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Influence of ultrasonic treatment on formation of amylose nanoparticles prepared by nanoprecipitation
Carbohydrate Polymers 157 (2017) 1413–1418
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Influence of ultrasonic treatment on formation of amylose nanoparticles prepared by nanoprecipitation Yanjiao Chang, Xiaoxia Yan, Qian Wang, Lili Ren, Jin Tong, Jiang Zhou ∗ Key laboratory of Bionic Engineering (Ministry of Education), College of Biological and Agricultural Engineering, Jilin University, Changchun 130022, China
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Article history: Received 17 June 2016 Received in revised form 3 November 2016 Accepted 6 November 2016 Available online 8 November 2016 Keywords: Amylose Ultrasonic treatment Viscosity Molecular weight Nanoparticles Precipitation
a b s t r a c t Amylose aqueous solutions (1 wt%, 3 wt% and 5 wt%) were treated with 100W ultrasound for various periods of time and used to prepare amylose nanoparticles (ANPs) via nanoprecipitation by adding the amylose solutions drop-wise into absolute ethanol. Viscosity average molecular weight and size distribution of the ultrasonic treated amylose were determined by measuring intrinsic viscosity and using size exclusion chromatography, respectively. The ANPs were characterized using dynamic light scattering, scanning electron microscopy, and X-ray diffraction. Results showed that the ultrasonic treatments led to decrease in viscosity of amylose solutions, scission of amylose chains, and narrowing of size distribution of amylose molecules, which gave rise to smaller ANPs with more uniform size. The effect of the ultrasonic treatments on crystalline structure of the ANPs was negligible. This study indicates that ultrasonic treatment can be utilized to prepare smaller starch nanoparticles through nanoprecipitation with higher efficiency and lower cost. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Starch is a mixture of two main components: amylose and amylopectin. Amylose is essentially a linear polymer consisting of glucose units linked by ␣-(1 → 4) glycosidic bonds, slightly branched by ␣-(1 → 6) linkages. Amylopectin is a highly branched polymer constituted of relatively short branches of ␣-d-(1 → 4) glycopyranose that are interlinked by ␣-d-(1 → 6) glycosidic linkages (Dufresne, 2014). In recent years, starch nanoparticles have been attracted considerable attention because of their great potential for applications in various aspects, such as reinforcements for nanocomposites, delivery systems for drugs, nanoencapsulations for food ingredients and additives, and adsorbents for heavy metal ions (Le Corre & Angellier-Coussy, 2014; Kim, Park, & Lim, 2015). Among the methods to produce starch nanoparticles, nanoprecipitation is a simple and fast one. The precipitation process involves a successive addition of a dilute starch solution into a nonsolvent or inversely. Previous works showed that the concentration of starch solution is a key factor to influence the size of precipitated nanoparticles (Dong, Chang, Wang, Tong, & Zhou, 2015; Tan et al., 2009): high starch concentration leads to the formation of highly viscous solution and high viscosity of starch solution hampers dif-
∗ Corresponding author. E-mail address: [email protected] (J. Zhou). http://dx.doi.org/10.1016/j.carbpol.2016.11.019 0144-8617/© 2016 Elsevier Ltd. All rights reserved.
fusion of starch solution toward non-solvent, which in turn results in larger particles. It was also found that the ratio of solvent to non-solvent influenced the size of the precipitated starch particles (Chin, Pang, & Tay, 2011). Thus, in order to synthesize smaller starch nanoparticles through precipitation, highly diluted starch solutions and larger volume of non-solvent have to be used, which inevitably decrease production efficiency and increase cost. Besides starch concentration, starch molecular weight also has contribution to the viscosity of the starch solution (Peres, Leite, & Silveira, 2015). Therefore, starch molecular weight should have effect on nanoparticle synthesis by precipitation. However, as far as our literature survey could ascertain, no investigation about the influence of starch molecular weight on the production of starch nanoparticles via precipitation has been reported. On the other hand, because molecular weights of starch are diverse and depend on the botanical origin of starch as well as the growth condition of the plants from which starch is obtained, understanding the effect of starch molecular weight on nanoprecipitation results could help to optimize and control the formation of starch nanoparticles. It was demonstrated that ultrasonic treatment of chitosan and starch aqueous solutions is an efficient procedure to reduce molecular weight of these polysaccharides due to the intense mechanical and chemical effects associated with cavitation (CzechowskaBiskup, Rokita, Lotfy, Ulanski, & Rosiak, 2005). It was reported that the viscosity of starch solution and molecular weight of starch were reduced by ultrasonic treatment (Iida, Tuziuti, Yasui, Towata,
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& Kozuka, 2008). The ultrasonic treatment for starch modification has advantages such as simple, environmentally friendly, and effective. Most physical and chemical effects caused by the ultrasound are generally attributed to cavitation, which is the collapse of microbubbles that burst and propagate as a sound wave through the solution (Langton, 1969; Price & Smith, 1993). The shear forces created due to the collapse of the bubbles may break covalent bonds in polymeric materials (Zhang et al., 2013; Farzi, Saffari, EmamDjomeh, & Mohammadifar, 2011; Peres et al., 2015). Compared to amylopectin, amylose is a relatively simple and linear molecule. It is better to start with amylose to investigate effect of starch molecular weight on starch nanoparticle formation via precipitation. In this paper, ultrasound was used to treat amylose solutions with different concentrations aimed at lowering viscosity of the solutions without decreasing concentration. Through examining the size, morphology and structure of the amylose nanoparticles (ANPs) prepared via precipitation by using ultrasonic treated amylose solutions, we hope to clarify the effect of ultrasonic treatment on formation of ANPs.
The size of the prepared ANPs was measured through dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK). The samples were prepared by dispersing the ANPs in deionized water at a concentration of 0.1 mg/ml. The Z-average size (d. nm) and polydispersity index (PDI) were measured and used to characterize the particle size and size distribution. The measurements were performed six times for each sample, and the mean values were reported. Morphologies of the ANPs were observed using a Field Emission Scanning Electron Microscope (ZEISS, MERLIN Compact, Germany). Powder samples were mounted on specimen stubs with carbon black tape and then sputter-coated with gold before observation. The crystalline structure of the ANPs was analyzed by using a Rigaku D/max-2500 X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) with Cu-Ka radiation (= 1.542 Å) at a target voltage of 40 kV and current of 250 mA. The scanning range and rate were 4.00–40.00◦ (2) and 2◦ /min, respectively.
2. Experimental
2.6. Characterization of molecular weight
2.1. Materials
Viscosity average molecular weight (Mv ) of the amylose with different ultrasonic treating times was calculated from measured intrinsic viscosity () using the classical Mark–Houwink relationship (Baxter, Zivanovic, & Weiss, 2005):
Amylose with a purity of 99.5% was purchased from Shaanxi Tianwei Biological Production Co. Ltd. (Xi’an, China). Absolute ethanol was purchased from Beijing Chemical Works (Beijing, China). Dimethyl-sulfoxide (DMSO) was produced by Merck Co. Inc. (Kilsyth, VIC, Australia). LiBr was purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Pullulan standards with known molecular weights were purchased from Polymer Standards Service (PSS) GmbH (Mainz, Germany). 2.2. Ultrasonic treatment of amylose solution Amylose suspension with a certain concentration was prepared by mixing amylose with distilled water in a round bottom flask. The suspension was heated to 100 ◦ C in a water bath and held at this temperature for 60 min with continuous stirring at 150 rpm. After the obtained amylose solution was cooled down to room temperature, certain amount (60 g) of the solution was transferred to a 100 mL plastic beaker and treated by ultrasound for different periods of time using a 22KHz ultrasound generator (HN-1000Y, Shanghai Hanno Instrument Corp., China) equipped with a tapered horn tip (10 mm end diameter). The power output of 100 W and 2/4 s on/off pulses were applied during the ultrasonic treatments to minimize heat generation. The temperature of the amylose solution after the ultrasonic treatments was approximately 50 ◦ C. 2.3. Viscosity measurements Apparent viscosity of the amylose solutions after ultrasonic treatments was measured at 20 ◦ C using a viscometer (RVDV-III, Brookfield Engineering Laboratories, Middleboro, USA) with the spindle rotation at 180 rpm. The measurements were done in triplicates for each sample and average values were reported. 2.4. Preparation of amylose nanoparticles (ANPs) ANPs were prepared by adding certain amount of the amylose solution drop-wise into absolute ethanol which was continually agitated with ultrasound. The resulting mixture was then centrifuged at 4000 × g for 5 min. The precipitated ANPs were obtained by removing the supernatant and rinsed 2 times by centrifugation with absolute ethanol. The final product was freeze dried.
2.5. Characterization of ANPs
= Km M␣ (1) where Km and ␣ are the Mark–Houwink parameters which depend on the temperature and solvent type. In this study, Km = 0.367 × 10−2 ml/g and ␣ = 0.829 were used (Cornell, Rix, & McGrane, 2002). The intrinsic viscosities were determined by applying Huggins extrapolation to zero concentration procedure (Perevyazko et al., 2012). The samples for the intrinsic viscosity measurements were prepared by dissolving the ANPs (obtained by using the 3 wt% amylose solutions with different ultrasonic treating times) in DMSO and heated in boiling water for 30 min then filtered using 0.08 m membrane. By using an Ubbelohde viscometer (SYD-265, Shanghai Changji Geological Instrument Crop., China), the flow time of the amylose DMSO solutions with four different concentrations was measured at 23± 0.1 ◦ C. From the flow time (t) of the amylose solutions and that (t0 ) of the solvent (DMSO), relative viscosity (r = t/t0 ) was obtained. Specific viscosity (sp ) was calculated from the relation sp = r - 1(Nayak & Singh, 2001). Then, the plots of the reduced viscosity (sp /C) versus the concentration (C) for the investigated samples were obtained. The intrinsic viscosity was determined from the intercept of the reduced viscosity by extrapolating the amylose concentration C to zero. Size-exclusion chromatography (SEC) was also used to analyze the molecular size distribution of the amylose before and after the ultrasonic treatments. The SEC system setup (Agilent Technologies 1260 Infinity) consisted of an isocratic pump, auto sampler without temperature regulation, an online degasser, a refractive index (RID) detector (Optilab T-Rex, Wyatt Technology Corp., USA) and a multi-angle laser light scattering (MALLS) detector (Wyatt DAWN Heleos II, Wyatt Technology Corp., USA). Astra 6 software was used for data processing. Freeze dried ANPs obtained from amylose solutions with different ultrasonic treating times were dissolved in DMSO containing 0.5% (w/w) LiBr at a concentration of 2 g/L in thermomixer at 80 ◦ C for 24 h during which the thermomixer was inverted by hand occasionally. The prepared solutions were centrifuged at 4000 × g for 10 min and the supernatants were transferred to SEC vials for analysis. The sample was injected into a GRAM precolumn with a flow rate of 0.3 mL/min and the separation was carried out with GRAM 100 and GRAM 1000 columns (Polymer
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Fig. 1. Viscosity of amylose aqueous solutions against ultrasonic treating time.
Standard Service, Mainz, Germany) set at 80 ◦ C. The relationship between SEC elution volume and hydrodynamic size, i.e., hydrodynamic volume Vh or hydrodynamic radius Rh , Vh = 4/3 Rh 3 (Vilaplana & Gilbert, 2010), was obtained using pullulan standards with molecular weights ranging from 342 to 2.35 × 106 , and molar mass of the amylose molecules was obtained from the hydrodynamic volume Vh or radius Rh using the Mark–Houwink equation (Vilaplana & Gilbert, 2010; Gilbert, Wu, Sullivan, Sumarriva, Ersch, & Hasjim, 2013; Wang et al., 2015). The used Mark–Houwink parameters Km and ␣ for pullulan in DMSO/LiBr solution at 80 ◦ C are 2.424 × 10−4 dL/g and 0.68, respectively (Wang et al., 2015; Cave, Seabrook, Gidley, & Gilbert, 2009).
3. Results and discussion 3.1. Effect of ultrasonic treatment on viscosity of amylose solutions Because starch solution with lower viscosity and higher concentration is beneficial to produce smaller nanoparticles at high efficiency via nanoprecipitation, it is desirable to reduce the viscosity of starch solutions with high concentration. Fig. 1 shows the changes in viscosity of amylose solutions with different concentrations against ultrasonic treating time. It was found that, for all the amylose solutions, viscosity decreased with increasing ultrasonic treating time. However, when concentration was low, such as 1 wt%, the effect of ultrasonic treatment was not significant, the viscosity only decreased about 50%, from 2.87 mPa s to 1.45 mPa s, after 30 min ultrasonic treatment. For the amylose solutions with higher concentration, the effect of ultrasonic treatment on viscosity was more significant. For instance, the viscosity of the 5 wt% amylose solution decreased about two orders of magnitude, from 1136 mPa s to 13.9 mPa s, after 30 min ultrasonic treatment. The higher the concentration of amylose solution was, the more significant the effect of ultrasonic treatment on viscosity. The above observations could be attributed to the contributions of entanglement and intermolecular interactions among amylose chains to viscosity of solution at high concentration. For the amylose solutions with high concentrations, the intense mechanical effect and heat induced by ultrasonic treatment could break the entanglements and intermolecular interactions of amylose chains which primarily result in the high viscosity of amylose solution,
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Fig. 2. Reduced viscosity of amylose DMSO solutions versus amylose concentration for the amyloses with different ultrasonic treating times.
therefore, the effect of ultrasonic treatment on viscosity reduction was more significant when concentration was high. Besides the above mechanism of viscosity decrease, amylose molecules in the solution may undergo degradation (chain scission) during ultrasonic treatment, which could also lead to decrease in solution viscosity. In the case of present study, both mentioned mechanisms may be operative. It was seen from Fig. 1 that, for the 30 min ultrasonic treatment, the decrease in viscosity in the initial 15 min was more significant than the later 15 min. This phenomenon was similar to the previous observation in ultrasonic treated maize starch solutions, the decrease of viscosity mainly occurred in the first 15 min treatment (Cheng, Chen, Liu, Ye, & Ke, 2010). These observations could be due to the characteristic feature of ultrasound-induced chain scission, i. e., there is a definite minimum chain length limiting the degradation process (Czechowska-Biskup et al., 2005). When the minimum chain length was reached, no further chain scission occurred, therefore the viscosity of amylose solution hardly decreased further. Although the properties of starch slurry subjected to ultrasonic treatments were described in the past (Iida et al., 2008; Zuo, Knoerzer, Mawson, Kentish, & Ashokkumar, 2009), much less is known on the mechanisms of ultrasound-induced starch chain scission in aqueous solution. 3.2. Molecular weight of amylose As mentioned above, ultrasonic treatment of amylose solution could cause scission of amylose chains. Viscosity average molecular weight, calculated from measured intrinsic viscosity, can be used to characterize the degradation of amylose molecules. Fig. 2 shows the changes of the reduced viscosity of amylose DMSO solutions against amylose concentration for the amyloses with 0 min, 2 min and 30 min ultrasonic treatments, respectively. The intrinsic viscosity of amylose without ultrasonic treatment was determined as 0.125, while the intrinsic viscosities of amylose with 2 min and 30 min ultrasonic treatments were 0.122 and 0.066, respectively. The viscosity average molecular weight of the amylose without ultrasonic treatment was 3.36 × 105 . After 2 min ultrasonic treatment, the viscosity average molecular weight of amylose decreased to 3.27 × 105 , and further down to 1.54 × 105 when the ultrasonic treatment was prolonged to 30 min. These results confirmed that the ultrasonic treatments did cause amylose chain scission.
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Fig. 3. Changes of molar masses versus hydrodynamic radius Rh of the amylose with different ultrasonic treatments.
Fig. 3 shows SEC molar mass distribution of the amyloses before and after ultrasonic treatments for 2 min and 30 min. The molar mass of the amylose without ultrasonic treatment ranged 2.4 × 106 − 4.5 × 108 g/mol, showing a broad distribution. After ultrasonic treatment, the distribution became narrower, and it was down to 3.2 × 105 − 6.0 × 106 g/mol when the ultrasonic treatment was carried out for 30 min. These results also indicated that the ultrasonic treatments led to amylose chain scission. The viscosity average molecular weight and SEC molar mass distribution contain different information, the exact consistency between the values obtained by the two different methods will not be necessary. However, both of the molecular weight measurements suggested that amylose chain scission, a degradation process, occurred during the ultrasonic treatments. It should be mentioned that the chain scission process indicated by the molecular weight reduction in this study showed a tendency similar to the previous work (Iida et al., 2008), in which high performance gel permeation chromatography (HPGPC) was used to measure molecular weight distribution of ultrasound treated starch. 3.3. Size of ANPs The viscosity of amylose solution is dependant on concentration and molecular weight of amylose, and it affects the size of ANPs prepared through precipitation. As presented above, the ultrasonic treatments decreased viscosity of amylose solution and reduced molecular weight of amylose. It is desirable to investigate the effect of the ultrasonic treatments to amylose solution on the size of ANPs formed in precipitation. Fig. 4 shows the mean sizes and PDIs of the ANPs prepared by precipitation (the volume ratio of amylose solution to absolute ethanol was 1:10) and using amylose solutions with different concentrations and treated with ultrasound for various periods of time. The data in Fig. 4 clearly indicated that the higher the concentration of amylose solution, the larger the ANPs and the greater the PDI. For the ANPs obtained from the 3 wt% and 5 wt% solutions without ultrasonic treatment, the values of PDI were around 0.7, suggesting that the samples had a very broad size distribution and might contain large particles or aggregates. The results in Fig. 4 also indicated that the effect of the ultrasonic treatment on the size of the ANPs was obvious. The particle size of ANPs decreased gradually with increasing ultrasonic treating time. For the ANPs obtained from the 1 wt% amylose solution, the mean size decreased from 206 nm to
Fig. 4. Mean sizes (column) and PDIs (solid square) of the ANPs prepared by using amylose solutions with different concentrations and ultrasonic treating times (the volume ratio of amylose solution to absolute ethanol was 1:10).
91 nm after the solution was treated with ultrasound for 15 min and further down to 82 nm after another 15 min ultrasonic treatment. For the ANPs obtained from the 5 wt% amylose solution, the mean size decreased from 337 nm to 196 nm and further to176 nm after the solution was treated 15 min and 30 min. The decrease in viscosity induced by the ultrasonic treatment might be responsible to the size reductions of the ANPs, because the viscosity decrease improved diffusion of amylose solution toward ethanol. However, the viscosity of the amylose solution was not the only parameter to control the size of the precipitated ANPs. Examining Figs. 1 and 4, it could be found that the viscosity of the 3 wt% amylose solution was very close to that of the 5 wt% amylose solution with 7 min ultrasonic treatment, but the mean sizes of the ANPs obtained from these solutions were 296 nm and 219 nm, respectively. It was thought that high concentration of polymer solution give rise to larger particle size not only because the high viscosity of the solution but also the number of polymer chains per unit volume of the solvent (Beck-Broichsitter, Rytting, Lebhardt, Wang, & Kissel, 2010; Legrand et al., 2007). As a consequence of the greater number of polymer chains per unit volume, the solvent diffusing into the non-solvent carries more polymer chains that aggregate and thus should form larger particles. Because high polymer concentration increases polymer–polymer interactions, this means more polymer chains remain associated during the diffusion process (Galindo-Rodriguez, Allemann, Fessi, & Doelker, 2004). However, the results of this study indicated that, through ultrasonic treatment, smaller ANPs can be obtained by using high concentration amylose solutions. This is probably because the ultrasonic treatment could break the interactions of amylose molecules and cut amylose chains. Scission of amylose chains reduces the probability of chain entanglement during precipitation, thus the shorter amylose chains aggregate and form smaller particles. In addition, the data in Fig. 4 also showed that the size of the ANPs obtained from the 1 wt% amylose solution without ultrasonic treatment was very close to that of the 5 wt% amylose solution with 30 min ultrasonic treatment, which means ultrasonic treatment could significantly raise production efficiency. Therefore, the ultrasonic treatment of amylose solution not only provides a route to prepare smaller ANPs but also an approach with higher production efficiency. Fig. 5 shows typical size distribution patterns of the ANPs prepared by precipitating the 3 wt% amylose solution with 15 min ultrasonic treatment into absolute ethanol at various volume ratios of amylose solution to absolute ethanol. It could be seen that the
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Fig. 5. Typical size distribution patterns of the ANPs obtained from the 3 wt% amylose solution with 15 min ultrasonic treatment at various volume ratios of amylose solution to absolute ethanol.
main peak shift to smaller size with increasing ethanol amount, but the shift was very small. The values of the mean size for the ANPs obtained at the volume ratios of 1:10, 1:5 and 1:3 were 126 nm, 131 nm and 138 nm, respectively. These results indicated that, after the ultrasonic treatment, the impact of the non-solvent amount on the size of the precipitated ANPs was not considerable, suggesting ANPs could be produced with less non-solvent. Therefore, the ultrasonic treatment of amylose solution provides a route to prepare ANPs via nanoprecipitation at lower cost. 3.4. Morphology of ANPs Fig. 6 shows the SEM micrographs as well as the corresponding size distribution patterns of the ANPs obtained from the 3 wt% amylose solution without ultrasonic treatment and the one with 30 min ultrasonic treatment, respectively. Although the ANPs were observed to be spherical in shape, the effect of ultrasonic treatment on morphological characteristics of the ANPs was significant. As shown in the SEM micrographs, the size of the ANPs obtained from the amylose solution with 30 min ultrasonic treatment was smaller than those prepared with the amylose solution without ultrasonic treatment. Moreover, the ANPs obtained from the ultrasonic treated solution was more uniform in size, there were few larger particles. These observations were consistent with the particle size distribution pattern measured by DLS which presented a main peak with narrower width. For the ANPs prepared using the amylose solution without ultrasonic treatment, the SEM micrograph showed that the particle size was not uniform and there were some large particles that were also reflected in the particle size distribution pattern (a strong peak at large size). This is understandable. Because the ultrasonic treatment broke the entanglements and interactions of amylose molecules and cut amylose chains, which could reduce nucleation of large aggregates from the entangled or interacted amylose chains and increase nucleation of small aggregates from shorter amylose chains during precipitation, thus the precipitated ANPs using the ultrasound treated solutions were smaller and more uniform in size.
Fig. 6. SEM micrographs and the corresponding size distribution patterns of the ANPs obtained from the 3 wt% amylose solution without ultrasonic treatment (a) and the one with 30 min ultrasonic treatment (b). The volume ratio of amylose solution to absolute ethanol was 1:10.
3.5. XRD analysis Fig. 7 shows X-ray diffraction patterns of the ANPs obtained from amylose solutions with different concentrations and ultra-
Fig. 7. X-ray diffraction patterns of the ANPs obtained from amylose solutions with different concentrations and ultrasonic treating times.
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sonic treating times. Examining the XRD patterns in Fig. 7, there were very weak diffraction peaks at 13.8◦ , 18.9◦ and 23.7◦ for all the ANPs, suggesting that the precipitated ANPs possess the V-type crystalline structure. Because there was no obvious difference in peak position and intensity for the XRD patterns of the ANPs obtained from the solutions with and without ultrasonic treatments, it could be concluded that the effect of the ultrasonic treatments on the structure and crystallinity of the precipitated ANPs was negligible. 4. Conclusions Ultrasonic treatment to amylose solution decreased solution viscosity and caused scission of amylose chains, which is beneficial to prepare ANPs through precipitation technique. The ANPs obtained from the solutions with ultrasonic treatments were smaller and more uniform compared to those from the solutions without ultrasonic treatment. The effect of the ultrasonic treatment on the structure and crystallinity of the precipitated ANPs was negligible. The findings of this study indicate that ultrasonic treatment of starch solution not only offers a convenient and easily controllable methodology to prepare smaller starch nanoparticles through precipitation, but also provides a route to raise production efficiency and lower cost of starch nanoparticles preparation by using high concentration solution and less amount of non-solvent. Acknowledgement The authors are grateful to financial support from the National Natural Science Foundation of China (51273083). References Baxter, S., Zivanovic, S., & Weiss, J. (2005). Molecular weight and degree of 310 acetylation of high-intensity ultrasonicated chitosan. Food Hydrocolloids, 19, 821–830. Beck-Broichsitter, M., Rytting, E., Lebhardt, T., Wang, X., & Kissel, T. (2010). Preparation of nanoparticles by solvent displacement for drug delivery: A shift in the ouzo region upon drug loading. European Journal of Pharmaceutical Sciences, 41, 244–253. Cave, R. A., Seabrook, S. A., Gidley, M. J., & Gilbert, R. G. (2009). Characterization of starch by size-exclusion chromatography: The limitations imposed by shear scission. Biomacromolecules, 10, 2245–2253. Cheng, W., Chen, J., Liu, D., Ye, X., & Ke, F. (2010). Impact of ultrasonic treatment on properties of starch film-forming dispersion and the resulting films. Carbohydrate Polymers, 81, 707–711. Chin, S. F., Pang, S. C., & Tay, S. H. (2011). Size controlled synthesis of starch nanoparticles by a simple nanoprecipitation method. Carbohydrate Polymers, 86, 1817–1819. Cornell, H. J., Rix, C. J., & McGrane, S. J. (2002). Viscometric properties of solutions of amylose and amylopectin in aqueous potassium thiocyanate. Starch-Stärke, 54, 517–526.
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Influence of ultrasonic treatment on formation of amylose nanoparticles prepared by nanoprecipitation Yanjiao Chang, Xiaoxia Yan, Qian Wang, Lili Ren, Jin Tong, Jiang Zhou ∗ Key laboratory of Bionic Engineering (Ministry of Education), College of Biological and Agricultural Engineering, Jilin University, Changchun 130022, China
a r t i c l e
i n f o
Article history: Received 17 June 2016 Received in revised form 3 November 2016 Accepted 6 November 2016 Available online 8 November 2016 Keywords: Amylose Ultrasonic treatment Viscosity Molecular weight Nanoparticles Precipitation
a b s t r a c t Amylose aqueous solutions (1 wt%, 3 wt% and 5 wt%) were treated with 100W ultrasound for various periods of time and used to prepare amylose nanoparticles (ANPs) via nanoprecipitation by adding the amylose solutions drop-wise into absolute ethanol. Viscosity average molecular weight and size distribution of the ultrasonic treated amylose were determined by measuring intrinsic viscosity and using size exclusion chromatography, respectively. The ANPs were characterized using dynamic light scattering, scanning electron microscopy, and X-ray diffraction. Results showed that the ultrasonic treatments led to decrease in viscosity of amylose solutions, scission of amylose chains, and narrowing of size distribution of amylose molecules, which gave rise to smaller ANPs with more uniform size. The effect of the ultrasonic treatments on crystalline structure of the ANPs was negligible. This study indicates that ultrasonic treatment can be utilized to prepare smaller starch nanoparticles through nanoprecipitation with higher efficiency and lower cost. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Starch is a mixture of two main components: amylose and amylopectin. Amylose is essentially a linear polymer consisting of glucose units linked by ␣-(1 → 4) glycosidic bonds, slightly branched by ␣-(1 → 6) linkages. Amylopectin is a highly branched polymer constituted of relatively short branches of ␣-d-(1 → 4) glycopyranose that are interlinked by ␣-d-(1 → 6) glycosidic linkages (Dufresne, 2014). In recent years, starch nanoparticles have been attracted considerable attention because of their great potential for applications in various aspects, such as reinforcements for nanocomposites, delivery systems for drugs, nanoencapsulations for food ingredients and additives, and adsorbents for heavy metal ions (Le Corre & Angellier-Coussy, 2014; Kim, Park, & Lim, 2015). Among the methods to produce starch nanoparticles, nanoprecipitation is a simple and fast one. The precipitation process involves a successive addition of a dilute starch solution into a nonsolvent or inversely. Previous works showed that the concentration of starch solution is a key factor to influence the size of precipitated nanoparticles (Dong, Chang, Wang, Tong, & Zhou, 2015; Tan et al., 2009): high starch concentration leads to the formation of highly viscous solution and high viscosity of starch solution hampers dif-
∗ Corresponding author. E-mail address: [email protected] (J. Zhou). http://dx.doi.org/10.1016/j.carbpol.2016.11.019 0144-8617/© 2016 Elsevier Ltd. All rights reserved.
fusion of starch solution toward non-solvent, which in turn results in larger particles. It was also found that the ratio of solvent to non-solvent influenced the size of the precipitated starch particles (Chin, Pang, & Tay, 2011). Thus, in order to synthesize smaller starch nanoparticles through precipitation, highly diluted starch solutions and larger volume of non-solvent have to be used, which inevitably decrease production efficiency and increase cost. Besides starch concentration, starch molecular weight also has contribution to the viscosity of the starch solution (Peres, Leite, & Silveira, 2015). Therefore, starch molecular weight should have effect on nanoparticle synthesis by precipitation. However, as far as our literature survey could ascertain, no investigation about the influence of starch molecular weight on the production of starch nanoparticles via precipitation has been reported. On the other hand, because molecular weights of starch are diverse and depend on the botanical origin of starch as well as the growth condition of the plants from which starch is obtained, understanding the effect of starch molecular weight on nanoprecipitation results could help to optimize and control the formation of starch nanoparticles. It was demonstrated that ultrasonic treatment of chitosan and starch aqueous solutions is an efficient procedure to reduce molecular weight of these polysaccharides due to the intense mechanical and chemical effects associated with cavitation (CzechowskaBiskup, Rokita, Lotfy, Ulanski, & Rosiak, 2005). It was reported that the viscosity of starch solution and molecular weight of starch were reduced by ultrasonic treatment (Iida, Tuziuti, Yasui, Towata,
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& Kozuka, 2008). The ultrasonic treatment for starch modification has advantages such as simple, environmentally friendly, and effective. Most physical and chemical effects caused by the ultrasound are generally attributed to cavitation, which is the collapse of microbubbles that burst and propagate as a sound wave through the solution (Langton, 1969; Price & Smith, 1993). The shear forces created due to the collapse of the bubbles may break covalent bonds in polymeric materials (Zhang et al., 2013; Farzi, Saffari, EmamDjomeh, & Mohammadifar, 2011; Peres et al., 2015). Compared to amylopectin, amylose is a relatively simple and linear molecule. It is better to start with amylose to investigate effect of starch molecular weight on starch nanoparticle formation via precipitation. In this paper, ultrasound was used to treat amylose solutions with different concentrations aimed at lowering viscosity of the solutions without decreasing concentration. Through examining the size, morphology and structure of the amylose nanoparticles (ANPs) prepared via precipitation by using ultrasonic treated amylose solutions, we hope to clarify the effect of ultrasonic treatment on formation of ANPs.
The size of the prepared ANPs was measured through dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK). The samples were prepared by dispersing the ANPs in deionized water at a concentration of 0.1 mg/ml. The Z-average size (d. nm) and polydispersity index (PDI) were measured and used to characterize the particle size and size distribution. The measurements were performed six times for each sample, and the mean values were reported. Morphologies of the ANPs were observed using a Field Emission Scanning Electron Microscope (ZEISS, MERLIN Compact, Germany). Powder samples were mounted on specimen stubs with carbon black tape and then sputter-coated with gold before observation. The crystalline structure of the ANPs was analyzed by using a Rigaku D/max-2500 X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) with Cu-Ka radiation (= 1.542 Å) at a target voltage of 40 kV and current of 250 mA. The scanning range and rate were 4.00–40.00◦ (2) and 2◦ /min, respectively.
2. Experimental
2.6. Characterization of molecular weight
2.1. Materials
Viscosity average molecular weight (Mv ) of the amylose with different ultrasonic treating times was calculated from measured intrinsic viscosity () using the classical Mark–Houwink relationship (Baxter, Zivanovic, & Weiss, 2005):
Amylose with a purity of 99.5% was purchased from Shaanxi Tianwei Biological Production Co. Ltd. (Xi’an, China). Absolute ethanol was purchased from Beijing Chemical Works (Beijing, China). Dimethyl-sulfoxide (DMSO) was produced by Merck Co. Inc. (Kilsyth, VIC, Australia). LiBr was purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Pullulan standards with known molecular weights were purchased from Polymer Standards Service (PSS) GmbH (Mainz, Germany). 2.2. Ultrasonic treatment of amylose solution Amylose suspension with a certain concentration was prepared by mixing amylose with distilled water in a round bottom flask. The suspension was heated to 100 ◦ C in a water bath and held at this temperature for 60 min with continuous stirring at 150 rpm. After the obtained amylose solution was cooled down to room temperature, certain amount (60 g) of the solution was transferred to a 100 mL plastic beaker and treated by ultrasound for different periods of time using a 22KHz ultrasound generator (HN-1000Y, Shanghai Hanno Instrument Corp., China) equipped with a tapered horn tip (10 mm end diameter). The power output of 100 W and 2/4 s on/off pulses were applied during the ultrasonic treatments to minimize heat generation. The temperature of the amylose solution after the ultrasonic treatments was approximately 50 ◦ C. 2.3. Viscosity measurements Apparent viscosity of the amylose solutions after ultrasonic treatments was measured at 20 ◦ C using a viscometer (RVDV-III, Brookfield Engineering Laboratories, Middleboro, USA) with the spindle rotation at 180 rpm. The measurements were done in triplicates for each sample and average values were reported. 2.4. Preparation of amylose nanoparticles (ANPs) ANPs were prepared by adding certain amount of the amylose solution drop-wise into absolute ethanol which was continually agitated with ultrasound. The resulting mixture was then centrifuged at 4000 × g for 5 min. The precipitated ANPs were obtained by removing the supernatant and rinsed 2 times by centrifugation with absolute ethanol. The final product was freeze dried.
2.5. Characterization of ANPs
= Km M␣ (1) where Km and ␣ are the Mark–Houwink parameters which depend on the temperature and solvent type. In this study, Km = 0.367 × 10−2 ml/g and ␣ = 0.829 were used (Cornell, Rix, & McGrane, 2002). The intrinsic viscosities were determined by applying Huggins extrapolation to zero concentration procedure (Perevyazko et al., 2012). The samples for the intrinsic viscosity measurements were prepared by dissolving the ANPs (obtained by using the 3 wt% amylose solutions with different ultrasonic treating times) in DMSO and heated in boiling water for 30 min then filtered using 0.08 m membrane. By using an Ubbelohde viscometer (SYD-265, Shanghai Changji Geological Instrument Crop., China), the flow time of the amylose DMSO solutions with four different concentrations was measured at 23± 0.1 ◦ C. From the flow time (t) of the amylose solutions and that (t0 ) of the solvent (DMSO), relative viscosity (r = t/t0 ) was obtained. Specific viscosity (sp ) was calculated from the relation sp = r - 1(Nayak & Singh, 2001). Then, the plots of the reduced viscosity (sp /C) versus the concentration (C) for the investigated samples were obtained. The intrinsic viscosity was determined from the intercept of the reduced viscosity by extrapolating the amylose concentration C to zero. Size-exclusion chromatography (SEC) was also used to analyze the molecular size distribution of the amylose before and after the ultrasonic treatments. The SEC system setup (Agilent Technologies 1260 Infinity) consisted of an isocratic pump, auto sampler without temperature regulation, an online degasser, a refractive index (RID) detector (Optilab T-Rex, Wyatt Technology Corp., USA) and a multi-angle laser light scattering (MALLS) detector (Wyatt DAWN Heleos II, Wyatt Technology Corp., USA). Astra 6 software was used for data processing. Freeze dried ANPs obtained from amylose solutions with different ultrasonic treating times were dissolved in DMSO containing 0.5% (w/w) LiBr at a concentration of 2 g/L in thermomixer at 80 ◦ C for 24 h during which the thermomixer was inverted by hand occasionally. The prepared solutions were centrifuged at 4000 × g for 10 min and the supernatants were transferred to SEC vials for analysis. The sample was injected into a GRAM precolumn with a flow rate of 0.3 mL/min and the separation was carried out with GRAM 100 and GRAM 1000 columns (Polymer
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Fig. 1. Viscosity of amylose aqueous solutions against ultrasonic treating time.
Standard Service, Mainz, Germany) set at 80 ◦ C. The relationship between SEC elution volume and hydrodynamic size, i.e., hydrodynamic volume Vh or hydrodynamic radius Rh , Vh = 4/3 Rh 3 (Vilaplana & Gilbert, 2010), was obtained using pullulan standards with molecular weights ranging from 342 to 2.35 × 106 , and molar mass of the amylose molecules was obtained from the hydrodynamic volume Vh or radius Rh using the Mark–Houwink equation (Vilaplana & Gilbert, 2010; Gilbert, Wu, Sullivan, Sumarriva, Ersch, & Hasjim, 2013; Wang et al., 2015). The used Mark–Houwink parameters Km and ␣ for pullulan in DMSO/LiBr solution at 80 ◦ C are 2.424 × 10−4 dL/g and 0.68, respectively (Wang et al., 2015; Cave, Seabrook, Gidley, & Gilbert, 2009).
3. Results and discussion 3.1. Effect of ultrasonic treatment on viscosity of amylose solutions Because starch solution with lower viscosity and higher concentration is beneficial to produce smaller nanoparticles at high efficiency via nanoprecipitation, it is desirable to reduce the viscosity of starch solutions with high concentration. Fig. 1 shows the changes in viscosity of amylose solutions with different concentrations against ultrasonic treating time. It was found that, for all the amylose solutions, viscosity decreased with increasing ultrasonic treating time. However, when concentration was low, such as 1 wt%, the effect of ultrasonic treatment was not significant, the viscosity only decreased about 50%, from 2.87 mPa s to 1.45 mPa s, after 30 min ultrasonic treatment. For the amylose solutions with higher concentration, the effect of ultrasonic treatment on viscosity was more significant. For instance, the viscosity of the 5 wt% amylose solution decreased about two orders of magnitude, from 1136 mPa s to 13.9 mPa s, after 30 min ultrasonic treatment. The higher the concentration of amylose solution was, the more significant the effect of ultrasonic treatment on viscosity. The above observations could be attributed to the contributions of entanglement and intermolecular interactions among amylose chains to viscosity of solution at high concentration. For the amylose solutions with high concentrations, the intense mechanical effect and heat induced by ultrasonic treatment could break the entanglements and intermolecular interactions of amylose chains which primarily result in the high viscosity of amylose solution,
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Fig. 2. Reduced viscosity of amylose DMSO solutions versus amylose concentration for the amyloses with different ultrasonic treating times.
therefore, the effect of ultrasonic treatment on viscosity reduction was more significant when concentration was high. Besides the above mechanism of viscosity decrease, amylose molecules in the solution may undergo degradation (chain scission) during ultrasonic treatment, which could also lead to decrease in solution viscosity. In the case of present study, both mentioned mechanisms may be operative. It was seen from Fig. 1 that, for the 30 min ultrasonic treatment, the decrease in viscosity in the initial 15 min was more significant than the later 15 min. This phenomenon was similar to the previous observation in ultrasonic treated maize starch solutions, the decrease of viscosity mainly occurred in the first 15 min treatment (Cheng, Chen, Liu, Ye, & Ke, 2010). These observations could be due to the characteristic feature of ultrasound-induced chain scission, i. e., there is a definite minimum chain length limiting the degradation process (Czechowska-Biskup et al., 2005). When the minimum chain length was reached, no further chain scission occurred, therefore the viscosity of amylose solution hardly decreased further. Although the properties of starch slurry subjected to ultrasonic treatments were described in the past (Iida et al., 2008; Zuo, Knoerzer, Mawson, Kentish, & Ashokkumar, 2009), much less is known on the mechanisms of ultrasound-induced starch chain scission in aqueous solution. 3.2. Molecular weight of amylose As mentioned above, ultrasonic treatment of amylose solution could cause scission of amylose chains. Viscosity average molecular weight, calculated from measured intrinsic viscosity, can be used to characterize the degradation of amylose molecules. Fig. 2 shows the changes of the reduced viscosity of amylose DMSO solutions against amylose concentration for the amyloses with 0 min, 2 min and 30 min ultrasonic treatments, respectively. The intrinsic viscosity of amylose without ultrasonic treatment was determined as 0.125, while the intrinsic viscosities of amylose with 2 min and 30 min ultrasonic treatments were 0.122 and 0.066, respectively. The viscosity average molecular weight of the amylose without ultrasonic treatment was 3.36 × 105 . After 2 min ultrasonic treatment, the viscosity average molecular weight of amylose decreased to 3.27 × 105 , and further down to 1.54 × 105 when the ultrasonic treatment was prolonged to 30 min. These results confirmed that the ultrasonic treatments did cause amylose chain scission.
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Fig. 3. Changes of molar masses versus hydrodynamic radius Rh of the amylose with different ultrasonic treatments.
Fig. 3 shows SEC molar mass distribution of the amyloses before and after ultrasonic treatments for 2 min and 30 min. The molar mass of the amylose without ultrasonic treatment ranged 2.4 × 106 − 4.5 × 108 g/mol, showing a broad distribution. After ultrasonic treatment, the distribution became narrower, and it was down to 3.2 × 105 − 6.0 × 106 g/mol when the ultrasonic treatment was carried out for 30 min. These results also indicated that the ultrasonic treatments led to amylose chain scission. The viscosity average molecular weight and SEC molar mass distribution contain different information, the exact consistency between the values obtained by the two different methods will not be necessary. However, both of the molecular weight measurements suggested that amylose chain scission, a degradation process, occurred during the ultrasonic treatments. It should be mentioned that the chain scission process indicated by the molecular weight reduction in this study showed a tendency similar to the previous work (Iida et al., 2008), in which high performance gel permeation chromatography (HPGPC) was used to measure molecular weight distribution of ultrasound treated starch. 3.3. Size of ANPs The viscosity of amylose solution is dependant on concentration and molecular weight of amylose, and it affects the size of ANPs prepared through precipitation. As presented above, the ultrasonic treatments decreased viscosity of amylose solution and reduced molecular weight of amylose. It is desirable to investigate the effect of the ultrasonic treatments to amylose solution on the size of ANPs formed in precipitation. Fig. 4 shows the mean sizes and PDIs of the ANPs prepared by precipitation (the volume ratio of amylose solution to absolute ethanol was 1:10) and using amylose solutions with different concentrations and treated with ultrasound for various periods of time. The data in Fig. 4 clearly indicated that the higher the concentration of amylose solution, the larger the ANPs and the greater the PDI. For the ANPs obtained from the 3 wt% and 5 wt% solutions without ultrasonic treatment, the values of PDI were around 0.7, suggesting that the samples had a very broad size distribution and might contain large particles or aggregates. The results in Fig. 4 also indicated that the effect of the ultrasonic treatment on the size of the ANPs was obvious. The particle size of ANPs decreased gradually with increasing ultrasonic treating time. For the ANPs obtained from the 1 wt% amylose solution, the mean size decreased from 206 nm to
Fig. 4. Mean sizes (column) and PDIs (solid square) of the ANPs prepared by using amylose solutions with different concentrations and ultrasonic treating times (the volume ratio of amylose solution to absolute ethanol was 1:10).
91 nm after the solution was treated with ultrasound for 15 min and further down to 82 nm after another 15 min ultrasonic treatment. For the ANPs obtained from the 5 wt% amylose solution, the mean size decreased from 337 nm to 196 nm and further to176 nm after the solution was treated 15 min and 30 min. The decrease in viscosity induced by the ultrasonic treatment might be responsible to the size reductions of the ANPs, because the viscosity decrease improved diffusion of amylose solution toward ethanol. However, the viscosity of the amylose solution was not the only parameter to control the size of the precipitated ANPs. Examining Figs. 1 and 4, it could be found that the viscosity of the 3 wt% amylose solution was very close to that of the 5 wt% amylose solution with 7 min ultrasonic treatment, but the mean sizes of the ANPs obtained from these solutions were 296 nm and 219 nm, respectively. It was thought that high concentration of polymer solution give rise to larger particle size not only because the high viscosity of the solution but also the number of polymer chains per unit volume of the solvent (Beck-Broichsitter, Rytting, Lebhardt, Wang, & Kissel, 2010; Legrand et al., 2007). As a consequence of the greater number of polymer chains per unit volume, the solvent diffusing into the non-solvent carries more polymer chains that aggregate and thus should form larger particles. Because high polymer concentration increases polymer–polymer interactions, this means more polymer chains remain associated during the diffusion process (Galindo-Rodriguez, Allemann, Fessi, & Doelker, 2004). However, the results of this study indicated that, through ultrasonic treatment, smaller ANPs can be obtained by using high concentration amylose solutions. This is probably because the ultrasonic treatment could break the interactions of amylose molecules and cut amylose chains. Scission of amylose chains reduces the probability of chain entanglement during precipitation, thus the shorter amylose chains aggregate and form smaller particles. In addition, the data in Fig. 4 also showed that the size of the ANPs obtained from the 1 wt% amylose solution without ultrasonic treatment was very close to that of the 5 wt% amylose solution with 30 min ultrasonic treatment, which means ultrasonic treatment could significantly raise production efficiency. Therefore, the ultrasonic treatment of amylose solution not only provides a route to prepare smaller ANPs but also an approach with higher production efficiency. Fig. 5 shows typical size distribution patterns of the ANPs prepared by precipitating the 3 wt% amylose solution with 15 min ultrasonic treatment into absolute ethanol at various volume ratios of amylose solution to absolute ethanol. It could be seen that the
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Fig. 5. Typical size distribution patterns of the ANPs obtained from the 3 wt% amylose solution with 15 min ultrasonic treatment at various volume ratios of amylose solution to absolute ethanol.
main peak shift to smaller size with increasing ethanol amount, but the shift was very small. The values of the mean size for the ANPs obtained at the volume ratios of 1:10, 1:5 and 1:3 were 126 nm, 131 nm and 138 nm, respectively. These results indicated that, after the ultrasonic treatment, the impact of the non-solvent amount on the size of the precipitated ANPs was not considerable, suggesting ANPs could be produced with less non-solvent. Therefore, the ultrasonic treatment of amylose solution provides a route to prepare ANPs via nanoprecipitation at lower cost. 3.4. Morphology of ANPs Fig. 6 shows the SEM micrographs as well as the corresponding size distribution patterns of the ANPs obtained from the 3 wt% amylose solution without ultrasonic treatment and the one with 30 min ultrasonic treatment, respectively. Although the ANPs were observed to be spherical in shape, the effect of ultrasonic treatment on morphological characteristics of the ANPs was significant. As shown in the SEM micrographs, the size of the ANPs obtained from the amylose solution with 30 min ultrasonic treatment was smaller than those prepared with the amylose solution without ultrasonic treatment. Moreover, the ANPs obtained from the ultrasonic treated solution was more uniform in size, there were few larger particles. These observations were consistent with the particle size distribution pattern measured by DLS which presented a main peak with narrower width. For the ANPs prepared using the amylose solution without ultrasonic treatment, the SEM micrograph showed that the particle size was not uniform and there were some large particles that were also reflected in the particle size distribution pattern (a strong peak at large size). This is understandable. Because the ultrasonic treatment broke the entanglements and interactions of amylose molecules and cut amylose chains, which could reduce nucleation of large aggregates from the entangled or interacted amylose chains and increase nucleation of small aggregates from shorter amylose chains during precipitation, thus the precipitated ANPs using the ultrasound treated solutions were smaller and more uniform in size.
Fig. 6. SEM micrographs and the corresponding size distribution patterns of the ANPs obtained from the 3 wt% amylose solution without ultrasonic treatment (a) and the one with 30 min ultrasonic treatment (b). The volume ratio of amylose solution to absolute ethanol was 1:10.
3.5. XRD analysis Fig. 7 shows X-ray diffraction patterns of the ANPs obtained from amylose solutions with different concentrations and ultra-
Fig. 7. X-ray diffraction patterns of the ANPs obtained from amylose solutions with different concentrations and ultrasonic treating times.
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sonic treating times. Examining the XRD patterns in Fig. 7, there were very weak diffraction peaks at 13.8◦ , 18.9◦ and 23.7◦ for all the ANPs, suggesting that the precipitated ANPs possess the V-type crystalline structure. Because there was no obvious difference in peak position and intensity for the XRD patterns of the ANPs obtained from the solutions with and without ultrasonic treatments, it could be concluded that the effect of the ultrasonic treatments on the structure and crystallinity of the precipitated ANPs was negligible. 4. Conclusions Ultrasonic treatment to amylose solution decreased solution viscosity and caused scission of amylose chains, which is beneficial to prepare ANPs through precipitation technique. The ANPs obtained from the solutions with ultrasonic treatments were smaller and more uniform compared to those from the solutions without ultrasonic treatment. The effect of the ultrasonic treatment on the structure and crystallinity of the precipitated ANPs was negligible. The findings of this study indicate that ultrasonic treatment of starch solution not only offers a convenient and easily controllable methodology to prepare smaller starch nanoparticles through precipitation, but also provides a route to raise production efficiency and lower cost of starch nanoparticles preparation by using high concentration solution and less amount of non-solvent. Acknowledgement The authors are grateful to financial support from the National Natural Science Foundation of China (51273083). References Baxter, S., Zivanovic, S., & Weiss, J. (2005). Molecular weight and degree of 310 acetylation of high-intensity ultrasonicated chitosan. Food Hydrocolloids, 19, 821–830. Beck-Broichsitter, M., Rytting, E., Lebhardt, T., Wang, X., & Kissel, T. (2010). Preparation of nanoparticles by solvent displacement for drug delivery: A shift in the ouzo region upon drug loading. European Journal of Pharmaceutical Sciences, 41, 244–253. Cave, R. A., Seabrook, S. A., Gidley, M. J., & Gilbert, R. G. (2009). Characterization of starch by size-exclusion chromatography: The limitations imposed by shear scission. Biomacromolecules, 10, 2245–2253. Cheng, W., Chen, J., Liu, D., Ye, X., & Ke, F. (2010). Impact of ultrasonic treatment on properties of starch film-forming dispersion and the resulting films. Carbohydrate Polymers, 81, 707–711. Chin, S. F., Pang, S. C., & Tay, S. H. (2011). Size controlled synthesis of starch nanoparticles by a simple nanoprecipitation method. Carbohydrate Polymers, 86, 1817–1819. Cornell, H. J., Rix, C. J., & McGrane, S. J. (2002). Viscometric properties of solutions of amylose and amylopectin in aqueous potassium thiocyanate. Starch-Stärke, 54, 517–526.
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