Effects of Degree of Polymerization on Size, Crystal Structure, and

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Effects of Degree of Polymerization on Size, Crystal Structure and Digestibility of Debranched Starch Nanoparticles and Their Enhanced Antioxidant and Antibacterial Activities of Curcumin Yang Qin, Jinpeng Wang, Chao Qiu, Yao Hu, Xueming Xu, and Zhengyu Jin ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00290 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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ACS Sustainable Chemistry & Engineering

Effects of Degree of Polymerization on Size, Crystal Structure and Digestibility of Debranched Starch Nanoparticles and Their Enhanced Antioxidant and Antibacterial Activities of Curcumin Yang Qin,†,‡,§ Jinpeng Wang,†,‡,§ Chao Qiu,†,‡,§ Yao Hu,†,‡ Xueming Xu,†,‡,§ and Zhengyu Jin*,†,‡,§ †State

Key Laboratory of Food Science and Technology, ‡School of Food Science and

Technology, and §Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China. * Corresponding Author, E-mail: [email protected]. ABSTRACT The versatility of debranched starch nanoparticles (DBS-NPs) has attracted considerable attention from the food, agricultural, and cosmetic industries. The aim of this study was to investigate the effect of DBSs with low (Degree of Polymerization (DP) < 10), moderate (10 > DP < 20), and high (20 > DP < 25) DPs as well as reaction temperatures (25C, 60C, and 90C) on size, morphology, crystal structure, and digestibility of the refined DBS-NPs via nanoprecipitation. Spherical DBS-NPs with a controlled size of 40200 nm were obtained; no nanoparticles were observed when the average DP was smaller than 10. The relative crystalline raised from 11.1% to 71.6% with increasing the average DPs. When the micro-sized DBSs were converted to nano-scale DBSs, the rapidly digestible starch levels of the DBS-NPs decreased, and the DBS-NPs showed remarkable increase in the sum of slowly digestible starch and resistant starch contents compared to the responding DBS. Furthermore, the maximum encapsulation efficiency of curcumin in DBS-NPs reached up to 92.49%. The antioxidant and antibacterial activities of curcumins in DBS-NPs were enhanced significantly, and their antioxidant stability was improved markedly when exposed to UV light or heat treatments.

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KEYWORDS: debranched starch, nanoparticles, in-vitro digestion, antioxidant activity, stability， cytotoxicity INTRODUCTION Starch is one of the most important and frequently used functional ingredients in the food processing industry and in other industrial applications because of its inexpensive, abundant, renewable, and biodegradable.1,2 Nanotechnology is used to enable a decrease in particle size, and improves the functional property of raw material.3 Due to potentially faster diffusion rates, higher solubilities, higher absorption capacities, and higher penetration rates through biological barriers,4 starch nanoparticles (SNPs), as a novel material, have great potential applications in a variety of ways: mimicking lipid micelles, stabilizing food ingredients, and acting as potential fillers, adsorbents, and agrochemicals or drug delivery carriers.5-7 The fabrication of nanostructures from native starch has attracted considerable attention in the field of food, agriculture, chemistry, and bio-pharmaceutical industry.8 SNPs can be produced using the techniques of precipitation, high-pressure homogenization, sonication, regeneration, combination of acid or enzymatic hydrolysis and sonication, nanoemulsion, etc.6 One of the most promising techniques for commercial application is nanoprecipitation.9 Previous works showed the preparation of a desired diameter and morphology of SNPs by controlling the native starch type, molecular weight (Mw), and concentration, non-solvent type, the solvent to non-solvent ratio, and reaction temperature, etc.11-14 The increasing ratio of starch solution to ethanol changed the morphology of sago SNPs from fibrous to a mixture of spherical and elongated fiber-like structures, and finally to mainly spherical particles with the size of 300–400 nm.10 As the concentrations of corn starch raised


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from 2.5% to 10%, the average diameter of the SNPs increased from 132 to 220 nm.15 A high viscosity of starch solution hampers the diffusion of starch solution toward non-solvent, and results in larger particles, thus, a high concentration also influenced the size of the precipitated nanoparticles.16 Using a low concentration of native starch solution (1%), Qin et al. obtained spherical and elliptical SNPs with a mean size of 30–75 nm by nanoprecipitation. Afterwards, they investigated the effect of starch types and amylose contents on the formation of the native SNPs.17 Chang et al. reported the scission of amylose chains and the decrease in solution viscosity by ultrasonic treatment, which are beneficial for preparing small and uniform amylose nanoparticles via nanoprecipitation.18 Starch is mainly composed of amylose (linear chain) and amylopectin (branched chain). Debranched starch (DBS), a short linear glucan, is separated from amylopectin, which is hydrolyzed by debranching enzymes.19 Researchers have confirmed that DBS displays remarkable new properties and functionalities, meeting the requirements of specific applications such as fat or protein replacement, drug formulation, and acting as a ingredient for the production of resistant starch (RS) and slowly digestible starch (SDS).20-22 To further improve the physicochemical properties of DBSs, nano-scale particles are obtained using hybrid DBSs as precursor materials via self-assembly,23 nanoprecipitation,24 and ultrasonication techniques.25 Qiu et al. preliminarily investigated the effect of the addition ratio of ethanol on the size, morphology, and thermal property of nano-scale DBSs using the DBS mixtures.24 Moreover, the nanoparticles—as fillers, adsorbents, and drug delivery carriers—have been investigated in the fields of packaging materials, functional foods, and pharmaceuticals.26,27 Therefore, functionalized DBS has become a topic of broad and current interest in starch science.23,28,29



However, in previous studies, the DBSs, or nano-scale DBSs, were still a mixture of mainly short and long linear glucans of a wide range of degree of polymerization (6 < DP < 60).23-29 To overcome the issue, the feasibility of separating DBS mixtures into different DBS fractions according to their Mw were evaluated by Chang et al. and Lu et al., 30,31 and we prepared the small and fine DBS-NPs using the refined DBSs via nanoprecipitation in this study. The rate of the double-helix formation depends on the chain length, concentration, and reaction temperature of amylose solution. Moreover, a double helix formed only when the chain length of the DP is above 10.31 However, in starch science, it is currently unclear which kind of the average DP of DBSs is more suitable to form small nanoparticles with single-helical structures via nanoprecipitation, or whether there is any correlation between the average DPs and SNPs by characterization of their morphology and crystal structure? To address the above problems, we focused on the investigation of the effect of the DBSs with low (DP < 10), moderate (10 > DP < 20), and high (20 > DP < 25) DPs on the size, morphology, crystal structure, and digestibility of the refined DBS-NPs via precipitation for the first time. Because DBSs have been used as raw material for RS and SDS productions,21 it is also important to understand the digestibility so that novel methods can be developed to produce SDS and RS that have practical applications in the food industry. Although there are a few reports of nano-scale DBSs of heterogeneous DPs by nanoprecipitation, as far as our literature survey could ascertain, no investigation about the effect of DPs on the digestion property of the refined DBS-NPs has been reported. Therefore, to fill the knowledge gap, the effects of the DP values and reaction temperatures on the size, morphology, crystal structure, and digestibility of DBS-NPs fabricated by nanoprecipitation from five refined DBSs with low (an average DP of 9), moderate (average DP of 13 and 17), and high (average DP of 20 and 24)


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degrees of debranching were investigated. Curcumin, a traditional Asian spice, is a component of the golden spice turmeric. Previous studies determined that curcumin has properties that are antioxidant, antimicrobial, anti-inflammatory, etc.32-33 In spite of this potential, the applications of curcumin are restricted mainly because of the low bioavailability, poor absorption, and rapid metabolism.33 Owing to the external hydrophilic structure and ultrafine size, NPs have been used as the adsorbing materials for bioactive compounds to improve bioactive compounds’ instability and enhance their bioavailability.34 Therefore, we also investigated the encapsulation, antibacterial activity, and antioxidative activity of curcumins in DBS-NPs as well as their antioxidant stability when exposed to the harsh conditions (UV light and heat treatment). MATERIALS AND METHODS Materials. Waxy maize starch (0.85% amylose content) was purchased from the National Starch Co. (Shanghai, China). Maize starch (26.9% amylose content) was offered by Ingredient China Ltd. (Guangdong, China). Curcumin was supplied by Aladdin Industrial Co. Ltd. (Shanghai, China). Pullulanase (4461.6 NPUN/g) was supplied by Novozymes Investment Co. Ltd.

(Beijing,

China).

2,2-Diphenyl-1-picrylhydrazyl

(DPPH)

was

purchased

from

Sigma-Aldrich (St. Louis, MO, U.S.A.). All the reagents used were of analytical grade. Preparation of Different DPs of DBS-NPs. The DBS powders with different DP values were obtained from native starches by enzymatic modification.30,31 According to the distribution of DBS specimens obtained from the debranching waxy and normal maize starch (Figure S1), the specimens were coded as DBS-24, DBS-20, DBS-17, DBS-13, and DBS-9, respectively. The DBS nanoparticles (DBS-NPs) were obtained by nanoprecipitation according to a previously



reported by Qin et al.17 with some modification, one gram of dried DBS powders was re-dispersed in deionized water (100 ml) and then heated at 150 °C for 40 min for complete gelatinization. Afterwards, the DBS solutions were maintained at different temperature (25 °C, 60 °C, and 90 °C) under constant stirring (350 rpm), certain volume of ethanol (the solvent: non-solvent volume ratio was 1:10) was added drop-wise into the DBSs solution using a BT100-2J syringe pump (Longer, Hebei, China) with a constant flow rate of 50 ml/min. Furthermore, the suspension was stirred continuously for another 60 min at the prepared temperature. Subsequently, centrifugation (10000 g for 10 min) was conducted and absolute ethanol was used to remove excessive water in the sediment. Finally, freeze-dried DBS-NPs were obtained by lyophilization (-80 C for 48 h). Characterization. The Molecular-weight-distribution for DBS specimens were determined by a LC-20A high-performance size-exclusion chromatograph (HPSEC, Shimadzu, Kyoto, Japan), as described by Chang et al.30 The morphology images of DBS-NPs were taken with a transmission electron microscope (TEM, 7650, Hitachi Tokyo, Japan) at 120 kV acceleration voltage. A drop of DBS-NPs suspension (0.5%, w/v) was placed on copper grid and lyophilized. The size distribution, mean size, and PDI of the DBS-NPs were determined by a dynamic light scattering (DLS) instrument (Malvan, Zetasizer NanoZS, U.K.). 2 ml of DBS-NPs dispersions (0.5% w/v) were added to cuvettes and measured. Fourier transform infrared (FTIR) spectra (4000 to 400 cm−1) of DBS-NPs were recorded on a Nicolet IS10 spectrometer (Thermo Nicolet, Waltham, MA, U.S.A.) at a resolution of 4 cm−1. The crystalline structure of DBS-NPs was performed by a X-ray diffractometer (D2 PHASER, Bruker., Germany) with Cu-Ka radiation source (λ = 1.542 Å). The operating voltage and current were set as 40 kV and 30 mA, and the


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scanning ranged from 5.0 to 40.0 (2) with a rate of 0.5/min. The relative crystallinity (RC) of DBS-NPs specimens was estimated by the equation, as described by Qin et al.17 The digestibility of native starches, DBS, and DBS-NPs specimen was measured according to the procedure of Chen et al.35, with minor modification. 500 mg of native starches, DBS, or DBS-NPs was dispersed in 15 mL sodium acetate buffer (0.2 mol/L, CaCl2 1 mM, pH 5.2), and the suspension was placed at 37 C for 5 min. Curcumin Loading. Adsorption experiment was carried out following the method described by Qiu et al.36 with minor modifications. Briefly, 100 microliter of curcumin stock solution (2.0 mg/ml) was added to 1 mL DBS-NPs solution (10 mg/ml). Afterward, the DBS-NPs solution containing curcumin was stirred for 24 h with a constant rate (200 rpm) in the dark at room temperature and then separated by centrifugation (10,000 g for 20 min) to collect supernatant. The DBS-NPs loaded with curcumin were rinsed (three times) with ethanol to remove unbound curcumin. The curcumin concentrations were determined using an UV-1800PC UV−vis spectrophotometer (Mapada, Shanghai, China) at a λmax wavelength of 419 nm. The equations of encapsulation efficiency (EE) and loading capacity (LC) were described as following:

DPPH Radical Scavenging Activity. According to the previous reported,37 the DPPH assay was used to determine the free radical scavenging activity of specimens. Two milliliters of the specimen solution was mixed with 2 mL of 0.2 mM DPPH ethanolic solution. Ultrapure water (2 mL) mixed with 2 mL of DPPH ethanolic solution served as the control. Pure curcumin



and freeze-dried curcumin loaded DBS-NPs with the equivalent concentration of curcumin were dissolved in ultrapure water. The reaction mixture was shaken well and then incubated in the dark (25 C for 30 min). After the incubation, the absorbance was measured at λmax wavelength of 517 nm. The equation of DPPH radical scavenging activity was described as following:

where Ab is the absorbance of the control and Ac represents the absorbance of the curcumin loaded DBS-NPs specimen. The DPPH radical scavenging activities of curcumin loaded DBS-NPs with ultraviolet (UV) light and temperature treatment were also determined. The pure curcumin and curcumin loaded DBS-NPs had undergone the UV radiation treatment (20 w for 30 min) or incubated at high temperature (80 °C for 30 min) and then cooled to 25 °C to evaluate the stability. Determination of Antibacterial Activity. The inhibitory effects of curcumin loaded DBS-NPs on the growth rate of Gram-negative E. coli and Gram-positive S. aureus were measured.38 Briefly, the growth curves of bacteria after exposure to the samples were plotted with the optical density (OD) versus time. 106 CFU of bacterium were monitored by counting the viable cells, which number of per milliliter was equivalent to 0.1 optical density at 600 nm (OD600). Each curcumin loaded DBS-NPs (10 mg) were added to the culture medium (100 mL) and the sample without curcumin was served as a control. Subsequently, each sample was incubated under gentle shaking in a thermostatic water bath (37 C). The growth of bacteria was determined by a UV-1800PC UV-vis spectrophotometer (Mapada, Shanghai, China). In-Vitro Cytotoxicity. The in-vitro cytotoxicity of DBS-NPs and curcumin loaded DBS-NPs were investigated through cell viability tests using undifferentiated Mouse embryonic


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fibroblast (MEF) (American Type Culture Collection (ATCC)) as described previously.26 Statistical Analysis. Each experiment was performed in triplicate at least independently prepared samples, and the data are expressed as mean  standard deviation. RESULTS AND DISCUSSIONS Morphology and Particle Size of DBS-NPs. Amylose can instantly form a helical complex with the complexing agent, and the single-helical structure will further align and fold into lamellar crystallites.39 The effects of the average DP values and reaction temperatures on the formation of DBS-NPs by nanoprecipitation and the morphology and size distribution of DBS-NPs were determined by TEM and DLS. When the DPs of DBS were higher than 10 (average DP = 1324), the DBS formed spherical NPs (40200 nm) with some aggregation (Figure 1). As a natural phenomenon, a similar aggregation structure of SNPs was previously reported by Qiu et al.24 Moreover, the results demonstrated that the morphology of the DBS-NPs did not seem to be related to the reaction temperature. When the average DP was 9, no nano-scale particles were observed in Figure S2. In a previous report, in pure oligosaccharide solution, a double helix formed only when the chain length of the DP is above 10.40 Therefore, we speculated that the DBS with a low DP also could not be developed into a nanostructured particle by themselves. This is probably because of many low-molecular-weight DBSs (DP < 6) and high solubility in ethanol solution leading to the decrease in the nucleation rate of DBSs, fewer particles were available for growth and more amorphous structures were formed. As the average DP reached up to 13–17, there were many microscopic ranges presented (Figure 1: a, e, and i). Similar microscopic ranges were previously reported, i.e., amylose-fatty acid complexes formed nano-scale lamellae interspersed in the amorphous regions to form submicron size spheroids, which tended to



aggregate into a microscopic range.41 Interestingly, compared with the DBS-NPs samples with an average DP of 13 and 17, the outline of the NPs with a higher DP became clearer, suggesting that the crystalline structure was more orderly and the particle had become denser. The particle size distribution of the DBS-NPs is presented in Figure 2, and the responding mean size and PDI are listed in Table 1. Although different DBS-NPs had different size distributions, all of the DBS-NPs exhibited a single peak, indicating the DBS-NPs had a uniform particle size distribution. PDI is a parameter defining the dispersion homogeneity of NPs: a smaller PDI means NPs have a narrower size distribution.42 Since values close to 1 indicate heterogeneity and less than 0.5 show more homogeneity, the results in Table 1 indicate that the DBS-NPs (0.1240.413 of PDI) could be well dispersed in water and could form a stable and uniform aqueous suspension. As shown in Table 1, owing to the increased frequency of collisions among particles leading to agglomeration, an increase in the mean size of the DBS-NPs that were prepared at a high temperature was observed.8 Moreover, a higher temperature could decrease in the degree of supersaturation upon mixing and slow down the rate of nucleation, leading to the formation of larger particles. At room temperature, the mean size and the PDI value of the DBS-NPs decreased from 118.8  9.3 nm to 46.3  12.2 nm and from 0.302 to 0.213 with increasing the average DPs from 13 to 20, whereas the particle sizes of NPs significantly increased, reaching up to 172.6  8.2 nm when the average DP was 24. The mean sizes of the DBS-NPs prepared at high temperatures (60C and 90C) also showed a similar trend. The results revealed that small and well-distributed DBS-NPs could be formed at an average DP of 20. X-Ray Diffraction Analysis. The gelatinized amylose can instantly form a single-helical complex with the complexing agent and exhibit a V-type crystallite.39 There were weaker


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diffraction peaks at 2 of approximately 13.8, and 20.7 for DBS-9 samples, suggesting that the precipitated DBS-9 had formed a few V-type single-helical structures. When the average DP of DBSs was larger than 10, the DBS-NPs exhibited a strong diffraction peak at 2 with values of around 13.8, 15.3, 17.1, 18.5, 20.7, and 23.5, which is an A+V-type X-ray pattern. Due to the quick retrograde of the refined DBSs, DBSs could retrograde in a short time to form A-type crystallization during the addition of ethanol. Generally, an A-type crystallite was formed at high temperature incubation, while a B-type crystallite was formed at low temperature (approximately 4C) incubation.43 Similar A-type crystallites of short-chain amylose were synthesized at higher temperatures.44 Furthermore, as the average DP of DBS increased from 13 to 20, the diffraction peaks of 13.8 and 20.7 were stronger, and subsequently were decreased when the average DP of DBS reached up to 24. However, the RC, which can be seen in Figure 3d, increased significantly with a maximum of 71.6% when the average DP of DBS was 20 compared to DBS-9 (11.1%) at 90C. Thus, we speculated that the DBS with an average DP of 20 was more favorable to form a single-helical structure and oriented easily to form a perfect crystalline structure within the range of the measured DP. Figure 3 demonstrates that, when the reaction temperature was increased from 25C to 90C, the intensity of the diffraction peaks was enhanced and the RC increased. This is probably because, at low complex temperatures, the nucleation rate was very high and the helices “froze” rapidly in a structure with little crystallographic order, while the slow nucleation by the crystal growth led to a more ordered crystalline structure at higher temperatures.45 Another, higher temperature strengthened the mobility of the molecules and then enhanced the diffusion of the ethanol molecules toward amylose to form a more ordered crystalline structure.46 As reported previously,



the amylose-fatty acid complex formed at a higher temperature also exhibited a higher crystallinity,47 which is consistent with the results of this work. Thus, it could be concluded that elevating the reaction temperature of nanoprecipitation contributed to form a higher crystallinity in the DBS-NPs. In addition, to reveal the relationship between the average DP of refined DBSs and RC of DBS-NPs, we created a hypothetical polynomial model (R2 = 0.98 for 25 C; R2 = 0.984 for 60 C; R2 = 0.958 for 90 C) within the test range of average DP (925) (Figure 4). We hope that these models could lay a theoretical foundation for the construction of the high and well-ordered crystalline DBS-NPs with a single-helical structure. FTIR Spectra Analysis. Figure 5 shows the FTIR spectra of the different DPs of DBS-NPs prepared at 25C, 60C, and 90C. The intensity and peak position of O−H stretching vibrations (3750−3000 cm−1) from DBS-NPs depended on the extent of the formation of inter- and intra-molecular hydrogen bonding. The peaks of the bound water in amorphous regions (2900 and 1650 cm−1) corresponded to the C-H stretching and O−H stretching vibration.48 The characteristic bands of glucose bond (1022, 1074, and 1156 cm−1) were assigned to the C-O ether-stretching vibrations.49 Owing to the C-O stretching vibrations and the C-O-C group in the anhydrous glucose ring, there are two peaks observed at 1150 cm−1 and 990 cm−1, respectively. As the average DP value and reaction temperature increased, the characteristic bands at 3750−3000 cm−1 of DBS-NPs shifted to a lower wavelength. Amylose molecules were favored to form additional hydrogen bonds at a high temperature.50 This could be due to the enhanced interaction force of intermolecular hydrogen bonds: the more orderly structure of DBS-NPs with higher DP values than that of DBS-NPs with lower DP values, which was in accord with the result of RC of DBS-NPs (as shown in Figure 3). Furthermore, a previous study reported that the peak position of


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3750−3000 cm−1 shifted to a shorter wavelength, determining an amorphous region decrease in the particles.51 In-Vitro Digestion. Because the digestibility of starch receives significant contributions from the particle size, type of starch, quantity of crystallinity, and structure, etc.,35 it is important to understand the digestibility (SDS and RS contents) of the DBS-NPs for food industry application. Table 2 shows the digested starch fractions (RDS (rapidly digestible starch), SDS, and RS) of native starch, DBS, and DBS-NPs specimens, respectively. Waxy maize starch displayed high SDS content (64.4%), whereas the SDS content of maize starch was only 13.0%, agreeing with the previous findings that native starches with low amylose were more resistant to enzyme digestion as compared with native starches with high amylose.52 Compared with native starch, the DBS specimens showed a remarkable increase in RS level owing to the hydrolysis of amylose into short chains. A similar result was reported when α-amylase was applied to favor the formation of RS.53 When the micro-sized DBSs were precipitated and converted to nano-scale DBSs, the RDS contents of DBS-NP specimens decreased, and the DBS-NPs showed remarkable increase in slow digestion properties compared to the responding DBS. For example, taking the DBS-NPs prepared at room temperature, the contents of SDS were 39.0%, 46.3%, 50.4%, and 47.6% for DBS-NPs-13, DBS-NPs-17, DBS-NPs-20, and DBS-NPs-24, respectively. And the contents of RS were 19.8%, 25.5%, 32.8%, and 27.3%, for the same four DPs of nanoparticles, respectively. From the X-ray diffraction data (Figure 3), the crystal type of DBS was determined to be the A-type or amorphous structure,30 which was changed to V+A-type after nanoprecipitation. It was previously reported by Zhan, Tian, and Tong that the presence of a V-type crystalline structure is beneficial to slow digestion property.54 This explains why the



DBS-NPs with single-helical structure are more resistant to enzyme digestion than DBS. As shown in Table 2, the RS content increased while the SDS content exhibited a decreased trend with the increase in reaction temperature. Regarding the DBS-NPs-20 samples, the RS content rapidly increased from 32.8% to 55.5% with the increase in temperature (2590C); however, SDS content showed a relatively smaller decrease from 50.4% to 35.3% compared to the RS content. Generally, the slow rate of digestion is proportionally correlated with high RC. Especially at high temperature, DBS-NPs-20 had the highest RS content and relatively low SDS content; however, it exhibited the highest RS+SDS level compared with all samples. The stronger anti-enzymatic hydrolysis may be due to the increased frequency of collisions among starch molecules at a high temperature, leading to a larger crystalline region and forming a perfect and rigid crystalline structure. Similarly, Miao et al. found that there is a parallel change between RS and RC, which is consistent with the higher degree of perfection of crystallites and the lower susceptibility to amylolytic hydrolysis.55 In addition, the mean sizes of DBS-NPs-20 were much smaller than the other DBP-NPs at the same temperature. Consequently, another explanation for the DBS-NPs was more resistance to enzyme hydrolysis, possibly because the smaller particle sizes had a more compact structure that was more resistant to enzymes, which is consistent with the description by Chen et al.35 These results revealed that the size, RC, and compactness of DBS-NPs were positively correlated with RS, and together influenced the digestive properties of starch. Encapsulation Efficiency (EE) and Loading Capacity (LC) of Curcumin. The EE and LC of curcumin in the DBS-NPs are listed in Table 3. Amylose and amylopectin molecules can form helical structures and interact with hydrophobic molecules by hydrophobic interaction.14


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Encapsulation mainly occurred by passive diffusion and was assisted by the intermolecular hydrogen bonding between the hydroxyl groups of curcumin and DBS-NPs.56 As the average DP values increased, the LC and EE of DBS-NPs displayed an initially increasing and then a decreasing trend, which was probably due to the difference of the formed particle size and single-helical structure of the DBS-NPs. Compared with the DBS-NPs-13 (82.21% of EE and 11.95% of LC), the EE and LC in the DBS-NPs-20 were achieved up to 92.49% and 20.41%, respectively. Moreover, the smaller the particle size and the more single-helical the structure of DBS-NPs-20 (as shown in Table 1 and Figure 3), the higher the EE and LC achieved. Moreover, the EE of DBS-NPs increased with increasing reaction temperature, and the maximum EE was more than 90%. Similarly, Gelders et al. reported that the yield of amylose-lipid complexes increased markedly with increasing reaction temperature.57 When the EE reached up to 80%, the nanocapsule was found to be an excellent nano-carrier.58 DPPH Radical Scavenging Activity. Owing to the phenolic hydroxyl (−OH) groups, curcumin had strong antioxidant activity, and the antioxidant capacity depended on the binding efficiency between free radicals and −OH groups.59 Therefore, the antioxidant activity of free curcumin and curcumin-loaded DBS-NPs was evaluated by a DPPH free radical scavenging activity (Figure 6). Significant increase (p < 0.05) in antioxidant activity of curcumin was efficiently displayed by the IC50 (50% inhibitory concentration) values for the encapsulated curcumin in DBS-NPs (Figure 6). When encapsulated with the DBS-NPs-20, in comparison to the pure curcumin (49.95  1.09 M), the IC50 value of curcumin decreased to 33.02  1.03 M. These results demonstrate that the encapsulation of curcumin by DBS-NPs exhibited more efficiently on scavenging free radicals than the free curcumin. This could be due to the low



solubility of curcumin lowering the quantity of reactant molecules.59 Additionally, the enhancement of dispersibility and surface area after curcumin encapsulation, which strengthened the interactions between curcumins and free radicals.60 A previous study has been reported by Huang et al., who found a similar effect in which the nano-carrier systems exhibited higher antioxidant activities than pure curcumin.61 Therefore, we hope that the antioxidant activity enhancement will be beneficial to improving the economic and health values of bioactive product in functional foods.34 Stability of Curcumin Antioxidant Activity. A variety of factors (ultraviolet radiation, temperature, etc.) have been reported to influence the chemical stability of curcumin.32 Therefore, to further reveal the physical stability of the encapsulated curcumin, a DPPH assay was used to evaluate the antioxidant activity of free and encapsulated curcumin after thermal and UV radiation treatment. Figure 7 depicts the antioxidant activities of the pure curcumin and curcumin-loaded DBS-NPs after thermal and UV radiation treatment. Owing to the continuous degradation of curcumin during thermal or UV irradiation treatment, the treated pure curcumin exhibited less efficient radical-quenching capacities than when untreated (Figure 7). Moreover, for encapsulated curcumin, increasing IC50 values were also observed following the treatment in thermal or UV irradiation environments. However, the IC50 values were still far below those of pure curcumin, suggesting the radical scavenging activity of curcumin-loaded DBS-NPs was significantly (p < 0.05) higher than those of free curcumin. These results demonstrated that DBS-NPs had a greater capacity to deliver and protect curcumin, which confirmed the protective effect of encapsulation on the chemical stability of bioactive compounds.34,62 Antibacterial Activity in Vitro. The antimicrobial activity of the curcumin loaded


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DBS-NPs against E. coli and S. aureus is shown in Figure 8. Three growth phases were existed for E. coli and S. aureu: lag phase, exponential phase, and stabilization phase. After exposure to the new external, the number of bacteria increased slowly. Moreover, because the released curcumins destroyed the cell structures of bacterium, partial bacterium was killed after exposure to the curcumin loaded DBS-NPs, and the OD600 exhibited a decreased trend with increasing the loaded curcumin contents in each given time. With increasing the incubation time, the bacteria population of the control showed an exponential increase and without any growth restrictions, while the curves for curcumin loaded DBS-NPs showed a remarkable decrease in the number of bacteria. Similar result was also reported that curcumin showed a good antimicrobial activity against gram-positive and gram-negative bacteria.63 Therefore, these results also suggested that the curcumin loaded DBS-NPs had an excellently antibacterial activity towards E. coli and S. aureus. The possible manners for curcumin to lyse bacterial cells could be destruction of the cell wall or membrane, or a specific mechanism may exist38. On the other hand, irreversible destruction occurred when nano-scale particles adhered to the surface of bacteria, followed by penetrating into cells and inhibiting the activity of proteins, thus bacteria apoptosis happened.64 Thus, it could be concluded that the antimicrobial effect of curcumin loaded DBS-NPs depended on the size of the particles and the loading content of curcumin in NPs. Furthermore, the curcumin loaded DBS-NPs exhibited better antimicrobial activity against S. aureus than E. coli. This could be due to the reason that the lipopolysaccharides in the cell wall of gram-negative bacteria could prevent bioactive molecules from reaching the cytoplasmic membrane.63 In-Vitro Cytotoxicity. The MTT assay was used to determine the cytotoxicity of DBS-NPs and curcumin loaded DBS-NPs; MEF cells were incubated with DBS-NPs and curcumin loaded



DBS-NPs in a range of concentration (0–500 g/mL) for 24 h, respectively. As shown in Figure 9, no significant differences between the viability of untreated cells and cells treated with DBS-NPs or curcumin loaded DBS-NPs were observed. Although the cell survival rate slightly decreased upon increasing the concentration of curcumin-loaded DBS-NPs, over 85% of the cells were viable after being incubated with the DBS-NPs or curcumin loaded DBS-NPs, indicating that DBS-NPs and curcumin loaded DBS-NPs did not exhibit visible cytotoxicity within the concentration tested. These results were consistent with previously reported that the nontoxic effect of SNPs on cells.26,65 Additionally, these results indicated that DBS-NPs and curcumin-loaded DBS-NPs had good biocompatibility and no toxicity. In summary, we successfully obtained the refined DBS-NPs with an average DP of 13, 17, 20, and 24 via nanoprecipitation. At the same time, the influence of the average DP values and reaction temperatures on the size, morphology, crystal structure, and digestibility of the refined DBS-NPs were evaluated, as well as the effects of their encapsulation as nano-carriers on the antibacterial and antioxidant activities of curcumin. When the DP was higher than 10, spherical DBS-NPs with a controlled size of 40200 nm were obtained; no nanoparticles were obtained. Furthermore, the morphology of the DBS-NPs did not seem to be related to the reaction temperature. Within the test range of average DP, we created a hypothetical polynomial model (R2 = 0.98 for 25 C; R2 = 0.984 for 60 C; R2 = 0.958 for 90 C) to reveal the relationship between the DP of refined DBS and RC of DBS-NPs. Moreover, the DBS with an average DP of 20 was more favorable to form a single-helical structure and oriented easily to form small and well-distributed DBS-NPs with a perfect crystalline structure within the range of the measured average DP. In comparison to the responding DBS, the DBS-NPs showed remarkable increase in


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the sum of SDS and RS levels. Furthermore, as nano-carriers, DBS-NPs significantly (p < 0.05) enhanced the antibacterial and antioxidant activities of curcumin, and improved the antioxidant stability of curcumin when exposed to UV light or heat treatments. We hope that the green and biodegradable DBS-NPs, as fillers, water-adsorbents, and carriers (bioactive compounds, pesticides, etc.), could be useful in the field of functional food, medicine agriculture, and environmental engineering. Supporting Information HPSEC chromatograms; TEM images of debranched starch nanoparticles (PDF) ACKNOWLEDGMENTS This work was supported by National Key Research and Development Program of China (No. 2018YFC1602101) and National First-Class Discipline Program of Food Science and Technology (JUFSTR20180203). Notes The authors declare no competing financial interest.



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Table Captions Table 1 the mean size and polydispersity index (PDI) of debranched starch nanoparticles (DBS-NPs) with different degree of polymerization that formed at 25C, 60C, and 90C, respectively. Table 2. Digestibility of waxy and normal maize starches, DBS, and DBS-NPs. Table 3. Encapsulation efficiency (EE) and loading capacity (LC) of DBS-NPs for curcumin.


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Figure Captions Figure 1. Transmission electron microscopic (TEM) images of debranched starch nanoparticles (DBS-NPs) with different average degree of polymerization (DP) that formed at 25C, 60C, and 90C, respectively. Figure 2. Size distribution patterns of debranched starch nanoparticles (DBS-NPs) with different average degree of polymerization that formed at 25C (a), 60C (b), and 90C (c), respectively. Figure 3. X-ray diffractogram and the relative crystallinity (RC) of DBS-NPs with different average DP (13-24) prepared at 25 C (a), 60 C (b), and 90 C (c). Figure 4. Relative crystallinity of DBS-NPs a function of DP values of DBS. DBS-NPs the prepared at 25C (A, R2 = 0.980), 60C (B, R2 = 0.984), and 90C (C, R2 = 0.958). Figure 5. FTIR patterns of DBS-NPs with different average DP (13-24) prepared at 25C (a), 60C (b), and 90C (c). Figure 6. IC50 values of DPPH radical scavenging activity of curcumin and curcumin loaded DBS-NPs with different average DP values. Figure 7. IC50 values of DPPH radical scavenging activity of curcumin and curcumin loaded DBS-NPs with different average DP values after treatments with temperature (a) and ultraviolet light (b). Figure 8. Growth curves of E. coli (A, B, and C) and S. aureus (a, b, and c) exposed to curcumin loaded DBS-NPs. A, a: DBS-NPs prepared at 25C (A, a), 60C (B, b), and 90C (C, c). Figure 9. Cytotoxicity of MEF treated with the different average DPs (a, 13; b, 17; c, 20; d, 24) of DBS-NPs and curcumin loaded DBS-NPs at different concentrations.



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Table 1 the mean size and polydispersity index (PDI) of debranched starch nanoparticles (DBS-NPs) with different average degree of polymerization that formed at 25C, 60C, and 90C, respectively. 25 C Samples DBS-NPs9 DBS-NPs13 DBS-NPs17 DBS-NPs20 DBS-NPs24

60 C

90 C

Mean size (nm)

PDI

Mean size (nm)

PDI

Mean size (nm)

PDI













118.89.3Ac

0.3020.08Cd

171.64.7Bc

0.2330.06Bc

190.86.8Cc

0.1650.14Ab

81.36.1Ab

0.2320.14Bab

143.69.2Bb

0.3230.04Cd

172.75.2Cb

0.1170.09Aa

46.312.2Aa

0.2130.10Ba

96.710.3Ba

0.2110.11Bab

143.37.5Ca

0.1590.09Ab

172.68.2Ad

0.2690.18Bc

348.97.9Cd

0.2050.10Ca

223.19.3Bd

0.1240.07Aa

“—” represents no endothermic peak detected. Values mean ± SD indicates the replicates of three experiments. Different letters in the same column (a–d) or row (A–C) indicate significant differences (upper case) (p < 0.05).


Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Digestibility of waxy and normal maize starches, DBS, and DBS-NPs. Reaction temperature

Control

25 C

60 C

90 C

Sample

RDS (%)

SDS (%)

RS (%)

Waxy maize starch

28.12.0i

64.41.2m

7.51.2a

Maize starch

76.81.1m

13.01.9a

10.20.8b

DBS-13

49.41.7l

33.11.4c

12.51.1c

DBS-17

16.60.8c

32.71.1c

48.71.3k

DBS-20

18.70.5e

28.90.9b

52.41.3l

DBS-24

20.20.9fg

27.50.7b

53.31.5lm

DBS-NPs-13

41.21.1k

39.00.7e

19.80.8d

DBS-NPs-17

28.20.6i

46.30.5i

25.50.7f

DBS-NPs-20

16.80.6c

50.40.7l

32.81.0h

DBS-NPs-24

25.10.4h

47.60.6j

27.30.3g

DBS-NPs-13

35.10.3j

42.30.5g

23.60.5e

DBS-NPs-17

19.41.0ef

48.30.8jk

32.30.6h

DBS-NPs-20

14.50.4b

40.30.7f

45.20.3k

DBS-NPs-24

20.40.7fg

40.30.4f

39.30.5i

DBS-NPs-13

28.41.0i

43.90.4h

27.70.5g

DBS-NPs-17

17.40.4cd

40.80.2f

41.80.7j

DBS-NPs-20

9.20.3a

35.30.4d

55.51.0n

DBS-NPs-24

18.40.3e

39.80.7e

41.80.4j

Data are expressed as the mean ± standard deviation (n=3). Values of means followed by different lowercase letters in the same column are significantly different (p < 0.05).



Page 32 of 42

Table 3. Encapsulation efficiency (EE) and loading capacity (LC) of DBS-NPs for curcumina Sample DBS-NPs13 DBS-NPs17 DBS-NPs20 DBS-NPs24

EE (%)

LC (%)

25 C

60 C

90 C

25 C

60 C

90 C

80.830.42Ad

82.210.45Bb

82.900.39Ba

10.890.31Aa

11.950.40Bb

12.110.49Ba

86.010.29Ab

87.130.62Bc

87.890.61Bb

15.900.42Ac

16.510.37ABc

17.080.27Bb

87.190.62Aa

92.490.55Bd

91.610.72Bd

16.530.40Acd

20.410.31Bd

20.170.61Bd

83.710.55Bc

78.031.11Aa

90.090.24Bc

12.880.36Bb

9.561.03Aa

18.290.71Cc

aValues represent the mean ± standard deviation of triplicate tests. Different letters in the same column (a–d) or row (A–C) indicate significant differences (upper case) (p < 0.05).


Page 33 of 42

a

c

d

25 C

b 200 nm

200 nm

200 nm

200 nm

g

h

200 nm

200 nm

200 nm

i

j

l

m

200 nm

200 nm

200 nm

200 nm

f

60 C

e

200 nm

90 C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DP = 13

DP = 17

DP = 20

DP = 24

Figure 1. Transmission electron microscopic (TEM) images of debranched starch nanoparticles (DBS-NPs) with different average degree of polymerization (DP) that formed at 25C, 60C, and 90C, respectively.



Page 34 of 42

DBS-NPs-13 DBS-NPs-17 DBS-NPs-20 DBS-NPs-24

a

1

10

100

1000

Size (nm) DBS-NPs-13 DBS-NPs-17 DBS-NPs-20 DBS-NPs-24

b

1

10

100

Size (nm)

1000 2000

DBS-NPs-13 DBS-NPs-17 DBS-NPs-20 DBS-NPs-24

c

100

150

200

250

300

Size (nm)

Figure 2. Size distribution patterns of debranched starch nanoparticles (DBS-NPs) with different average degree of polymerization that formed at 25C (a), 60C (b), and 90C (c), respectively.


Page 35 of 42

DBS-NPs- 9 DBS-NPs- 13 DBS-NPs- 17 DBS-NPs- 20 DBS-NPs- 24

5

10

15

20

25

30

35

b Intensity (%)

Intensity (%)

a

5

40

10

15

2 ()

d

c

20

25

2 ()

30

35

70

RC (%)

50 40 30 20 10 0

5

10

15

20

2 ()

25

30

35

40

40

25 C 60 C 90 C

80

60

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9

13

17

20

24

DP

Figure 3. X-ray diffractogram and the relative crystallinity (RC) of DBS-NPs with different average DP (13-24) prepared at 25C (a), 60C (b), and 90C (c).



a

70 60

RC (%)

50 40 30 20 y = -102.8641 + 15.30429x + (-0.36959)x2

10 0

b

2

(R = 0.98001)

5

10

15

c

70 60

25

80 70 60

RC (%)

40 30 20 y = -107.38146 + 16.57263x + (-0.4051)x2

10 0

20

DP

50

RC (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 42

(R2=0.98369)

5

10

15

20

25

50 40 30 20

y = -112.3751 + 17.65217x + (-0.43311)x2

10 0

2

(R = 0.95765)

5

DP

10

15

20

DP

Figure 4. Relative crystallinity of DBS-NPs a function of average DP values of DBS. DBS-NPs the prepared at 25C (A, R2 = 0.980), 60C (B, R2 = 0.984), and 90C (C, R2 = 0.958).


25

Page 37 of 42

DBS-NPs-13 DBS-NPs-20

a

b


3204.03

T (%)

3308.19 T (%)

3309.78

3205.87 3308.14

3312.63

3309.60

3314.76

4000

3500

3000

2500

2000

1500

1000

4000

500

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

Wavenumber (cm-1)

c 3299.98

T (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3302.62 3305.07 3306.72

4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1)

Figure 5. FTIR patterns of DBS-NPs with different average DP (13-24) prepared at 25C (a), 60C (b), and 90C (c).



60

25 C

a c c

IC50(M)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

40

d f

e

Page 38 of 42

60 C

90 C

c fg

f

hi h

b

i

20

0 Curcumin DBS-NPs-13 DBS-NPs-17 DBS-NPs-20 DBS-NPs-24

Figure 6. IC50 values of DPPH radical scavenging activity of curcumin and curcumin loaded DBS-NPs with different average DP values.


Page 39 of 42

a

100

a

25 C

60 C

90 C

80

IC50(M)

c

b

cd e

60

ef

g

h

g

ig hi

g

hi

40 20 0 Curcumin DBS-NPs-13 DBS-NPs-17 DBS-NPs-20 DBS-NPs-24

b

120 a 100

IC50(g/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60

b b bc

b d de e

e

f f

e

f

40 20 0 Curcumin DBS-NPs-13 DBS-NPs-17 DBS-NPs-20 DBS-NPs-24

Figure 7. IC50 values of DPPH radical scavenging activity of curcumin and curcumin loaded DBS-NPs with different average DP values after treatments with temperature (a) and ultraviolet light (b).



Control DBS-NPs-20

1.2


DBS-NPs-17

a

1.2

1.0

1.0

0.8

0.8

OD600

OD600

A

0.6

0.4

0.2

0.2 0.0 0

5

10

15

20

25

30

35

40

45

Control DBS-NPs-20


50

0

5

10

15

20

Time (h)

Control DBS-NPs-20

1.2


DBS-NPs-17

b

1.2

1.0

1.0

0.8

0.8

0.6

0.4

0.2

0.2 0.0 0

5

10

15

20

25

30

35

40

45

Control DBS-NPs-20

Control DBS-NPs-20

1.2

50


DBS-NPs-17

c

0

5

10

15

20

0.8

OD600

0.8 0.6

0.4

0.2

0.2

10

15

20

25

30

45

50

35

40

45

50

DBS-NPs-17

25

30

35

40

45

50


DBS-NPs-17

0.6

0.4

5

Control DBS-NPs-20

1.2 1.0

0

40

Time (h)

1.0

0.0

35


Time (h)

C

30

0.6

0.4

0.0

25

Time (h)

OD600

OD600

B

DBS-NPs-17

0.6

0.4

0.0

OD600

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 42

0.0

0

5

10

Time (h)

15

20

25

30

35

40

45

50

Time (h)

Figure 8. Growth curves of E. coli (A, B, and C) and S. aureus (a, b, and c) exposed to curcumin loaded DBS-NPs. A, a: DBS-NPs prepared at 25C (A, a), 60C (B, b), and 90C (C, c).


Page 41 of 42

DBS-NPs Curcumin loaded DBS-NPs

110 100 90 80 70 60 50 40 30 20 10 0

b

110 100

Cell viability (%)

Cell viability (%)

a

90 80 70 60 50 40 30 20 10

0

50

100

150

300

0

500

110 100 90 80 70 60 50 40 30 20 10 0

d Cell viability (%)

c

0

50

100

150

300

Concentration (μg/mL)

500

110 100 90 80 70 60 50 40 30 20 10 0

100

50

0

150

300

500



Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

50

100

150

300


500

Figure 9. Cytotoxicity of MEF treated with the different average DPs (a, 13; b, 17; c, 20; d, 24) of DBS-NPs and curcumin loaded DBS-NPs at different concentrations.



Page 42 of 42

Graphic Abstract Synopsis Average DPs influence on size and morphology of DBS-NPs, and the nontoxic and biodegradable nanoparticles markedly enhance curcumin antioxidant activity. Ethanol

Enzymolysis

DP = 9 Dropwise

Curcumin

200 nm

Antioxidant activity Encapsulation

DP = 17 Ethanol precipitation Native starch DP = 24

200 nm


Stability

Effects of Degree of Polymerization on Size, Crystal Structure, and

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