Morphology and structural properties of novel short linear glucan

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Morphology and structural properties of novel short linear glucan/protein hybrid nanoparticles and their influence on the rheological properties of starch gel Xiaojing Li, Na Ji, Man Li, Shuangling Zhang, Liu Xiong, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02800 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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Journal of Agricultural and Food Chemistry

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Morphology and structural properties of novel short linear glucan/protein

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hybrid nanoparticles and their influence on the rheological properties of starch

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gel

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Xiaojing Li† Na Ji† Man Li†

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

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Province, 266109, China)

Shuangling Zhang† Liu Xiong † Qingjie Sun*, †

ACS Paragon Plus Environment


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ABSTRACT: Starch nanoparticles were potential texture modifiers. However, they have strong

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tendency to aggregate and poor water dispersibility, which limited their application. The

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interaction between glucan (prepared from starch by enzymatic modification) and protein could

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significantly improve the dispersity of starch nanoparticles, and thus enhance the rheological

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properties of food gels. In this work, glucan/protein hybrid nanoparticles were successfully

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developed for the first time using short linear glucan (SLG) and edible proteins (soy protein

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isolate (SPI), rice protein (RP), and whey protein isolate (WPI)). The results showed that the

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SLG/SPI hybrid nanoparticles exhibited hollow structures, the smallest size of which was

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approximately 10–20 nm, when the SLG/SPI ratio was 10:5. In contrast, SLG/RP nanoparticles

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displayed flower-like superstructures, and SLG/WPI nanoparticles presented stacked-lamellar

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nanostructures with a width of 5–10 nm and a length of 50–70 nm. Compared with bare SLG

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nanoparticles, SLG/SPI and SLG/WPI hybrid nanoparticles had higher melting temperatures. The

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addition of all nanoparticles greatly increased the storage modulus of corn starch gels and

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decreased loss tangent values. Importantly, the G' value of starch gels increased by 567% with the

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addition of flower-like SLG/RP superstructures.

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KEYWORDS: hybrid nanoparticles; short linear glucan; protein; superstructures; rheological

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properties


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INTRODUCTION

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Consumers' desire for foods with better texture, taste, and health-promoting properties is

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continually increasing. Hydrocolloids have versatile functionalities when added to food, such as

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thickening, gelling, holding water, dispersing, stabilizing, forming a film, and foaming. Therefore,

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they have been widely used as texture modifiers in many processed food products.1 It is reported

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that about 260,000 tons of hydrocolloids are needed per year by the year 2014, and they are

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composed of approximately 70% starch, 12% gelatin, 5% pectin, 5% carrageenan, and 4% xanthan

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gum, followed by locust bean gum, alginates, carboxymethylcellulose, and guar gum, among other

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agents.2 In addition, the market requirement of hydrocolloids is increasing year after year.

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Technical features, price, and safety are necessary elements in evaluating whether a hydrocolloid

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is suitable as an addition to food products. There is little doubt that starch is one of the most

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frequently used thickeners, as it is both inexpensive and abundant. However, some food gums,

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including carrageenan, xanthan gum, and konjac glucomannan, may still be the first choice due to

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their unmatched rheological properties, even though they are more expensive than starch.

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The rheological properties of hydrocolloids are particularly important when hydrocolloids are

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used in the formulation of any food for their effects on the food texture.3 Native starch has

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unsatisfactory rheological properties in the preparation of food products, such as gel syneresis,

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weak shearing resistance, and easy retrogradation.4 Therefore, much attention has been paid to

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starch modifications, including chemical and physical methods that could overcome the

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shortcomings of native starch. However, the use of chemical modifiers may have potential risks,

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as the residual chemical reagents can migrate into foods.5

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Conventional physical modification of starch includes pregelatinization, heat–moisture



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treatment, dry heating, ultrahigh pressure treatments, and blending with non-starch

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polysaccharides. Recently, it has been suggested that the incorporation of nanoparticles could

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significantly improve the rheological properties of starch. Valencia et al. (2015) reported that the

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addition of laponite to cassava starch gel could extend its linear viscoelastic range. Cassava starch

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gel started thinning at 0.2 Pa, whereas cassava starch gel with 20% laponite was stable when stress

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was increased to 4 Pa.6 El Miria et al. (2015) found that the addition of cellulose nanocrystals

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significantly improved the dynamic storage, and the increase in magnitude correlated positively

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with the concentration of cellulose nanocrystals.7

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Natural polysaccharides and proteins are the most promising materials for preparing

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nanoparticles due to their excellent characteristics, such as renewability, degradability, and

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biocompatibility.8 Starch nanoparticles (SNPs) have attracted increasing interest in food-based

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nanoparticle production as they are derived from abundant and inexpensive starch. In most

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published literature on this topic, the focus is on the use of prepared SNPs as reinforcing polymers

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in composites9-10 or delivery systems11-12 or as emulsion stabilizers.13-14 Therefore, potential

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applications of SNPs should be explored, particularly their use in food as thickeners or rheology

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modifiers.15 However, SNPs have a strong tendency to aggregate and have poor water

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dispersibility, which are huge limitations in the attempt to improve the rheological properties of

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food. Complexation with proteins can greatly improve water dispersibility and the stability of

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poorly soluble biopolymers.16 It was hypothesized that the interaction of SNPs with protein chains

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could improve the water dispersibility and the stability of SNPs and thus enhance the rheological

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properties of food.

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Therefore, in this work, hybrid nanoparticles of short linear glucan (SLG) /protein were


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fabricated and used as a food additive to improve the rheological properties of native starch. The

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morphological characteristics, structure, and thermal properties of the hybrid nanoparticles

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fabricated with different edible proteins in variable weight ratios of SLG to protein were

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investigated. The driving forces for glucan/protein hybrid formation were also investigated, and

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the storage modulus and loss tangent of starch gels with or without SLG/protein hybrid

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nanoparticles were determined.

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MATERIALS AND METHODS

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Materials

Corn starch (28.5% amylose) and waxy corn starch (2% amylose) were supplied

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by Tianjin Tingfung Starch Development Co., Ltd (Tianjin, China). Pullulanase (E.C.3.2.1.41,

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6000 ASPU/g, 1.15 g/mL; ASPU is defined as the amount of enzyme that liberates 1.0 mg glucose

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from starch in 1 min at pH 4.4 and 60 °C) was obtained from Novozymes Investment Co. Ltd.

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(Bagsvaerd, Denmark). Soybean protein isolate (SPI), whey protein isolate (WPI), and rice starch

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(RS) were purchased from Tianshen Bioprotein Co., Ltd. (Linyi, China). All other reagents were

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of analytical grade.

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Preparation of glucan/protein hybrid nanocomposites

SLG powder was prepared from

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waxy corn starch by enzymatic modification according to the method of Sun et al. (2014).17 The

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main low and high degree of polymerization of SLG were 9.51 (DP) and 38.23 (DP).18 SLG

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powder was dissolved in deionized water (10%, w/v) by heating in a sealed tube at 120 °C for 30

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min. After cooling down to 25 °C, the pH value of the solution was adjusted with the addition of

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NaOH solution (1.0 M) to pH 11 (for SPI), 9.0 (for RP), and 7.0 (for WPI and control). Then the

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different proteins were added into these SLG solutions (10%, w/v) with different SLG-to-protein

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weight ratios (10:1, 10:2.5, 10:5 and 10:7.5). The suspension was vigorous stirred until the



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proteins were completely dissolved. After that the solutions were incubated at 25 °C for 12 h with

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continuously mechanical stirring at 300 r/m for the retrogradation of the SLG and protein. Next,

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the suspensions were washed several times with distilled water until neutrality was achieved. They

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were then vacuum freeze-dried (0.1 MPa, -86 °C, 72 h) to obtain SLG/protein hybrid

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nanoparticles or SLG nanoparticles.

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Determining the morphologies of the nanoparticles

The morphologies of the SLG

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nanoparticles and SLG/protein hybrid nanoparticles were determined using a Hitachi (Tokyo,

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Japan) 7700 transmission electron microscope with an acceleration voltage of 80 kV. The

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treatment condition of the samples was in accordance with the previous report.18

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Differential scanning calorimeter (DSC) The thermal behaviors of SLG nanoparticles

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and SLG/protein nanoparticles were determined using a differential scanning calorimeter (DSC1,

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Mettler-Toledo, Schwerzenbach, Switzerland), as described by Sun et al. (2014).17

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Fourier transform infrared spectra The infrared spectra of samples were recorded using

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a Fourier transform infrared (FTIR) spectrophotometer (NEXUS-870, ThermoNicolet Corporation,

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American), as described by Kunal et al. (2008).19 Second derivation spectra were obtained using

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PeakFit software (version 4.12).20

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UV-visible spectroscopy

The SLG nanoparticles and SLG/protein hybrid nanoparticles

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(0.1 g) were dissolved in 100 ml distilled water and then treated in an ultrasonic bath

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(KQ-500TDE, Kunshan, China). Next, 3 mL of each sample was used to record UV-visible

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spectra using a UV-visible spectrophotometer (Persee UV-1810, Beijing, China) from 200 to 450

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nm, and the spectra obtained for the SLG nanoparticles and SLG/protein hybrid nanoparticles

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were analyzed.


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Hybrid nanoparticles dissociation test

SLG or hybrid nanoparticle dispersions were

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mixed with an equal volume of each dissociating reagent (urea, sodium dodecyl sulfate (SDS), and

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dithiothreitol (DTT)) at various concentrations. These mixed dispersions were adjusted to pH 7

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and stood overnight. The turbidity of the suspensions was then determined using a UV-visible

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spectrophotometer at 600 nm.

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Rheological properties

Freshly prepared corn starch pastes (12%, w/v) were mixed with

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an equal volume of SLG nanoparticles or SLG/protein hybrid nanoparticles (4%, w/v) at 50 °C.

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The test parameters, such as storage modulus (G') and loss tangent (tanδ), were determined

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according to the report by Qiu et al. (2015).5

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Statistical analysis Each measurement was performed using at least three independently

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prepared samples. All statistical analysis was executed using one-way analysis of variance

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SPSS17.0 (SPSS Inc., Chicago, United States). Measurement data were expressed as mean values

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± standard deviations. Significant differences between means were determined by Duncan’s

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multiple range test, and p values < 0.05 was considered statistically significant.

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RESULTS

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Morphological analysis of nanostructures The morphology of three SLG/protein hybrid

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nanoparticles was determined using transmission electron microscopy (TEM). As shown in

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Figures 1A–D, SLG/SPI hybrids were spherical with clear inner cavities when the ratio of

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SLG/SPI went from 10:1 to 10:5. The appearance of cavities could be attributed to hydrogen

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bonding between SLG and SPI. The hydrogen-bond interaction drives more hydrophobic groups

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of SPI toward the inside, thus forming cavities. In addition, outer hydrophilic groups make hybrid

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nanostructures combine with more water molecules, thus increasing their dispersity in the aqueous



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solution. The hollow micellar-like framework was also reported in previous literatures. Das &

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Paul found that p-toluidinium chloride (PTOL) in water created a micellar-like framework in

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which the hydrophobic small tail part of most of the PTOL molecules directed toward the inside,

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whereas in order to make favorable contact with water molecules its hydrophilic ammonium group

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pointed outward.21 SLG/SPI hybrid nanoparticles with the SLG/SPI ratio of 10:1 were 60–90 nm

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in size, and the size decreased with the decreasing SLG/SPI ratio (except for 10:7.5). When the

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SLG/SPI ratio was 10:5, the hybrid nanoparticles were smallest at 10–20 nm. Interestingly, these

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nanoparticles further self-assembled to form nanoclusters, and there were no overlapping and

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aggregation phenomena among nanoparticles. This self-assembly of hybrid nanoparticles could be

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attributed to the hydrogen bonding among them. Recently, Men et al. (2015) reported that

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autobiotinylated ferritin nanoparticles self-assembled superstructures through a biotin–streptavidin

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interaction.22 However, the excessive SPI addition (the SLG/SPI ratio was above 10:5) contributed

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to the aggregation of nanoparticles.

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However, irregularly diamond-shaped SLG/RP hybrid nanoparticles were formed at the low

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ratio (10:1) with a size of 40–80 nm, as shown in Figure 2A. Interestingly, the SLG/RP

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nanoparticles showed flower-like superstructures when the ratio of SLG/RP was over 10:1. In

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addition, the TEM image of the superstructure became darker due to the aggregation of

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nanoparticles with the increasing RP content. The flower-like structure is probably attributable to

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the self-assembly of SLG/RP hybrid nanoparticles (as shown in the red circle in Figure 2C-D).

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The flower-like structure of hybrid nanoparticles constitutes a new topological shape that has not

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been previously observed, and the driving forces of self-assembly need to be investigated further.

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Being different from the above SLG/SPI and SLG/RP hybrids, all SLG/WP hybrid


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nanoparticles showed stacked lamellas as the SLG/WP ratio varied from 10:1 to 10:7.5. The width

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of the lamellae was 5–10 nm and their length was 50–70 nm when the SLG/WP ratio was 10:1.

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The lamellae became wider with the increasing SLG/WP ratios. There were many extra small

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nanoparticles (as shown in the red circle in Figure 3A) throughout the lamellae; it is therefore

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thought that the formation of lamellae was due to the aggregation of nanoparticles.

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Thermal properties The thermal behaviors of SLG nanoparticles and SLG/protein hybrid

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nanoparticles were determined using DSC analysis, and the results are shown in Tables 1–3. The

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onset and peak melting temperatures of SLG/SPI nanoparticles with a ratio of 10:1 were 75.14 °C

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and 93.56 °C, respectively, quite a bit higher than those of SLG nanoparticles (Table 1). This was

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most likely due to reduced water availability to SLG in the presence of SPI and the improved

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crystallite stability,23 which hindered gelatinization of SLG. In addition, the absolute value of ∆H

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of hybrids was higher than that of SLG nanoparticles and increased with the increasing SLG/SPI

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ratio (except for 10:7.5). This could be because the interaction binding energy between SLG and

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SPI nanoparticles was higher than that among SLG nanoparticles. However, the peak temperature

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and absolute value of ∆H of the SLG/SPI hybrid nanoparticles with a ratio of 10:7.5 decreased,

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which could be because excess SPI could not interact with SLG to form a firm nanostructure. Liu

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et al. (2015) also reported that excess pure SPI in nanoparticle systems caused a decrease in hybrid

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nanoparticles at peak temperature and absolute value of ∆H.24

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The peak temperature and absolute value of ∆H of SLG/RP hybrid nanoparticles with a ratio

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of 10:1 were significantly higher than those of SLG nanoparticles (Table 2), suggesting that

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nanoparticles with a firmer structure were formed. However, the melting temperatures of SLG/RP

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nanoparticles with a ratio of over 10:1 decreased significantly, indicating that they were more



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easily melted than SLG nanoparticles; this could be related to their flower-like superstructures

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(Figure 2). In addition, the melting temperatures of SLG/RP hybrid were significantly lower than

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those of SLG/SPI hybrid, which could be also related to their morphology. Unfolded structures

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were easier to interact with water molecules than closed sphere, which accelerated their

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destruction with thermal treatment. The SLG/WPI hybrid nanoparticles had higher melting

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temperatures and absolute value of ∆H (Table 3) than the SLG/SPI and SLG/RP hybrid

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nanoparticles, which could be because the stacked lamellas formed by the oriented arrangement of

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hybrid nanoparticles had a more compact crystallite structure.

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Fourier transform infrared spectra

FTIR spectroscopy is a useful technique for

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investigating protein and starch interactions. The FTIR spectra of SLG/SPI nanoparticles with

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different SLG/SPI ratios are shown in Figure 4. The characteristic adsorption peaks of SLG

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nanoparticles were observed at 3500–3300 cm−1 (O-H stretching), and the peak position was

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related to the extent of the formation of inter- and intra-molecular hydrogen bonding. The peaks at

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2930–2940 cm−1 (C-H stretching) and 1630–1640 cm−1 (O-H stretching) reflected tightly bound

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water present in the SLG nanoparticles, similar to those reported earlier.25-26 The characteristic

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bands at around 1022 cm−1, 1074 cm−1, and 1156 cm−1 in the fingerprint region corresponded to

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the C-O ether-stretching vibration in glucose bonds.27 Moreover, 960 cm−1 was attributed to

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skeletal mode vibrations of the α-1, 4 glycosidic linkage (C–O–C).

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The characteristic bands at 3500–3300 cm−1 of SLG/SPI nanoparticles with the 10:1 ratio of

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SLG/SPI shifted to a lower wavelength, indicating that the interaction force of intermolecular

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hydrogen bonds between SLG and SPI molecules was enhanced. According to Spada et al. (2015)

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and Liu et al. (2015), the characteristic peaks at 1648 cm−1 and 1543 cm−1 (also as shown in


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Figure 4A of this work) are assigned, respectively, to the amide groups I and II of the soy

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protein.28-29 Amide I corresponds to C=O stretching vibrations (80%) with a minor contribution by

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C-N stretching vibrations, and amide II arises from N-H bonding (60%) and C-N stretching

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vibrations (40%). The peak of SLG nanoparticles at 1640 cm−1 was attributed to the O-H

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stretching. However, the peak of SLG/SPI hybrid nanoparticles with a 10:1 ratio of SLG/SPI

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shifted to 1643 cm−1 due to the peaks' overlap of SLG and SPI. When the ratio of SPI further

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increased, the peak shifted to 1648 cm−1, corresponding to C=O stretching vibrations. Similarly,

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the characteristic peaks corresponding to amide II appeared at a higher SPI ratio.

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The interaction force of intermolecular hydrogen bonds between SLG and RP or WP

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molecules was also enhanced, which was indicated by a shift to a lower wavenumber at 3500–

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3400 cm−1. However, the disappearance of wavenumbers in the amide II band could be due to the

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formation of hydrogen bonds between the NH group of the protein backbone and SLG.

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Deconvolution and curve fitting

The secondary structural changes of proteins was

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determined by analyzing the deconvoluted spectra (1700–1600 cm-1).30 Table 4 shows that the

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SLG/SPI nanoparticles with an SLG/SPI ratio of 10:1 contained 19.76% α-helix contents (1650–

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1660 cm-1). The β-sheet absorption was in the frequency region of 1610–1640 cm-1, and other

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peaks were at 1640–1650 cm-1, 1660–1680 cm-1, and 1682–1685 cm-1, corresponding to the

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random coil, β-turn, and β-antiparallel, respectively. The random coil, β-turn, and β-antiparallel

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structure percentage decreased with increasing SPI content, while the percentages of α-helix and

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β-sheet structures increased. Fragoso et al. (2012) found that the addition of sodium sulfosuccinate

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increased the α-helix structures with a concomitant loss of the β-turn structure of soy proteins.31

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The secondary structure of pure RP was made up of 9.92% α-helix, 19.68% β-sheet, 20.53%



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β-turn, 44.23% random coil, and 5.64% β-antiparallel. However, due to the interaction between

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the SLG and RP, the hybrid nanoparticles with an SLG/RP ratio of 10:1 increased α-helix by

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20.56%, β-sheet by 132.42% and β-turn by 24.26%, while it decreased random coil by 70.86%,

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and β-antiparallel by 30.85%. However, the calculated percentages of α-helix, β-sheets, β-turns,

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and random coil in SLG/WPI hybrid nanoparticles differed somewhat from those in WPI, which

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contained 13.64% α-helix, 15.57% β-sheet, 26.87% β-turn, 30.19% random coil, and 13.73%

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β-antiparallel structures. The SLG/WPI nanoparticles with an SLG/WPI ratio of 10:1 contained

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19.48% α-helix, 40.54% β-sheet, 29.84% β-turn, 7.62% random coil, and 2.46% β-antiparallel

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structures. This indicates that the interaction of SLG and WPI caused a 82.08% reduction in

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β-antiparallel structure and a 74.76% reduction in random coil, which was converted into α-helix,

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β-sheet, and β-turn structures to form a more ordered structure. In addition, the percentage of

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random coil decreased with the increasing WPI content in the hybrid.

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UV-visible spectra

The UV-visible absorption spectra of SLG/SPI nanoparticles with

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different ratios of SLG to SPI are shown in Figure 5A. According to Kang et al. (2016), the broad

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absorption band of SPI appears at 275 nm.32 The maximum absorption peak of SLG/SPI hybrid

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nanoparticles with lower SPI content showed a blue shift at 260 nm, and the maximum peak

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absorption increased with the increasing concentration of SPI. This may have been caused by the

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energy change of the π-π transition due to the conjugation of SLG to the amine or sulfhydryl

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groups of peptide side chains.33

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Figure 5B shows the typical UV-visible spectroscopy of SLG/RP nanoparticles with different

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ratios of SLG to RP. The characteristic absorption band of tyrosine, tryptophan, and phenylalanine

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was at 275 nm, 279 nm, and 257 nm, respectively.34 In this work, the absorbance intensity of the


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SLG/RP hybrid was mainly at 270 nm, probably due to the exposure of the buried hydrophobic

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groups or the secondary structure modification.35 Another characteristic absorption of SLG/RP

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nanoparticles was at 235 nm, indicating that the secondary structure of RP was altered after

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binding with SLG. The characteristic absorption band of SLG/WPI hybrid nanoparticles was at

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278 nm, consistent with the report of Qi et al. (2017).36 Furthermore, the maximum peak

249

absorption increased with the increasing concentration of WPI.

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Interactions mechanism

The dissociation reagents, urea, SDS, and DTT, can disrupt

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hydrogen bonds, hydrophobic interactions, and the disulfide bond, respectively. To further analyze

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the interaction forces involved in SLG nanoparticles and SLG/protein hybrid nanoparticles, the

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turbidity at different concentrations of dissociation reagents was determined; the results are shown

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in Figures 6–8. The turbidity of SLG nanoparticles decreased slightly in the presence of SDS and

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DTT. However, the turbidity decreased significantly with the increasing urea concentration. This

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indicates that the main interaction force inside SLG nanoparticles was hydrogen bonding.

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However, the turbidity of SLG/SPI hybrid nanoparticles decreased sharply in the presence of 1 M

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urea or 0.1% SDS, suggesting that hydrogen bonds and hydrophobic interactions were the most

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important interactions in stabilizing SLG/SPI nanoparticles. These results are in agreement with

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the findings of Zhang et al. (2012).37 Compared with SLG/SPI hybrid nanoparticles, the turbidity

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of SLG/RP and SLG/WPI nanoparticles decreased sharply in the presence of 1 M urea, 0.1% SDS,

262

and 20 mM DTT. This indicates that the main interaction forces inside SLG/RP and SLG/WPI

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nanoparticles consisted of hydrogen bonding, hydrophobic interaction, and disulfide bonding.

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Rheological properties Figures 9–11 show the variation in G' and tanδ of corn starch gel

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incorporated with different nanoparticles as a function of frequency. The presence of SLG



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nanoparticles increased the G' of corn starch but showed significantly lower curves than those

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exhibited by the addition of SLG/SPI hybrid nanoparticles with various ratios of SLG to SPI. The

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outer hydrophilic groups of hybrid nanoparticles could combine with more water molecules, thus

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increasing the dispersity of hybrids in the aqueous solution. Furthermore, the free OH groups of

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hybrid nanoparticles could participate in the formation of corn starch hydrogen bonding, which in

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turn improved the gel properties of corn starch. These results are in agreement with previous

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reports stating that the loaded nanofiller in the matrix causes substantial improvement in gel

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characteristics due to the enhanced matrix–nanofiller interaction.38 However, the G' of corn

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starch-SLG/SPI nanocomposites with an SLG/SPI ratio of 10:7.5 was the lowest among the corn

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starch–SLG/SPI samples. This could be due to the aggregation characteristic of hybrid

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nanocomposites (as shown in Figure 1D). Figure 9B shows that all the samples exhibit a typical

277

gel network where tanδ values are less than 1. The tanδ of corn starch–SLG/SPI nanohybrid gels

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was obviously lower than that of both corn starch gels and corn glucan/SLG nanoparticle gels,

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indicating that a much stronger corn starch gel network could be formed in the presence of hybrid

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nanocomposites.

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The G' of all corn starch–SLG/RP nanoparticle gels with a SLG/RP ratio of over 10:1

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increased by over 567% compared to the corn starch gels. This significant increase could be due to

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their flower-like superstructures (Figure 2B). Li et al. (2016) reported that only if the nanofibers

284

can be broomed efficiently and entangled together with polymers to form a network structure can

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a real enhancement effect of polymers on gel characteristics be obtained.39 The flower-like

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superstructures of SLG/RP nanoparticles were similar to the assembly of nanofibers, and the

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extended structures were more beneficial to their interaction with the matrix. The G' of all corn


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starch–SLG/WPI nanoparticle gels increased by a lesser extent than that of corn starch gels

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incorporated with SLG/SPI and SLG/RP nanoparticles. This could be because the stacked lamellas

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of SLG/WPI (Figure 3A–C) were more difficult to become entangled with corn starch compared

291

to the other two hybrid nanoparticles. However, the introduction of hydrophilic WPI improved the

292

dispersibility of hybrid nanoparticles so that the G' of all corn starch–SLG/WPI nanohybrid gels

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was higher than that of corn starch–SLG nanoparticle gels.

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In conclusion, we successfully prepared SLG/protein hybrid nanoparticles and investigated

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the effect of protein type and SLG/protein ratios on the morphology and thermal properties of

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hybrid nanoparticles. Hollow SLG/SPI nanoparticles, a flower-like SLG/RP superstructure, and

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stacked lamellar SLG/WPI nanostructures were obtained due to the differences in proteins and the

298

interaction between SLG and protein. Compared with SLG nanoparticles, SLG/SPI and SLG/WPI

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nanoparticles had higher melting temperatures. Furthermore, the introduction of the hybrid

300

nanoparticles greatly improved the rheological properties of corn starch. These newly developed

301

glucan/protein hybrid nanoparticles have potential for improving the rheological properties of

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starch paste and the potential for broad applicability in modifying food texture.



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AUTHOR INFORMATION

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Corresponding Authors

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*E-mail: [email protected]

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Acknowledgment

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The study was supported by the National Natural Science Foundation of China (Grant No.

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31671814).

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Notes

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The authors declare no competing financial interest.


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REFERENCES

312

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415

1206-1215.



Page 22 of 37

416

Table 1 Thermal characteristics of short linear glucan (SLG)/soy protein isolate (SPI) hybrid

417

nanoparticles

Weight of

Onset

Peak

Conclusion ∆H/Jg-1

SLG to SPI

Temperature/°C

Temperature/°C

Temperature/°C

StNPs

64.32±0.76d

89.92±0.35d

104.03±1.77c

-12.40±0.13e

10:1

75.14±0.23b

93.56±0.82c

102.93±0.56c

-14.67±0.11c

10:2.5

77.09±2.47b

96.86±1.59b

108.15±1.18b

-15.06±0.10b

10:5

85.26±0.94a

102.19±0.77a

112.29±1.37a

-17.83±0.18a

10:7.5

71.97±1.05c

92.07±1.66c

101.79±0.99d

-14.18±0.15d

418

Values represent the mean ± standard deviation of triplicate tests. Values in column having

419

different superscripts (a, b, c, d) were significantly different (p < 0.05). StNPs represents starch

420

nanoparticles prepared by self-assembled of SLG.


Page 23 of 37


421

Table 2 Thermal characteristics of short linear glucan (SLG)/rice protein (RP) hybrid

422

nanoparticles

Ratio of SLG

Onset

Peak


to RP

Temperature/°C

Temperature/°C

Temperature/°C

StNPs

64.32±0.76a

89.92±0.35b

104.03±1.77a

-12.40±0.13c

10:1

64.36±0.47a

92.39±1.76a

104.52±1.41a

-14.08±0.09a

10:2.5

57.42±0.98b

80.92±1.94c

98.55±1.09c

-13.42±0.13b

10:5

54.78±0.87c

78.92±2.15c

100.09±1.55c

-12.23±0.10c

10:7.5

52.18±1.05d

73.07±1.66d

97.21±1.79d

-11.93±0.14d

423


424


425




Page 24 of 37

426

Table 3 Thermal characteristics of short linear glucan (SLG)/whey protein isolate (WPI)

427

hybrid nanoparticles Ratio of SLG

Onset

Peak


to WPI

Temperature/°C

Temperature/°C

Temperature/°C

StNPs

64.32±0.76d

89.92±0.35d

104.03±1.77d

-12.40±0.13e

10:1

79.38±1.53b

96.17±1.01c

110.62±1.08b

-15.22±0.14c

10:2.5

80.16±1.29b

103.88±0.32b

117.45±2.14a

-16.14±0.12b

10:5

87.63±3.05a

112.49±1.73a

120.43±3.17a

-18.23±0.10a

10:7.5

75.11±0.97c

91.26±2.01d

107.26±1.32c

-13.58±0.13d

428


429


430



Page 25 of 37


431

Table 4 Changes of protein secondary-structure in the short linear glucan (SLG) and protein

432

binding process Samples

α-helix/%

β-sheet/%

β-turn/%

Randon

β-antiparallel/%

coil/% SLG/SPI Pure SPI

11.58±0.56d

24.12±0.43d

23.79±0.74b

30.55±1.38a

9.96±0.47b

10:1

19.76±0.87c

25.25±0.12c

33.57±1.08a

8.09±0.25b

13.33±0.59a

10:2.5

21.80±0.94b

32.04±1.35b

32.08±0.55a

7.28±0.37c

5.80±0.22d

10:5

22.07±0.32b

39.80±0.49a

22.28±1.11b

6.77±0.18c

8.45±0.41c

10:7.5

24.83±0.99a

40.89±1.86a

22.23±0.63b

6.63±0.32c

5.42±0.16d

Pure RP

9.92±0.32e

19.68±0.79d

20.53±1.03b

44.23±2.13a

5.64±0.21a

10:1

11.96±0.18d

45.74±2.04b

25.51±1.11a

12.89±0.36b

3.90±0.12c

10:2.5

13.99±0.46c

47.52±1.75a

23.81±0.89a

12.29±0.58b

2.39±0.09e

10:5

19.13±0.87b

45.15±1.03b

20.55±0.75b

10.77±0.37c

4.36±0.17b

10:7.5

25.42±1.13a

40.35±1.86c

21.25±0.96b

9.65±0.15d

3.31±0.06d

Pure WPI

13.64±0.55d

15.57±0.24c

26.87±0.37b

30.19±0.96a

13.73±0.17a

10:1

19.48±0.93c

40.54±1.46a

29.84±1.05a

7.62±0.33d

2.46±0.06c

10:2.5

23.45±0.87a

42.82±1.32a

20.41±1.03c

10.96±0.49b

2.33±0.09c

10:5

24.50±0.46a

37.99±1.57b

20.71±0.98c

9.42±0.34c

3.08±0.14b

10:7.5

21.69±0.71b

40.66±0.99a

18.28±0.74d

6.16±0.28e

3.18±0.08b

SLG/RP

SLG/WPI

433

Values represent the mean ± standard deviation of triplicate tests. Values in column (for same

434

protein) having different superscripts (a, b, c, d) were significantly different (p < 0.05). The weight

435

ratio of SLG to protein (SPI: soy protein isolate, RP: rice protein, WPI: whey protein isolate) was

436

10:1, 10:2.5, 10:5 and 10:7.5.



Page 26 of 37

A

B

C

D

437

Figure 1. TEM images of short linear glucans (SLG)/soy protein isolate (SPI) hybrid

438

nanoparticles with different weight ratio of SLG to SPI (A 10:1, B 10:2.5, C 10:5 and D

439

10:7.5).


Page 27 of 37


A

B

C

D

440

Figure 2. TEM images of short linear glucans (SLG)/rice protein (RP) hybrid nanostructures

441

with different weight ratio of SLG to RP (A 10:1, B 10:2.5, C 10:5 and D 10:7.5).



Page 28 of 37

A

B

C

D

442

Figure 3. TEM images of short linear glucans (SLG)/whey protein isolate (WPI) hybrid

443

nanostructures with different weight ratio of SLG to WPI (A 10:1, B 10:2.5, C 10:5 and D

444

10:7.5).


Page 29 of 37


A

1533 3296

10:7.5

1648

3292

10:5 10:2.5

1648 1647 1643

T%

3317 3329

10:1 StNPs

1640

3330 SPI

1634 1648

3500

3000

2500

2000

1500

1000

Wavenumber (cm-1)

B

C 10:7.5

3290

10:5

3294

10:2.5

3288

10:1

T%

T%

3301

10:7.5 10:5 10:2.5 10:1

3283 3285

3303

3301 3330

StNPs WPI

StNPs

3330

RP

3500

3000

2500

2000

1500

Wavenumber (cm-1)

1000

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

445

Figure 4. FITR spectra of (A) short linear glucan (SLG)/soy protein isolated (SPI), (B)

446

SLG/rice protein (RP), and (C) SLG/ whey protein isolate (WPI) hybrid nanostructures with

447

different weight ratio of SLG to protein (10:1, 10:2.5, 10:5 and 10:7.5).



2.5

A

2.0

Absorbance

SLG/SPI (10:7.5) SLG/SPI (10:5)

1.5

SLG/SPI (10:2.5) 1.0

SLG/SPI (10:1) StNPs

0.5

0.0 200

250

300

350

400

450

Wavelength (nm)

B

3.0

2.5

SLG/RP(10:7.5)

Absorbance

2.0

SLG/RP(10:5)

1.5

SLG/RP(10:2.5) 1.0

SLG/RP(10:1) StNPs

0.5

0.0 200

250

300

350

400

450

Wavelength (nm)

C

3.0

Absorbance

2.5

SLG/WPI (10:7.5)

2.0

SLG/WPI (10:5)

1.5

SLG/WPI (10:2.5) SLG/WPI (10:1)

1.0

StNPs 0.5

0.0 200

250

300

350

400

450

Wavelength (nm)

448

Figure 5. The UV–vis spectra of (A) short linear glucan (SLG)/soy protein isolate (SPI), (B)

449

SLG/rice protein (RP), and (C) SLG/whey protein isolate (WPI) hybrid nanostructures with

450

different weight ratio of SLG to protein. Starch nanoparticles (StNPs) are prepared by

451

self-assembly of SLG.


Page 30 of 37

Page 31 of 37


StNPs SLG/SPI (10:1) SLG/SPI (10:2.5) SLG/SPI (10:5) SLG/SPI (10:7.5)

A

1.2

Turbidity

1.0 0.8 0.6 0.4 0.2 0

1

2

3

4

Urea (M)

1.20

StNPs SLG/SPI (10:1) SLG/SPI (10:2.5) SLG/SPI (10:5) SLG/SPI (10:7.5)

B

1.05

1.4 StNPs SLG/SPI (10:1) SLG/SPI (10:2.5) SLG/SPI (10:5) SLG/SPI (10:7.5)

C

Turbidity

Turbidity

1.2

0.90 0.75 0.60

1.0

0.45 0.8

0.30 0.15 0.0

0.2

0.4

0.6

0.8

1.0

0

10

20

30

40

50

60

DTT (mM)

SDS (w/v, %) 452

Figure 6. The influence of dissociate reagents on the turbidity of short linear glucan

453

(SLG)/soy protein isolate (SPI) hybrid nanoparticles with different weight ratio of SLG and

454

SPI.



1.2

StNPs SLG/RP(10:1) SLG/RP(10:2.5) SLG/RP(10:5) SLG/RP(10:7.5)

A

1.0

Turbidity

Page 32 of 37

0.8 0.6 0.4 0.2 0

1

2

3

4

Urea (M) StNPs SLG/RP(10:1) SLG/RP(10:2.5) SLG/RP(10:5) SLG/RP(10:7.5)

B

1.2

0.8

0.6

StNPs SLG/RP(10:1) SLG/RP(10:2.5) SLG/RP(10:5) SLG/RP(10:7.5)

C

1.2

Turbidity

Turbidity

1.0

1.4

1.0 0.8 0.6 0.4

0.4

0.2 0.0

0.2

0.4

0.6

0.8

1.0

0

10

20

30

40

50

60

DTT (mM)

SDS (w/v, %)

455


456

(SLG)/rice protein (RP) hybrid nanostructures with different weight ratio of SLG and RP.


Page 33 of 37


1.2

A

StNPs SLG/WPI (10:1) SLG/WPI (10:2.5) SLG/WPI (10:5) SLG/WPI (10:7.5)

Turbidity

1.0 0.8 0.6 0.4 0.2 0

1

2

3

4

Urea (M) 1.2


B

1.4


C

1.2

Turbidity

Turbidity

1.0 0.8 0.6

1.0 0.8 0.6 0.4

0.4

0.2

0.2 0.0

0.2

0.4

0.6

0.8

1.0

0

10

SDS (w/v, %)

20

30

40

50

60

DTT (mM)

457


458

(SLG)/whey protein isolate (WPI) hybrid nanostructures with different weight ratio of SLG

459

and WPI.



CS CS/StNPs

1000

CS/(SLG/SPI) hybrid a

A

CS/(SLG/SPI) hybrid b CS/(SLG/SPI) hybrid c

G' (Pa)

CS/(SLG/SPI) hybrid d

100

10 1

10

100

ω (rad/s) CS CS/StNPs

0.5

CS/(SLG/SPI) hybrid a

B

CS/(SLG/SPI) hybrid b CS/(SLG/SPI) hybrid c

tanδ

0.4

CS/(SLG/SPI) hybrid d

0.3

0.2

0.1

1

10

100

ω (rad/s)

460

Figure 9. The G'-ω curves (A) and the tanδ-ω (B) curves of corn starch (CS), CS/starch

461

nanoparticle (StNP) mixtures, and CS/short linear glucan (SLG)/soy protein (SPI) hybrids

462

with different weight ratio between SLG and SPI (a-d, 10:1, 10:2.5, 10:5 and 10:7.5) mixture.


Page 34 of 37

Page 35 of 37


CS CS/StNPs CS/(SLG/RP) hybrid a CS/(SLG/RP) hybrid b CS/(SLG/RP) hybrid c CS/(SLG/RP) hybrid d

1000

G' (Pa)

A

100

10 0.1

1

10

ω (rad/s) CS CS/StNPs

0.5

CS/(SLG/RP) hybrid a

B

CS/(SLG/RP) hybrid b CS/(SLG/RP) hybrid c

0.4

CS/(SLG/RP) hybrid d

tanδ

0.3

0.2

0.1

1

10

100

ω (rad/s) 463


464

nanoparticles (StNPs) mixture and CS/short linear glucan (SLG)/rice protein (RP) hybrid

465

with different weight ratio (a-d, 10:1, 10:2.5, 10:5 and 10:7.5) mixture.



CS

1000

CS/StNPs CS/(SLG/WPI) hybrid a

A

CS/(SLG/WPI) hybrid b CS/(SLG/WPI) hybrid c

G' (Pa)

CS/(SLG/WPI) hybrid d

100

10 1

10

100

ω (rad/s) CS

0.5

CS/StNPs CS/(SLG/WPI) hybrid a

B

CS/(SLG/WPI) hybrid b CS/(SLG/WPI) hybrid c

0.4

CS/(SLG/WPI) hybrid d

tanδ

0.3

0.2

0.1

1

10

100

ω (rad/s)

466


467

nanoparticles (StNPs) mixture and CS/short linear glucan (SLG)/whey protein (WPI) hybrid

468

with different weight ratio (a-d, 10:1, 10:2.5, 10:5 and 10:7.5) mixture.


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Page 37 of 37


TOC


Morphology and structural properties of novel short linear glucan

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