Preparation of a Strong GelatinâShort Linear Glucan Nanocomposite

Article pubs.acs.org/JAFC

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Preparation of a Strong Gelatin−Short Linear Glucan Nanocomposite Hydrogel by an in Situ Self-Assembly Process Shengju Ge,† Man Li,† Na Ji,† Jing Liu,‡ Hongyan Mul,† Liu Xiong,† and Qingjie Sun*,† †

College of Food Science and Engineering and ‡Central Laboratory, Qingdao Agricultural University Qingdao, Shandong Province 266109, China ABSTRACT: Gelatin hydrogels exhibit excellent biocompatibility, nonimmunogenicity, and biodegradability, but they have limited applications in the food and medical industries because of their poor mechanical properties. Herein, we first developed an in situ self-assembly process for the preparation of gelatin−short linear glucan (SLG) nanocomposite hydrogels with enhanced mechanical strength. The microstructure, dynamic viscoelasticity, compression behavior, and thermal characteristics of the gelatin−SLG nanocomposite hydrogels were determined using scanning electron microscopy (SEM), dynamic rheological experiments, compression tests, and texture profile analysis tests. The SEM images revealed that nanoparticles were formed by the in situ self-assembly of SLG in the gelatin matrix and that the size of these nanoparticles ranged between 200 and 600 nm. The pores of the nanocomposite hydrogels were smaller than those of the pure gelatin hydrogels. Transmission electron microscopy images and X-ray diffraction further confirmed the presence of SLG nanoparticles with spherical shapes and B-type structures. Compared with pure gelatin hydrogels, the nanocomposite hydrogels exhibited improved mechanical behavior. Notably, the hardness and maximum values of the compressive stress of gelatin−SLG nanocomposites containing 5% SLG increased by about 2-fold and 3-fold, respectively, compared to the corresponding values of pure gelatin hydrogels. KEYWORDS: starch, gels, rheological properties, nanoparticles, mechanical strength, short chain amylose

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INTRODUCTION Natural polymer hydrogels, which contain three-dimensional biopolymer networks and can absorb and retain large amounts of water, have attracted attention from a wide range of researchers. Because of their renewability, biocompatibility, and biodegradability, natural hydrogels have potential applications in the food industry.1−3 The structures of many foods, such as yogurt, frankfurters, cheese, processed meats, cooked egg whites, and confectionery products, are dependent on the formation of gel networks.4 Because of the high complexity of these foods, biopolymer gels are commonly employed as model systems for mechanical investigations. An ideal hydrogel needs to have high strength and flexibility, as well as reversible deformation under high strain.5,6 However, conventional natural hydrogels are usually weak and brittle and break easily under stress because of the restricted molecular motion of their biopolymer chains, an effect caused by large amounts of randomly arranged cross-links. These characteristics greatly limit their practical applications.7 To maintain their structural integrity during manufacturing, gelatin-based food products (e.g., processed meats, jelly, gelatin desserts, frankfurters, and confectionery) need to have high gel strength against severe deformation or even final fracture.4 To overcome these limitations, several types of extremely tough hydrogels have been developed, including nanocomposite hydrogels,8,9 hydrogen-bonding-enhanced hydrogels,10 ionically cross-linked hydrogels,11 chemically and physically double-cross-linked hydrogels,12,13 and double-network hydrogels.14,15 Among these enhanced hydrogels, nanocomposite hydrogels synthesized by incorporating nanoparticles in the hydrogel matrixes have been attracting more research interest because of their simple preparation method. For example, the incorporation of © XXXX American Chemical Society

nanosilica particles as mechanical reinforcement significantly improves the mechanical strength of hyaluronic acid hydrogels.16 Recently, Huang et al. reported that the mechanical properties of nanocomposite hydrogels composed of sodium alginate and chitin whiskers were significantly improved compared to those of alginate hydrogel.17 The compressive modulus of composite hydrogels with 2% chitin whiskers increased by about 1.7-fold over that of pure alginate gels. However, when the concentration of chitin whiskers was 2.5%, the distribution of nanoparticles was heterogeneous within the hydrogels because of excessive whisker aggregation, which detracted from the desired mechanical improvements. The preparation of nanocomposite hydrogels through in situ nanoparticle formation has attracted great interest as a solution for the poor mechanical strength of hydrogels. It is notable that, because of the effectiveness of in situ nanoprecipitation in generating uniformly distributed nanoparticles, the mechanical and physical properties of nanocomposite hydrogel systems are significantly enhanced. For example, a very tough chitosancomposite hydrogel was prepared by the in situ precipitation formation of uniformly distributed calcium phosphate nanoparticles in a chitosan matrix.18 Short linear glucan (SLG), debranched from amylopectin, can easily form starch nanoparticles by self-assembly in aqueous solution.19 Starch nanoparticles have many potential applications in various fields, such as reinforcing nanofillers,20 carriers for the delivery of bioactive compounds,21 and biodegradable composites. However, the in Received: Revised: Accepted: Published: A

October 15, 2017 December 3, 2017 December 18, 2017 December 18, 2017 DOI: 10.1021/acs.jafc.7b04684 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

additional 10 min until fully homogenized. The concentration of gelatin was 10% (w/v) in all of the samples, and the SLG concentrations in the mixture solutions were 1, 3, and 5% (w/v) for the samples prepared using 0.2-, 0.6-, and 1.0-g aliquots, respectively. All of the solutions were stored at 10 °C overnight to form gelatin hydrogels and gelatin−SLG nanocomposite hydrogels. To observe the nanoparticles formed in situ, the as-prepared nanocomposite hydrogels with 5% SLG were dissolved by water-bath heating at 45 °C. Then, the samples were immediately centrifuged at 2000g for 5 min to remove the dissolved gelatin, and the precipitates were washed three times with distilled water. Subsequently, the samples were dried by lyophilization for 48 h to obtain isolated SLG nanoparticles. For comparison, bare SLG nanoparticles were also prepared through the previously reported method.20 In brief, SLG powder was dissolved in deionized water (10%, w/v) by heating at 120 °C for 30 min. Then, the solution was stored at 10 °C for 12 h. The suspensions were washed with distilled water and were then vacuumfreeze−dried to obtain bare SLG nanoparticles. Scanning Electron Microscopy (SEM). The morphologies of the gelatin−SLG nanocomposite hydrogels were observed using a scanning electron microscope (S-4800, Hitachi Instruments Ltd., Tokyo, Japan). To maintain the network structure originally formed in the gel state, the hydrogels were frozen in liquid nitrogen, snapped immediately, and then vacuum-freeze−dried for 2 days to obtain dried samples. The surfaces and fractured sections were sputtered with gold for SEM observations. Transmission Electron Microscopy (TEM). The morphologies of SLG nanoparticles isolated from a gelatin matrix were observed with an HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). One drop of nanoparticle dispersion was spread onto a 400-mesh copper grid and lyophilized for TEM observations. For comparison, a drop of gelatin solution was also spread onto a grid and freeze-dried for TEM observation. Dynamic Light Scattering (DLS) Measurements. The apparent average size and size distribution of isolated SLG nanoparticles were measured by dynamic laser scattering using a Malvern Zetasizer Nano instrument (Malvern Instruments Ltd., Malvern, U.K.) equipped with a helium−neon (He−Ne) laser (0.4 mW, 633 nm). Measurements were made at 25 °C and at a 90° scattering angle. All samples were dispersed in Milli-Q water without filtering.32 X-ray Diffraction (XRD) Patterns. The crystal structures of the SLG, isolated SLG nanoparticles, and bare SLG nanoparticles were analyzed using an X-ray diffractometer (AXS D8 ADVANCE, Bruker, Karlsruhe, Germany) in a manner described by Qin et al.33 The powders were equilibrated to 20% moisture content at ambient temperature for 24 h prior to analysis. All samples were tightly packed into the sample holder, and X-ray diffraction patterns were recorded in an angular (2θ) range from 4° to 40° in 0.01° steps. Rheological Properties of the Nanocomposite Hydrogels. Dynamic rheological measurements of the nanocomposite hydrogels were carried out with a strain-controlled rheometer (MCR102, Anton Paar, Graz, Austria) using a parallel-plate system with a diameter of 50 mm and a gap of 1 mm. To record the storage modulus (G′) and loss modulus (G″), frequency sweeps were performed over an angular frequency range of 0.1−100 rad/s at 10 °C with constant deformation and a strain amplitude of 1% (within the linear viscoelastic region). To observe the maturation of the gelling samples, time sweeps were performed. For each measurement, a sample was placed in the sample holding region and cooled from 40 to 10 °C at a rate of 2 °C/min. Subsequently, the temperature was maintained at 10 °C for 2.5 h. Values of the G′ and G″ were recorded over a fixed time period at a frequency of 1 Hz and a constant strain of 1%. Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectra of the samples were recorded using a NEXUS-760 FTIR spectrometer (Thermo Nicolet Corp., Madison, WI) with a resolution of 4 cm−1 in the wavenumber range of 4000−400 cm−1, as described by Zou et al.34 FTIR spectra were measured using dried samples obtained from SLG, bare and isolated SLG nanoparticles, pure gelatin hydrogels, and gelatin−SLG nanocomposite hydrogels. All samples were analyzed

situ formation of SLG nanoparticles in a hydrogel matrix has not been reported. Gelatin, as a commonly used natural hydrogel matrix, is a fibrous protein that is derived from collagen-containing materials. Gelatin has a helix-to-coil transition temperature. Upon cooling below this temperature, the disordered peptide chains of gelatin return to a collagen triple-helix structure and act as gel junction zones stabilized by intermolecular hydrogen bonds, thereby forming a gel network. Because of their biocompatibility, nonimmunogenicity, and biodegradability, gelatin-based hydrogels are widely used in the food industry (e.g., in clear dessert jellies, mousses, and fruit gums),4 drug delivery systems,22,23 and pharmaceuticals.24 However, the applications of gelatin-based hydrogels remain limited because of their poor mechanical performance. Therefore, various crosslinking agents including glutaraldehyde, transglutaminase, and genipin have been developed to cross-link gelatin chains to improve the mechanical properties of gelatin gels.13,25−28 However, the applications of gelatin hydrogels with various cross-linking systems are still limited because of the potential toxicity of chemical cross-linkers or the high cost of naturally occurring cross-linking agents. Recently, gelatin nanocomposite hydrogels that bring about further mechanical improvements by introducing different nanoparticles have been proposed.8 Previous research on graphene oxide−gelatin nanocomposite hydrogels demonstrated that the presence of graphene oxide improved the mechanical properties.29 In this article, we report a novel in situ self-assembly process using SLG for the preparation of composite hydrogels of gelatin and nanoparticles. Nanoparticles were formed mainly by the self-assembly of SLG in a gelatin matrix. The objective of this stud was to improve the mechanical properties of gelatin hydrogels through the in situ formation of SLG nanoparticles. The effects of different contents of SLG (1−5%, w/v) on the microstructure, rheological properties, and compression behavior of the gelatin hydrogels were systematically investigated. These newly developed gelatin hydrogels, reinforced with in situ SLG nanoparticles, have potential applications in the food, pharmaceutical, and photographic industries.

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MATERIALS AND METHODS

Materials. Waxy corn starch (98.8% amylopectin) was obtained from Zhucheng Xingmao Corn Starch Co., Ltd. (Weifang, China). Pullulanase (EC 3.2.1.41, 6.17 × 104 kat/g) was purchased from Novozymes Investment Co., Ltd. Gelatin isolated from porcine skin (type B, 200 Bloom) was supplied by Sigma-Aldrich Company Ltd. (Steinheim, Germany). All other reagents used were of analytical grade, and deionized water was used throughout unless otherwise stated. Gelatin−SLG Nanocomposite Hydrogel Preparation. The SLG was fabricated according to a previously described method.30 Briefly, waxy corn starch slurry was gelatinized, cooled to 58 °C, and debranched with pullulanase for 8 h. The reaction was stopped by heating to inactivate the pullulanase, and then the solution was centrifuged to remove the precipitate. The supernatant was precipitated using four times as much absolute ethanol, washed with distilled water, and then freeze-dried. The weight-average degree of polymerization (DPw) of the SLG was approximately 38. To achieve a uniform solution of SLG, aliquots of the SLG (0, 0.2, 0.6, and 1.0 g) were dissolved in 10 mL of deionized water by heating the mixtures in a sealed tube at 120 °C and then cooling them to 45 °C. Solutions of gelatin were prepared by allowing 2.0 g of gelatin to swell in 10 mL of distilled water for 30 min at room temperature and then heating the mixtures at 45 °C for 30 min to dissolve the gelatin.31 Tenmilliliter samples of solutions containing various amounts of SLG were then added to the gelatin solutions, and the mixtures were stirred for an B

DOI: 10.1021/acs.jafc.7b04684 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Scheme 1. Schematic Diagram of the Fabrication of Gelatin−SLG Nanocomposite Hydrogels by an in Situ Self-Assembly Process

Figure 1. Surface morphology images illustrating the microstructures of (A) gelatin hydrogels and (B−D) nanocomposite hydrogels reinforced with SLG at concentrations of (B) 1%, (C) 3%, and (D) 5% (w/v). texture profile analysis (TPA). TPA tests were carried out using a texture analyzer (TA-XTplus, Stable Micro Systems, Surrey, U.K.) fitted with a P 0.5 probe (0.5 in probe). The test speed was 0.5 mm/s, and the nanocomposite hydrogels were compressed twice to a strain of 75%. The textural parameters recorded were hardness (g), springiness,

using the KBr method, and pure KBr powders were used for the background. Mechanical Properties of the Nanocomposite Hydrogels. The gelatin solution or a mixture solution of gelatin and SLG was poured into a vessel with an inner diameter of 20 mm and a height of 10 mm. The vessel was kept in a refrigerator at 10 °C overnight before C


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Figure 2. Cross-sectional morphologies at different magnifications of (A,a) gelatin hydrogels and (B−D,b−d) nanocomposite hydrogels reinforced with SLG at concentrations of (B,b) 1%, (C,c) 3%, and (D,d) 5% (w/v). compressed with a P36R probe at a strain rate of 1 mm s−1. Strain and stress data were recorded during the experiments. Differential Scanning Calorimetry (DSC). The thermal properties of SLG, bare and isolated SLG nanoparticles, gelatin hydrogels, and gelatin−SLG nanocomposite hydrogels were monitored using a

cohesiveness, gumminess (g), chewiness (g), and resilience. These parameters were measured in six samples from each batch. Compressive measurements of the hydrogels were performed using the same texture analyzer. A cylindrical hydrogel sample (1.5 cm in diameter and 1.5 cm in height) was placed on the lower plate and D


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Figure 3. (A,B) Typical TEM images of (A) a dilute suspension of pure gelatin and (B) isolated SLG nanoparticles. (C) Enlarged view of isolated SLG nanoparticles. (D,E) Size distributions of SLG nanoparticles. differential scanning calorimeter (DSC1, Mettler-Toledo International Inc., Greifensee, Switzerland). Powder samples (10 mg) and 20 mg of water were added to an aluminum pan, and the hydrogels (20 mg) were added directly; then, the container was hermetically sealed. After incubation at 25 °C for 12 h, the samples were scanned at 10 °C/min from 25 to 125 °C. Rescanning of the sample was performed in the same manner after the DSC pan had been allowed to cool. The endothermic enthalpy change (ΔH) and the onset (To), peak (Tp), and conclusion (Tc) temperatures were recorded. Statistical Analysis. Each measurement was carried out using at least three fresh, independently prepared samples. The data analyses were performed using an analysis of variance (ANOVA) procedure in SPSS 17.0 software (SPSS Inc., Chicago, IL). Duncan’s multiple range test was also applied to determine the difference of means from ANOVA using a significance test level of 5% (p < 0.05)

hydrogels with different SLG contents (1, 3, and 5%) are shown in Figure 1. As a control, the pure gelatin hydrogel displayed a smooth surface (Figure 1A). After the incorporation of SLG, the surface of the composite hydrogels became uneven, and spherical particles appeared on the surface with sizes ranging between 200 and 600 nm. With increasing SLG content, the quantities of particles on the surface of the gelatin−SLG nanocomposite hydrogels increased, and the size of the nanoparticles decreased markedly. This observation suggested that nanoparticles formed through the in situ self-assembly of SLG as the mixed solutions of gelatin and SLG were cooling. It has been reported that SLG that has been debranched from waxy maize starch is very prone to crystallization and is widely used to fabricate starch nanoparticles.19,35 In terms of cross-sectional morphology, the pure gelatin hydrogels typically exhibited porous microstructures, with pore sizes of 10−50 μm, as shown in Figure 2A. The gelatin−SLG nanocomposite hydrogels containing different contents of SLG also exhibited porous microstructures with interconnected channels, implying that the network structure of the hydrogels was maintained after the in situ process (Figure 2B,C). However, at a higher magnification, significant differences in the cross-sectional morphologies of the pure gelatin hydrogels and the gelatin−SLG nanocomposite hydrogels emerged (Figure 2a−d). The nanocomposite hydrogels with SLG showed a dense distribution of spherical nanoparticles within the gelatin matrix. Moreover, when the concentration of the SLG was increased from 1% to 5%, the quantities of SLG nanoparticles increased, and the particle sizes became smaller. The formation and growth of self-assembled SLG nanoparticles should exhibit three stages: the appearance of nuclei by the retrogradation of SLG, the growth and coagulation of the nuclei

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RESULTS AND DISCUSSION Nanocomposite Hydrogel Formation. A schematic diagram of the in situ self-assembly process for fabricating gelatin−SLG nanocomposite hydrogel is illustrated in Scheme 1. We first prepared SLG by debranching waxy corn starch with pullulanase. Then, SLG was introduced into the gelatin solution, and the mixture was stored at 10 °C overnight to form gelatin− SLG nanocomposite hydrogels. During this gelation process, SLG formed starch nanoparticles through in situ self-assembly in the gelatin matrix. The hydrogen bonding between the hydroxyl groups in the SLG nanoparticles and the amino groups in the gelatin strengthened the gelatin−SLG hydrogels. To the best of our knowledge, this is the first report on the introduction of SLG nanoparticles through in situ self-assembly inside a gelatin hydrogel matrix. Microstructure of Gelatin−SLG Nanocomposite Hydrogels. Surface morphology images of nanocomposite E


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nanoparticles with a B-type X-ray pattern.36 This result further confirmed the formation of self-assembled SLG nanoparticles at 10 °C in the gelatin hydrogels. The relative crystallinity (θ) of the isolated SLG nanoparticles (49.3%) was slightly higher that of the bare SLG nanoparticles (44.8%). This result indicates that a better B-type structure could be formed by the crystallization of SLG in the gelatin matrix. Rheological Properties of Gelatin−SLG Nanocomposite Hydrogels. The effects of the SLG content on the rheological properties of gelatin hydrogels were explored using a dynamic rheometer. Figure 5 shows the G′ and G″ curves of the nanocomposite hydrogels as a function of frequency. The magnitudes of the G′ values were larger than those of the corresponding G″ values over the entire frequency range, indicating that all of the hydrogels behaved as typical gel-like substances. Furthermore, the G′ values of all of the gelatin hydrogels remained unchanged as the angular frequency was increased from 0.1 to 100 rad/s, which indicated that the gel strength of the gelatin hydrogel was strong. In general, the G′ and G″ values of the gelatin−SLG nanocomposite hydrogels containing different contents of SLG were significantly higher than those of the pure gelatin hydrogels within the determined range. As the SLG content was increased to 5 wt %, G′ increased steadily, as shown in Figure 5A. The addition of 5% SLG enhanced the solid-like behavior of the gelatin hydrogel, with the G′ value of the nanocomposite hydrogels increasing by 98.3% at 20 rad/s as compared with that of the pure gelatin hydrogel. These results indicate that the SLG nanoparticles were strongly integrated with the gelatin matrix after in situ formation. In a recent study of the frequency dependence of G′ in hyaluronic acid−calcium phosphate nanocomposite hydrogels through in situ precipitation, the elastic properties of the hydrogels improved markedly as a result of stronger binding between the hyaluronic acid chains and the calcium phosphate particles, where the G′ values of the nanocomposite hydrogels exhibited frequency-independent behavior. In contrast, the G′ values of pure hyaluronic acid hydrogels changed as a function of frequency.37 Figure 6 presents the time sweep of the gelatin−SLG nanocomposite hydrogels upon cooling from 40 to 10 °C at 1 °C/min and maintaining at 10 °C for 2.5 h. In the first cooling period of 30 min, all hydrogels showed a profile in which there was an initial, rapid increase of G′, followed by a slow increase. When the temperature was held at 10 °C for 2.5 h, both G′ and G″ showed a continuous increase. Compared to those of the

to reach an appreciable size (nanoparticles), and the aggregation of the nanoparticles.36 In the presence of gelatin, the aggregation of nanoparticles is restricted with increasing SLG concentration. To further investigate the characteristics of the SLG nanoparticles formed in situ, the as-prepared nanoparticles were isolated from the gelatin−SLG nanocomposite hydrogels by heating the gel phase into a sol phase following centrifugation. TEM micrographs (Figure 3A,B) showed the presence of spherically shaped nanoparticles with diameters that mainly ranged from 200 to 600 nm, whereas smaller nanoparticles with sizes of about 30 nm were also observed (Figure 3C). The size distribution of the SLG nanoparticles was also measured by DLS. The nanoparticles were mostly between 100 and 600 nm, with small quantities of nanoparticles about 30 nm in size (Figure 3D,E), in agreement with the TEM results. Thus, the TEM images confirmed that SLG nanoparticles formed in the gelatin matrix, although the particle size distribution of the SLG nanoparticles was not uniform. The crystalline structure of the self-assembled SLG nanoparticles in the gelatin hydrogels was determined by X-ray diffraction. As shown in Figure 4, the SLG nanoparticles isolated

Figure 4. X-ray diffractograms of SLG, bare SLG nanoparticles, and SLG nanoparticles isolated from gelatin nanocomposite hydrogels. RC indicates relative crystallinity.

from the gelatin composite hydrogels displayed a typical B-type crystalline structure of starch with main diffraction peaks at 2θ = 5.9°, 17.1°, 22.5°, and 24.3°. Similarly, the debranched waxy corn starch solution incubated at 4 °C resulted in starch

Figure 5. Frequency dependence of the G′ and G″ values of gelatin nanocomposite hydrogels with different contents of SLG at 10 °C. F


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Additionally, the freeze-dried gelatin hydrogels exhibited characteristic amide A, amide I, and amide III peaks at 3520− 3320, 1663−1649, and 1335 cm−1, respectively. The presence of nanoparticles in the gelatin−SLG nanocomposite hydrogels was confirmed by the characteristic peak at 1022 cm−1, which was specific only to the samples containing SLG nanoparticles. The high peak intensity at 1022 cm−1 means that there was strong hydrogen bonding between the starch molecular chains.41 The intensity of this characteristic peak increased with increasing SLG content, and this band of the nanocomposite hydrogels tended to shift to lower frequency, indicating that the shortrange structure of the SLG nanoparticles was more ordered.42 Further, when the concentration of the SLG was increased from 1% to 5%, the characteristic bands at 3500−3400 cm−1 and the amide A band of the gelatin−SLG nanocomposite hydrogels shifted to lower wavelengths, indicating that the interaction force of the intermolecular hydrogen bonds between SLG nanoparticles and gelatin was enhanced. Similarly, Li et al. reported that the peaks at 3500−3300 cm−1 (O−H stretching) of SLG/protein hybrid nanoparticles with an SLG/protein ratio of 10:1 shifted to a lower wavelength because of the enhancement of the hydrogen-bonding interactions.43 Mechanical Properties of Gelatin−SLG Nanocomposite Hydrogels. The textural parameters of the gelatin−SLG nanocomposite hydrogels with different concentrations of SLG were determined by TPA, and the results are shown in Table 1. The hardness of the gelatin hydrogel was significantly increased (p < 0.05) by incorporating SLG through the in situ formation of nanoparticles, with the hardness increasing commensurately with increases in the concentration of the SLG. Notably, the hardness of the gelatin−SLG nanocomposites with 5% SLG increased approximately 2-fold compared to that of the pure gelatin hydrogels (168.53 versus 77.34 g). Moreover, the gumminess and chewiness of the gelatin hydrogels also increased with the addition of SLG. On the other hand, the springiness values of all of the hydrogels were close to 1, indicating that they were all highly elastic. These results show that the nanocomposite hydrogels are less likely to be damaged than pure gelatin hydrogels during mastication. To further investigate the effects of SLG on the mechanical properties of the nanocomposite hydrogels, compression tests were performed on the nanocomposites, and the compressive modulus was evaluated from the engineering stress−strain curve (Figure 8). Under compression, the stress values at fracture of nanocomposite hydrogels increased in comparison with those of pure gelatin hydrogels. When the concentration of SLG was 5%, the nanocomposite hydrogels’ maximum values of compressive stress were almost 3 times higher than that of gelatin hydrogels (0.0299 versus 0.0101 MPa). Comprehensively, the gelatin− SLG nanocomposite hydrogels exhibited a marked improvement in their mechanical properties compared to pure gelatin hydrogel. These results suggest the important role of SLG in the

Figure 6. Time dependence of G′ and G″ during holding at 10 °C for gelatin hydrogels with different concentrations of SLG.

initial samples, the G′ and G″ values of all of the samples were 9−10 times higher after incubation for 3 h. Still, gelation of the hydrogels was not completed in 3 h. In addition, the slope of the time-response curve indicates the rate of the gelation process, and the slopes of the time-response curves of the nanocomposite hydrogels increased with increasing SLG content. This suggests that the SLG nanoparticles play a vital role in the gelation process and that the rate of gelation increases with increasing amount of self-assembled SLG nanoparticles in the gelatin hydrogels. FTIR Spectra. The FTIR spectra of gelatin hydrogels and gelatin−SLG nanocomposite hydrogels are shown in Figure 7.

Figure 7. FTIR spectra of SLG, gelatin, bare SLG nanoparticles, and gelatin nanocomposite hydrogels with different SLG contents.

For comparison, the spectra of SLG, bare SLG nanoparticles, and isolated SLG nanoparticles were also determined. SLG and bare SLG nanoparticles exhibited characteristic bands at 3500− 3400 cm−1 (O−H stretching); 2930−2940 cm−1 (C−H stretching); 1630−1640 cm−1 (O−H stretching); and about 1022, 1074, and 1156 cm−1 (C−O ether stretching).38−40

Table 1. Textural Properties of Gelatin Nanocomposite Hydrogels with Different Levels of Short Linear Glucan (SLG)a,b SLG (%, w/v)

hardness (g)

springiness

0 1 3 5

77.34 ± 2.14 90.64 ± 3.76c 135.90 ± 2.27b 168.53 ± 5.96a

1.030 ± 0.022 0.940 ± 0.019a 0.948 ± 0.008a 0.939 ± 0.013a

c

cohesiveness a

0.880 ± 0.012 0.929 ± 0.018a 0.824 ± 0.009b 0.790 ± 0.014b a

gumminess (g)

chewiness (g)

resilience

68.09 ± 1.74 84.20 ± 3.01c 111.90 ± 2.77b 133.15 ± 4.33a

70.16 ± 2.74 79.18 ± 2.15c 106.13 ± 3.21b 125.02 ± 3.57a

0.628 ± 0.031b 0.727 ± 0.012a 0.645 ± 0.024ab 0.576 ± 0.012b

d

d

a Data are expressed as the mean ± standard deviation (n = 3). bValues of means followed by different lowercase letters in the same column are significantly different (p < 0.05) by Duncan’s multiple range test.

G


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larger hysteresis loops than the gelatin hydrogels at different rates of maximum compression. This shows that the energy dissipation of the nanocomposite hydrogels with SLG was more efficient during the loading/unloading cycles. This could be due to the destruction and delayed restoration of the hydrogenbonding interactions between the SLG nanoparticles and the gelatin at different strain values during the cycles.12 Thermal Properties. Table 2 summarizes the To, Tp, and Tc temperatures and the ΔH values of SLG, bare SLG nanoparticles, isolated SLG nanoparticles, gelatin hydrogel, and gelatin−SLG nanocomposites hydrogels. The gelatin hydrogels displayed no peak (data not shown), indicating that crystallinity was not present. However, the gelatin−SLG nanocomposite hydrogels exhibited a single thermal transition with melting temperature and ΔH values similar to those of the isolated SLG nanoparticles. These results support the in situ formation of SLG nanoparticles in the gelatin matrix. The ΔH value of the isolated SLG nanoparticles (7.30 J/g) was lower than that of the bare SLG nanoparticles (12.32 J/g), which could be due to the partial crystallization of SLG in the gelatin matrix. Moreover, the Tp value of the gelatin−SLG nanocomposites (86.29 °C) was similar to that of the bare SLG nanoparticles (86.43 °C), indicating that the gelatin had no effect on the self-assembly of SLG for a long period of 12 h. The Tp and ΔH values of the isolated SLG nanoparticles (86.43 and 7.30 J/g, respectively) were similar to those of the gelatin−SLG nanocomposites. These results are consistent with the results of relative crystallinity (Figure 4). All of the samples were rescanned, and Figure 10 presents the rescanned thermograms of SLG, bare SLG nanoparticles, isolated SLG nanoparticles, and gelatin−SLG nanocomposites.

Figure 8. Compressive stress−strain curves of gelatin nanocomposite hydrogels with different SLG contents.

mechanical stiffness of gelatin−SLG nanocomposite hydrogels. We suppose that the strong hydrogen-bonding interactions between the SLG nanoparticles and gelatin also contributed to the mechanical enhancement, which is consistent with the FTIR results (Figure 7). Similarly, Liu et al. reported that starch nanoparticles showed a strong reinforcing effect on the starch matrix due to the hydrogen-bonding interactions between the nanoparticles and the starch matrix.44 The compressive stress−strain curves of nanocomposite hydrogels at different rates of maximum compression are shown in Figure 9. All of the hydrogels exhibited excellent shape recovery behavior. When the strain was increased from 40% to 60%, all of the hydrogels exhibited an increased hysteresis loop. Moreover, the gelatin−SLG nanocomposite hydrogels exhibited

Figure 9. Compressive stress−strain curves of pure gelatin hydrogels and nanocomposite hydrogels with different contents of SLG with varying maximum compression. H


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Table 2. Thermal Characteristics of SLG, Bare SLG Nanoparticles, Isolated SLG Nanoparticles, and Gelatin Nanocomposite Hydrogelsa,b gelatinization temperatures (°C) sample SLG SLG gelatin nanocomposite hydrogel gelatin nanocomposite hydrogel isolated SLG nanoparticles isolated SLG nanoparticles bare SLG nanoparticles bare SLG nanoparticles

(rescan) (rescan) (rescan) (rescan)

To

Tp

Tc

ΔH (J/g)

66.33 ± 0.11f 75.19 ± 0.07c 73.31 ± 0.21d 79.40 ± 0.21a 75.14 ± 0.12c 78.44 ± 0.21b 65.19 ± 0.05g 70.90 ± 0.17e

85.96 ± 0.12f 86.77 ± 0.21de 86.29 ± 0.05ef 92.79 ± 0.22b 87.12 ± 0.07d 94.12 ± 0.22a 86.43 ± 0.15ef 89.23 ± 0.33c

102.10 ± 0.39b 93.90 ± 0.28d 95.47 ± 0.23c 103.94 ± 0.22a 96.15 ± 0.12c 104.47 ± 0.24a 103.89 ± 0.05a 101.66 ± 0.16b

−2.19 ± 0.05a −8.92 ± 0.01e −8.30 ± 0.05d −4.33 ± 0.11b −7.30 ± 0.02c −4.30 ± 0.11b −12.32 ± 0.04f −18.24 ± 0.13g

Data are expressed as the mean ± standard deviation (n = 3). bValues of means followed by different lowercase letters in the same column are significantly different (p < 0.05) by Duncan’s multiple range test. a

enhanced the hardness, stress at fracture, and fracture strain of the composite hydrogels. Furthermore, the introduction of SLG nanoparticles greatly improved the G′ and G″ values of the gelatin hydrogels. The newly developed gelatin−SLG nanocomposites therefore have high potential for use in the food, nutraceutical, and medical industries.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Qingjie Sun: 0000-0002-7371-1052 Notes

The authors declare no competing financial interest.

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Figure 10. DSC curves of SLG, gelatin−SLG nanocomposite hydrogel (gelatin−SLG), bare SLG nanoparticles, and isolated SLG nanoparticles. Rescans are indicated by “re”.

ACKNOWLEDGMENTS This work was supported by the Special Funds for Taishan Scholars Project of Shandong Province.

The rescanning of the gelatin hydrogels revealed no peaks, indicating the absence of crystalline structure (data not shown). The rescanned SLG, bare SLG nanoparticles, isolated SLG nanoparticles, and gelatin−SLG nanocomposites each exhibited a single endothermic peak. These results suggest that a crystalline structure was formed by SLG self-assembly during cooling. Similarly, it has been reported that the melting of starch nanoparticles is highly reversible when those nanoparticles are prepared from recrystallized SLG.35 The Tp and ΔH values of bare SLG nanoparticles were increased after rescanning (Table 2). Gong et al. also found that the Tp and ΔH values of the rescanned starch nanoparticles were increased.35 However, the ΔH value of the gelatin−SLG nanocomposite hydrogels was reduced from 8.30 to 4.33 J/g after rescanning, which is similar to the results for the isolated SLG nanoparticles. We supposed that the interactions of gelatin and melted SLG after the first scanning might have led to a decreased efficiency of SLG selfassembly and, thus, reduced the relative crystallinity. Similarly, the ΔH value of starch nanoparticles with Span 80 was lower than that of the bare starch nanoparticles, which could be because the surfactant inhibited the formation of hydrogen bonds between SLG nanoparticles.36 In summary, we have successfully developed gelatin−SLG nanocomposite hydrogels with significantly enhanced mechanical strength by introducing the in situ self-assembly of SLG within a gelatin hydrogel matrix. Small-sized SLG nanoparticles (200−600 nm) formed in situ were observed in gelatin−SLG nanocomposites. Increasing the content of SLG markedly

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