SIF) containing 50 mM KH2PO4 and 1%



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(w/v) pancreatin (pH 6.8). After 40 min incubation at 37 °C with shaking, the sample

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was taken out for further TEM observation.

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Preparation of EGCG-loaded Nanoparticles. Different amounts of EGCG were

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added into 10 mL of CRG or LMP (0.5 mg mL−1) solution at a final concentration of

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0.2, 0.5, and 1 mM. Subsequently, the mixed solution was added dropwise to 10 mL

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of dissolved CDBS at 25 °C with constant stirring at 500 rpm in a dark condition.

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After incubation for 2 h, EGCG-loaded nanoparticles were formed. Then, the

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nanoparticle suspensions were centrifuged at 12000 g for 30 min with ultrafiltration

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centrifuge tubes with a molecular weight cut-off (MWCO) of 5 kDa. The supernatant

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was obtained and the sediments were washed three times. The total supernatant was

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

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(LC). The amount of free EGCG in the samples was determined by the Folin–

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Ciocalteu method.28 Finally, the sediments were lyophilized for determination of in

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vitro release of EGCG. The EE and LC of EGCG in the nanoparticles were calculated

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according to the following equations, respectively: 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 ― 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑓𝑟𝑒𝑒 𝐸𝐺𝐶𝐺

× 100

(3)

× 100

(4)

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EE (%) =

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LC (%) =

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In Vitro Release of EGCG. In vitro release experiments were carried out with

𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐸𝐺𝐶𝐺 ― 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑓𝑟𝑒𝑒 𝐸𝐺𝐶𝐺 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠

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the equilibrium dialysis method described by Shtay et al.29 with some modifications.

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For this purpose, 5 mL of EGCG-loaded nanoparticles in SGF (pH 1.5) was placed

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into a dialysis bag (MWCO: 5 KDa) and dialyzed against 30 mL of release medium

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(SGF, pH 1.5) under gentle stirring (100 r/min) at 37 °C. At each time point, 1 mL of


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external release medium was withdrawn and replaced with an equal volume of the

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fresh medium. The amount of EGCG released from the nanoparticle solution was

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determined at various times. The same procedure was applied to the release of EGCG

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in SIF (pH 6.8). Pure EGCG was used as a control.

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Statistical Analysis. All of the experiments were conducted in triplicate. The

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experimental data were subjected to analysis of variance (ANOVA) using SPSS 17.0

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(SPSS Inc., Chicago, USA), and the results were expressed as the mean values ± the

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standard deviations. Differences were considered at a significance level of 95% (p

Characterization of Cationic Modified Debranched Starch and

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