Chitin Microfibers Reinforce Soy Protein Gels ... - ACS Publications

Article pubs.acs.org/JAFC

Chitin Microfibers Reinforce Soy Protein Gels Cross-Linked by Transglutaminase Yang Yuan,† Ying-En Sun,† Zhi-Li Wan,† Xiao-Quan Yang,*,†,‡ Jun-Feng Wu,† Shou-Wei Yin,† Jin-Mei Wang,† and Jian Guo† †

Research and Development Center of Food Proteins, College of Light Industry and Food, South China University of Technology, Guangzhou 510640, PR China ‡ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China ABSTRACT: To improve the gel strength, we attempt to introduce the microcomposite concept into the food gel system. A stable positively charged chitin microfibers (CMFs) suspension was fabricated by a facile microfluidizer approach without changing its chemical structure. The obtained CMFs bearing width of about 0.5−5 μm and length of more than 500 μm were then developed in a transglutaminase cross-linked β-conglycinin (7S) gel. The morphological and rheological characterizations of the 7S-CMF composited gels were done as a function of the protein and CMFs concentrations. Results showed that the presence of the CMFs network improved the gel strength significantly. This effect was CMFs content dependent and was related to the formation of a sponge-like porous microstructure. We inferred that the CMFs provided an initial framework for gel formation and added structural rigidity to the protein gel. The role of protein was to participate in network development as an electrostatic coating and gelation component. KEYWORDS: β-conglycinin, chitin, microfiber, reinforce, composite, gel

■

absorption.11 However, chitin is insoluble in all common solvents due to its rigid crystalline structure, which is a major problem in developing its uses. Even though it can be dissolved in some specific solvents such as saturated calcium solvent,12 using them by this means in food industry can be challenging. Recently, for promotion of the use of chitin, fibrillar chitin provides another interesting pathway because a lot of novel functional properties have been reported after the formation of chitin and chitosan nano- or microfibers.4,10,13−15 Araki et al.4 reported that the presence of chitin nanowhiskers significantly enhanced the gel strength of the cross-linked chitosan hydrogel. Zhou et al.14 found that the chitosan nanofibers played a role like a cross-linker and a reinforcing agent in the polyacrylamide hydrogel, resulting a significant increase in compression strength and storage modulus. Most recently, reports on using chitin nanofibers suspensions as metal sorbents have been first published.15 However, as far as we know, fibrillar chitin as food compositions have seldom been investigated until now. The applications of nano- or microfibers in the abovementioned investigations provide a novel route, and we attempt to introduce this nano- or microcomposite concept into a food system for reinforcing the food gel. Soy proteins (SP) are the most important representative of legume proteins and have been widely used to formulate foods to improve their nutritional and functional qualities. Microbial transglutaminase (MTGase), because of its low-cost mass production and is “Generally Recognized As Safe”, has been largely utilized as a food processing tool in the past few years.16

INTRODUCTION Foods should be understood as soft materials.1 It is no surprise that food technologists are getting or will be getting inspiration from other scientific fields such as material science. Novel functional properties are expected to be created by high performance composite materials in polymer-based food systems. Recently, the application of the nano- or microstructured composite concept has been observed in biopolymer-based films, hydrogels, and scaffolds.2−6 As novel functional renewable materials, natural cellulose and chitin nano- or microfibers have received increasing interest because of their abundance, biocompatibility, and specific properties.7 In addition to their high mechanical strength, they have several useful advantages such as high aspect ratio and facile chemical modification, showing a potential application in the material and food industry.4 According to previous research, the top-down method is applicable to the production of nano- or microfibers, involving physical, enzymatic, chemical, and mechanical ways.8 Compared to other approaches, microfluidization, as a novel mechanical method, has attracted a great deal of interest in recent years because it is convenient. Lee et al.9 reported that cellulose nanofibers can be prepared by the application of a microfluidizer at 138 MPa. Liu et al.10 also found that the joint treatment of wet-grinding and high-pressure homogenization can effectively disassemble chitosan particles into nanofibers. Chitin (poly-β-(1,4)-N-acetyl-D-glucosamine) is the second most abundant natural polysaccharide next to cellulose. As the waste products of the seafood industry, crab and shrimp shells are the main commercial sources of chitin, implying that there is a great profit if chitin can be used as a food ingredient. Chitin has shown a great deal of functional and nutritional benefits such as the reduction of plasma cholesterol and intestinal lipid © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4434

February April 26, April 26, April 27,

21, 2014 2014 2014 2014

dx.doi.org/10.1021/jf500922n | J. Agric. Food Chem. 2014, 62, 4434−4442

Journal of Agricultural and Food Chemistry

Article

Figure 1. Observation and viscosity of the raw suspension and treated suspension of chitosan (CS) and chitin (CTI), respectively (0.1 wt % dry sample weight). and 0.5% (w/w) dry sample weight. After 10 min of stirring, the slurries were homogenized using an Ultraturrax (T25, IKA, Staufen, Germany) at 10 000 rpm for 5 min. The obtained predispersed slurries were then passed through a homogenizer (Microfluidizers M-110 EH Processor Co., Lampertheim, Germany) under a constant pressure of 150 MPa and at a flow rate of 100 mL/min for 10 passes. The obtained chitin and chitosan fiber suspensions were stored at 4 °C prior to further analysis. Preparation of 7S-CMF Composite Gels. The gel solutions were prepared by dissolving the 7S powders into the chitin fiber suspensions directly. A certain amount of 7S powders was dissolved into a certain amount of chitin fiber suspensions (0.1%, 0.25%, and 0.5%, w/w) under continuous stirring (200 rpm) for 3 h. The pHs of mixtures were adjusted to 7.0, and then the mixtures were stored overnight at 4 °C to allow a complete hydration. For the gelation step, a MTGase concentration of 30 unit/g of 7S was used in each reaction according to the pretest. In all mixtures, 7S reached a final concentration of 4%, 6%, and 8% (w/v), respectively. After vacuum degassing, the mixtures were incubated in a water bath (45 °C, 4 h) for gel formation. ζ-Potential. The ζ-potentials of the samples were determined according to previous publications by using a Nanosizer ZS instrument (Malvern Instruments Ltd., Worcestershire, UK).10,23 The electrophoretic mobility was determined by Laser Doppler Velocimetry, and the maximum working voltage on the electrode was ±160 V. The Henry equation was then applied for calculating the ζ-potential. All measurements were conducted in triplicate. Fourier Transform Infrared Measurements (FTIR). FTIR measurements were performed on flakes of sample powder mixed with KBr. The sample quantity (about 5 mg) in KBr (200 mg) was chosen in order to optimize the flake transmittance and to obtain a well detectable absorption in the spectral region of 4000−400 cm−1. All spectra were recorded at 25 °C, with a resolution of 4 cm−1, using a Vector-33 interferometer (Bruker Optics, Ettlingen, Germany) working under a vacuum to avoid intense spectral components. Low Amplitude Dynamic Oscillatory Measurements. Steady shear viscosities and dynamic rheological measurements were conducted by a HAAKE RheoStress 600 rheometer (Thermo electron GmbH, Karlsruhe, Germany) equipped with a parallel plates geometry (plate sensor PP35 Ti, 35 mm diameter, 1 mm gap) and a circulating system for temperature control. In the steady shear experiment, the apparent viscosity was measured across a range of shear rates (0.01− 300 s−1) at 25 °C. As for dynamic rheological analyses, time-sweep analysis was used to monitor the gelation kinetics of gel samples, which were induced by MTGase. The sample solutions were placed on the plate immediately after the addition of MTGase. Subsequently, the test at a frequency of 1 Hz and a strain of 0.5% (within linear

There have been several reports on the gelation of SP induced by MTGase.17,18 Tang et al.18 found that MTGase cross-linked glycinin-rich SP gel showed significantly higher gel strength than β-conglycinin-rich SP gel. In recent years, MTGase induced SP-polysaccharide composite gels, which showed notably improved gel strength, are attracting increased attention. Guo et al.19 introduced a novel method regarding MTGase induced SP-based hydrogel by complexing SP with dextran sulfate. Most recently, Zhang et al.20 investigated the combined cross-linking of MTGase and Maillard cross-linking, indicating that denser and finer networks of combined-treated gels were formed compared to those of the SP gels. β-Conglycinin (7S), one of the main fractions of SP, is a glycoprotein, showing a great potential in delivery of hydrophilic nutrients. However, 7S gels show poor gel strength, which could limit its uses. In this study, we prepared a chitin microfibers (CMFs) network in an aqueous system by a facile mechanical top-down approach and investigated the morphology and structure of the obtained CMFs network. Subsequently, use of CMFs for reinforcement of MTGase crosslinked 7S gels was investigated to reveal its potentially attractive application in food. Small and large deformation rheological analyses were used to investigate gelation kinetics and mechanical properties. The microstructures of the gels were characterized by using scanning electron microscopy.

■

MATERIALS AND METHODS

Materials. Defatted soybean flakes were purchased from Shandong Xinjiahua Industrial and Commercial Co. Ltd., China. The βconglycinin-rich SP was prepared according to Nagano’s procedure.21 Protein content of 7S lyophilized powder was 89.7 ± 0.4% determined by the Dumas method (N × 5.71) in a Rapid N Cube (Elementar France, Villeurbanne, France). The chitosan (molecular weight about 300 kDa; degree of deacetylation of 95%; moisture 8.0%; ash content 0.7%) and chitin (degree of deacetylation ≤50%; moisture 9.0%; ash content 0.9%) used for experiment were purchased from Shandong Aokang Industrial and Commercial Co. Ltd., China. MTGase was purchased from Ajinomoto Co., Japan. The activity of the enzyme product was 100 U/g as determined according to the method proposed by Folk et al.22 All chemicals used were of analytical or better grade. Preparation of Chitin and Chitosan Fiber Suspensions. A certain amounts of raw chitin and/or chitosan powders were suspended into distilled water to obtain a slurry with 0.1%, 0.25%, 4435



Article

Figure 2. SEM micrographs of the raw chitin sample and high-pressure homogenized treated sample. viscoelastic region) was started when the temperature was stable at 45 °C. Uniaxial Compression. The gel samples (cubes with an edge length of 10 mm) were compressed between lubricated plates fitted to a TA.TX2 texture analyzer (Stable 194 Micro System, Surrey, U.K.) with a cylinder measuring probe (P/20a) which had a diameter of 20.0 mm. The breakdown pattern of the gels was observed by compressing the gels at a constant speed of 0.2 mm/s to 20% of their initial height at room temperature. Stress and strain were calculated according to the following equations: stress = (F/Ai) × ((Li − ΔL)/Li); strain = | ln(Li/(Li − ΔL))|, where F(N) is the force measured during compression; Ai (m2) is the initial cross sectional area of the specimen; Li (m) is the initial specimen height; ΔL (m) is the deformation. At least two gel samples were tested per treatment, and each treatment was replicated two times. Scanning Electron Microscopy (SEM) Observations. According to a previous publication,18 chitin microfibers and gel samples were immersed in liquid nitrogen for 5 min to minimize the impact of ice crystals and then freeze-dried. Lyophilized samples were cut carefully and stuck onto an SEM aluminum plate through the double-sided conductive carbon tabs. Samples were coated with gold in a E-1010 ion sputter (Hitachi High-Technologies Corporation, Tokyo, Japan) before the test. The SEM images were recorded at 25 °C using a TM3000 tabletop low vacuum scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 15 kV. Statistical Analysis. All the data were subjected to one-way analysis of variance, and correlations between the results were carried out using Statistical Analysis System Software (SAS version 9.0, SAS Institute, Cary, NC); significant differences were determined by Duncan’s multiple range test and accepted at P < 0.05.24

Figure 3. FTIR spectra of the raw chitin sample and its microfibers after mechanical treatment.

■

RESULTS AND DISCUSSION Characterization of Chitin Microfibers. Figure 1 shows the observations and viscosities of the raw suspensions and treated suspensions of chitosan and chitin, respectively. As shown in photograph, raw chitosan and chitin particles, without any treatment, both settled quickly in water (less than 2 min). After being processed by high-pressure homogenization, chitosan and chitin suspensions displayed totally different stabilities in water. Large and millimeter-scale fibers were obtained in treated chitosan suspension coupled with the settlement of these fibrils after 5 min. Treated chitin, however, was macroscopically well suspended in water and did not precipitate during 2 months of storage at 4 °C. The viscosities of these samples also can be seen in Figure 1. As a control, samples showed very low shear viscosities before high-pressure homogenization treatment. No significant increase in viscosity can be found in treated chitosan suspension. In contrast, chitin fiber suspension showed a significant increase in viscosity, suggesting that the internal structure of chitin spread out after the treatment and the increased steric hindrance effect resulted

Figure 4. Effects of pH on ζ-potential of chitin microfibers (CMFs) suspension (1 mg/mL), β-conglycinin (7S) suspension (5 mg/mL), and 7S-CMFs mixture (5 mg/mL, 7S/CMFs ratio = 5/1).

in a higher viscostiy.25 Chitin fiber suspension showed a decrease in viscosity for higher shear rates, meaning that the samples were shear thinning. It could indicate that the shear reduced the connections between chitin fibers and made the suspension flow more easily.25,26 Figure 2 shows the SEM micrographs of raw chitin powder and high-pressure treated chitin sample. Large and irregularshaped particles which were millimeter sized were observed in Figure 2A. After treatment, the irregular-shaped chitin particles disassembled into chitin fibers. These fibers intertwined with each other, and the diameter of these fibers were distributed over a range from approximately 0.5 to 5 μm (Figure 2B), 4436



Article

characteristic bands of the CMFs are similar to those of the raw chitin particle, indicating the original chemical and molecular structures of chitin were retained in the case of mechanical treatment. Liu et al.10,14 also reported that the chemical compositions of both chitosan and chitin were unchanged during the mechanical treatments at neutral pH. Stability of 7S-CMF Mixture. Electrostatic forces have been shown to have a significant effect on colloid stability in many studies regarding protein and polysaccharide interaction.28 There appears to be no investigation on the ζ-potential of chitin colloid suspension, which is probably because measurements of ζ-potential of insoluble chitin particles are unreliable. However, the ζ-potentials were successfully measured after the chitosan and chitin particles were disassembled into micro- or nanofibers.10,29 The ζ-potential− pH patterns of CMFs are shown in Figure 4, which show that CMFs were positively charged microfibers at all tested pHs (from pH 5.0 to 8.0). The pKa of amino groups was reported as 6.2−7.0.30 At a pH below the pKa, chitin microfibers carry a positive charge due to the protonation of amino groups, and the charged groups provide electrostatic repulsion between fibers.29 With pH elevation, the ζ-potentials of CMFs decreased from 39.6 ± 5.5 mV at pH 5.0 to 5.5 ± 1.9 mV at pH 8.0. It is noteworthy that CMFs showed high ζ-potentials above 27 mV during a long pH region from 5.0 to 7.0 and then decreased to 5.5 mV rapidly. This phenomenon was due to the deprotonation of the amino groups. Concomitantly, the suspended CMFs were stable at a long pH region and then settled down in 5 min at high pH (pH 8.0), suggesting that CMFs were also electrically stabilized and aggregates were obtained at high pH due to the decrease electrostatic repulsion. The ζ-potential−pH patterns of 7S are shown in Figure 4 as well. It is well-known that 7S particles carry a negative charge at pH above their pI (pH 4.3). Electrostatic interaction between 7S and CMFs should occur at all tested pH values because they carry a complementary charge. The strength of the electrostatic interaction (SEI) between oppositely charged polyelectrolytes can be estimated by calculating the absolute value of the product of the measured ζ-potential of both polymers at each pH.28 The SEI−pH curves indicated that the interactions between 7S and CMFs should be strongest at pH 6.5 and coacervates should be obtained (data not shown). However, the 7S-CMFs mixture was stable, and no coacervates were found at all tested pHs. The ζ-potential curve of 7S-CMFs mixture suggested that the charge of the mixture was protein charge dominant because it showed similar values as the 7S ζ-potential curve. Moreover, the thermal stability of CMFs was also investigated by macroscopic observation. Macroscopic observation suggested that CMFs suspension was stable at 45 °C and became unstable at the temperature above 70 °C. This phenomenon can be ascribed to the increasing attractive interaction between the chitin fiber when increasing the system temperature.25 As a result, cold-set MTGase cross-linked gelation method was used to form the 7S-CMF composite gel because it can effectively avoid the impact of heat treatment on the stability of the microfibers 7S-CMF Composite Gel. In this study, we attempted to investigate the effect of CMFs on gelation rate and extent of soy 7S globulin. Therefore, dynamic viscoelastic properties of the 7S-CMF composite gels were investigated by small deformation rheological measurements. Storage modulus (G′) and loss modulus (G″) values represent the elastic and viscous

Figure 5. Time sweep rheological profiles of 7S-CMF composite gels as a function of 7S concentrations (A: 4%, B: 6%, and C: 8%).

meaning that chitin microfibers (CMFs) were obtained. Many research studies have clarified several advantages regarding high-pressure homogenization for obtaining chitin nano- or microfibers such as high mechanical strength, high aspect ratio, and adsorption of protein and metal ions.15 Most recently, Tzoumaki et al.27 successfully fabricated an oil-in-water Pickering emulsion which was stabilized by chitin nanocrystal. Overall, high-pressure homogenization successfully disassembled the chitin particles into stable water suspensible chitin microfibers, which could be used in food as a thickening and reinforced filler. The chemical composition of chitin was obviously unchanged during the high-pressure homogenization treatments, which can be seen in Figure 3. The chemical composition analysis of chitin was based on the identification of bands related to the functional groups present in chitin: the OH stretching band at 3480 cm−1, NH stretching band at 3270 cm−1, amide band I at 1660 and 1620 cm−1, and amide II band at 1560 cm−1. Both the position and relative intensity of 4437



Article

Figure 6. SEM images of 7S-CMF composite gels as a function of 7S concentrations (A and D: 4%; B and E: 6%; C and F: 8%; A, B, and C: 7S gels; D, E, and F: CMFs composited 7S gels).

components in the network.31 As shown in Figure 5, 7S dispersion showed different rheological profiles at different protein contents. Irregular scatter diagrams were obtained at 7S concentration of 4%, suggesting that the 7S dispersion was still in the liquid state, and no gelation networks were obtained at such a low concentration. As long as the protein content (6% and 8%, w/v) exceeded the critical gelation concentration, G′ and G″ of 7S dispersion developed similarly, and the G′ values were significantly greater in magnitude than the G″ values. As for 7S-CMF composite gels, significant enhancements of gel strength were observed in Figure 5. As shown in Figure 5A, compared to the 7S gel, the 7S-CMF composite gel displayed a more organized pattern even though it was still liquid state in appearance. This phenomenon may be related to the presence of CMFs network that provided an initial framework and the G′ values were stable around 1 Pa (CMFs gel in Figure 5). However, the lack of protein reacting sites resulted in a low elastic and viscous modulus. Significant increases of viscoelastic modulus were found at a higher protein concentration, representing an evident increase in G′ values of the 7S-CMF composite gel of about 10 times compared with those of the 7S gel at both protein content of 6% and 8%. Meanwhile, the gelation time (tgel), which is generally accepted as the time when the value of G′ becomes greater than that of G″ and is greater than 10 Pa,32 was significantly (p < 0.05) shortened in the presence of CMFs. In detail, the values of tgel at protein

content of 6% and 8% decreased from 2000 and 1900 s (7S gel) to 800 and 300 s (7S-CMF composite gel), respectively. The changes in rheological and mechanical properties of gels are highly related to the changes in microstructure. One way to look at the inside of a gel is with SEM. Typically, SEM showed better resolution than confocal laser scanning microscopy (CLSM), but the sample used in SEM requires rather a severe and time-consuming sample preparation including freezedrying.33 The porous and cellular microstructures observed with SEM are probably induced by ice crystal formation during the freezing step in sample preparation.34 As presented in Figure 6, the results of SEM showed that microstructural modifications occur in 7S-CMF composite gels, in particular, changes in porosity, including an increase in the amount and a decrease in size. In detail, 7S gels showed disordered and lamellar structures at 4%, while showed typical interconnected macroporous morphologies with pore sizes ranging from 10 to 40 μm at 6% and 8%, indicating that the more protein the gel had the more compact and homogeneous the microstructure was. Sponge-like pore structures were formed as seen in 7SCMF composite gels (Figure 6D−F). The size of the pores is around 3−9 μm. Beginning with the thin and shallow networks at low protein content, thicker and smoother networks and deeper pores were observed with an increase in protein content. Such changes in microstructure could affect gel rheological properties and explain the increase in gel viscoelastic modulus. 4438



Article

into a hydrogel, showing a similar decreases in pore size and increases in gel strength. Figure 7A showed that the elastic modulus increased while the tgel decreased significantly (p < 0.05) with increased concentrations of CMFs, suggesting that the formation of gel was significantly influenced by the CMFs content. Previous papers have clarified the effect of polysaccharide concentration on the formation of protein−polysaccharide mixed gel. Guo et al.19 showed that the gelation of soy protein was accelerated by adding dextran sulfate, and the strengths of the formed gels were dextran sulfate amount dependent. Le et al.35 found the βlactoglobulin and xanthan gum ratio strongly affected gelation kinetics and was a main factor controlling the gelation process and the gel structure. Uniaxial compression measurements were performed to investigate the fracture properties (Figure 7B). 7S gel fractured at relatively low strain and stress values which coincided with previous investigations,36 suggesting a weak and brittle gel was formed. 7S-CMF composite gel showed different stress−strain curves. As expected, increasing CMFs concentration caused an increase in both fracture stress and fracture strain. The value of the fracture stress increased from 5.8 to 17.0 kPa, 24.7 and 28.4 kPa, while the fracture strain increased from 0.315 to 0.551, 0.553, and 0.614 with CMFs content elevation. These agreed with the small deformation rheological investigations that showed a similar trend of G′ values for 7SCMF composite gels. To further confirm the data of small and large deformation tests, SEM images of 7S-CMF composite gels were recorded as a function of CMFs contents. As shown in Figure 8, a significantly different microstructure can be found between the 7S-CMF composite gels and control sample, and then smaller and regular pores were obtained when the CMFs concentration increased. The overall data suggested that the great enhancement in gel strength may be related to the modification of gel microstructure, and this modification was ratio dependent. General Discussion. In this work, a stable chitin microfibers suspension was obtained by passing chitin particles through a high-pressure homogenization equipment. The

Figure 7. (A) G′, tgel, and (B) stress−strain curves of 7S-CMF composite gels as a function of CMFs concentrations at a 7S concentration of 6%.

Aouada et al.5 also found a three-dimensional, well-oriented porous microstructure when they added cellulose nanofibers

Figure 8. SEM images of 7S-CMF composite gels as a function of CMFs concentrations at 7S concentration of 6% (A: 0%, B: 0.1%, C: 0.25%, and D: 0.5%). 4439



Article

Figure 9. Schematic illustration of the formation of 7S-CMF composite gel (A: the formation of MTGase induced 7S gel; B: the formation of CMFs; C: the formation of 7S-CMF composite gel).

obtained novel fiber material was used in 7S gel as a thickening and reinforced filler because the advantages such as high viscosity, high aspect ratio, and facile electrostatic modification (Figures 1, 2, and 4). Compared to MTGase induced 7S gel, the addition of CMFs enhanced the gel strength and modified the gel structure. To clarify a reasonable understanding of this improvement, a schematic illustration of the formation of 7SCMF composite gel is proposed and shown in Figure 9. β-Conglycinin is one of the main fractions of SP. MTGase induced 7S gelation has been fully investigated in past 20 years, concluding that transglutaminase catalyzed an acyl-transfer reaction between the γ-carbonyl group of a glutamine residue and the ε-amino group of a lysine residue. The resultant covalent connections between the protein molecules formed the three-dimensional networks of the gel.16 If protein underwent a heat treatment, more groups were exposed from the hydrophobic core of the molecules, and this treatment favored the MTGase cross-linking reaction.19 In this paper, heating cannot be used, and this type of MTGase cross-linked unheated 7S gel showed typical interconnected macroporous protein backbone and relatively weak gel strength which were proven by means of SEM and rheology technique (Figure 9A). As shown in Figures 1 and 2, high-pressure homogenization disassembled the chitin particles into stable water suspensible chitin microfibers (Figure 9B). Most interestingly, the result of ζ-potentials showed that the obtained CMFs still carried a positive charge at neutral pHs. The reason for this phenomenon is probably because the release, from within the chitin particles, of some unacetylated amino groups increased the protonation on the microfiber surfaces.37 Liu et al.10 reported a similar result using chitosan and chitin suspension. The adsorption of metal ions by these materials have been reported due to the microfibrous structure and electrostatic attraction.15 Most interestingly, some researchers suggested that the presence of nano- or microfiber significantly increased the mechanical and swelling properties of biomaterial hydro-

gel.38,39 From this viewpoint, a hypothesis is proposed in Figure 9C. When a 7S-CMF mixture was obtained at neutral pH, the presence of a CMFs network provided an initial framework, and then the protein coated on the CMFs network via electrostatic attractive force because protein carried a complementary charge in this case. However, the presence of excessive protein in the aqueous phase might exist around the CMFs framework and produce a net negative charge of the whole system (Figure 4). After MTGase induced covalent cross-linking, the 7S-CMF composite gel system was achieved. The CMFs played a key role as a gel backbone, while the role of the 7S protein was to participate in network development as a coating and gelation component. Thanks to the presence of CMFs, a more organized and rigid microstructure was remodeled, resulting a shorter gelation time and stronger gel strength. This reinforced behavior indicated that CMFs in this work had a similar reinforcing effect with other nano- or microfillers such as nanoparticles and nanocrystals, which can indicate that the uniformly dispensed nano- or microfillers can transfer the load from the polymer chain to themselves and stop the growing of microcracks.38−40 Moreover, Zhou et al.39 suggested that the mechanical properties of composite hydrogels depend not only on the reinforced properties of fillers, but also on interactions between the filler and the matrix, resembling the electrostatic coating of SP on rthe CMFs network in this paper. In conclusion, a stable chitin microfibers suspension was successfully fabricated by passing chitin particles through a microfluidizer. As shown in the morphology analysis, highpressure treatment disassemble the chitin particles into chitin microfibers. As a result, the enlarged steric structure and released amino groups improved the colloidal stability by a steric stabilizing and electrostatic repulsion effect. The most interesting finding in this paper was that the presence of the CMFs network improved the gel strength of the MTGase 4440



Article

(14) Zhou, C. J.; Wu, Q. L. A novel polyacrylamide nanocomposite hydrogel reinforced with natural chitosan nanofibers. Colloid. Surface. B 2011, 84, 155−162. (15) Liu, D. G.; Zhu, Y.; Li, Z. H.; Tian, D. L.; Chen, L.; Chen, P. Chitin nanofibrils for rapid and efficient removal of metal ions from water system. Carbohyd. Polym. 2013, 98, 483−489. (16) Motoki, M.; Seguro, K. Transglutaminase and its use for food processing. Trends Food Sci. Technol. 1998, 9, 204−210. (17) Tang, C. H.; Li, L.; Wang, J. L.; Yang, X. Q. Formation and rheological properties of ‘cold-set’ tofu induced by microbial transglutaminase. LWT–Food Sci. Technol. 2007, 40, 579−586. (18) Tang, C. H.; Wu, H.; Chen, Z.; Yang, X. Q. Formation and properties of glycinin-rich and β-conglycinin-rich soy protein isolate gels induced by microbial transglutaminase. Food Res. Int. 2006, 39, 87−97. (19) Guo, J.; Zhang, Y.; Yang, X. Q. A novel enzyme cross-linked gelation method for preparing food globular protein-based transparent hydrogel. Food Hydrocolloids 2012, 26, 277−285. (20) Zhang, J. B.; Wu, N. N.; Yang, X. Q.; He, X. T.; Guo, J.; Zhu, J. H. Improvement of microbial transglutaminase-induced gelation of βconglycinin by conjugation with dextran. Int. J. Food Sci. Technol. 2014, 49, 976−982. (21) Nagano, T.; Hirotsuka, M.; Mori, H.; Kohyama, K.; Nishinari, K. Dynamic viscoelastic study on the gelation of 7S globulin from soybeans. J. Agric. Food Chem. 1992, 40, 941−944. (22) Folk, J. E.; Cole, P. W. Structural requirements of specific substrates for guinea pig liver transglutaminase. J. Biol. Chem. 1965, 240, 2951−2960. (23) Yuan, Y.; Wan, Z. L.; Yang, X. Q.; Yin, S. W. Associative interactions between chitosan and soy protein fractions: Effects of pH, mixing ratio, heat treatment and ionic strength. Food Res. Int. 2014, 55, 207−214. (24) Duncan, D. B. Multiple Range and Multiple F Tests. Biometrics 1955, 11, 1−42. (25) Tzoumaki, M. V.; Moschakis, T.; Biliaderis, C. G. Metastability of nematic gels made of aqueous chitin nanocrystal dispersions. Biomacromolecules 2009, 11, 175−181. (26) Kuijk, A.; Koppert, R.; Versluis, P.; van Dalen, G.; Remijn, C.; Hazekamp, J.; Nijsse, J.; Veikov, K. P. Dispersions of attractive semiflexible fiberlike colloidal particles from bacterial cellulose microfibrils. Langmuir 2013, 29, 14356−14360. (27) Tzoumaki, M. V.; Moschakis, T.; Kiosseoglou, V.; Biliaderis, C. G. Oil-in-water emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids 2011, 25, 1521−1529. (28) Espinosa-Andrews, H.; Enríquez-Ramírez, K. E.; GarcíaMárquez, E.; Ramírez-Santiago, C.; Lobato-Calleros, C.; VernonCarter, J. Interrelationship between the zeta potential and viscoelastic properties in coacervates complexes. Carbohyd. Polym. 2013, 95, 161− 166. (29) Li, J.; Revol, J.; Naranjo, E.; Marchessault, R. Effect of electrostatic interaction on phase separation behaviour of chitin crystallite suspensions. Int. J. Biol. Macromol. 1996, 18, 177−187. (30) Hejazi, R.; Amiji, M. Chitosan-based gastrointestinal delivery systems. J. Controlled Release 2003, 89, 151−165. (31) Leksrisompong, P. N.; Foegeding, E. A. How micro-phase separation alters the heating rate effects on globular protein gelation. J. Food Sci. 2011, 76, 318−327. (32) Abbasi, S.; Dickinson, E. Influence of sugars on high-pressure induced gelation of skim milk dispersions. Food Hydrocolloids 2001, 15, 315−319. (33) Van den Berg, L.; Rosenberg, Y.; Van Boekel, M. A.; Rosenberg, M.; Van de Velde, F. Microstructural features of composite whey protein/polysaccharide gels characterized at different length scales. Food Hydrocolloids 2009, 23, 1288−1298. (34) Van Kleef, F. S. M. Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels. Biopolym. 1986, 25, 31−59.

cross-linked 7S gel significantly. The amount of CMFs was found to be a factor that affected the gel properties as well. The more CMFs the gel contained, the faster and the tougher the gel formed. The fracture stress and fracture strain also increased as a function of CMFs content. We inferred that the presence of CMFs provided an initial framework for gel formation and added structural rigidity to the 7S gel. The role of protein was to participate in network development as an electrostatic coating and gelation component. These findings will provide a new path for application of chitin in food.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: (+86 20) 87114262. Fax: (+86 20) 87114263. E-mail: [email protected]; [email protected]. Funding

This work is part of the research projects of Chinese National Natural Science Fund (Serial Number: 31371744 and 31130042, sponsored by the NSFC) and National High Technology Research and Development Program of China (863 Program: 2013AA102208-3). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Mezzenga, R.; Schurtenberger, P.; Burbidge, A.; Michel, M. Understanding foods as soft materials. Nat. Mater. 2005, 4, 729−740. (2) Shams, M. I.; Nogi, M.; Berglund, L. A.; Yano, H. The transparent crab: preparation and nanostructural implications for bioinspired optically transparent nanocomposites. Soft Matter 2012, 8, 1369−1373. (3) Fernandes, S. C. M.; Oliveira, L.; Freire, C. S. R.; Silvestre, A. J. D.; Neto, C. P.; Gandini, A.; Desbriéres, J. Novel transparent nanocomposite films based on chitosan and bacterial cellulose. Green Chem. 2009, 11, 2023−2029. (4) Araki, J.; Yamanaka, Y.; Ohkawa, K. Chitin-chitosan nanocomposite gels: reinforcement of chitosan hydrogels with rod-like chitin nanowhiskers. Polym. J. 2012, 44, 713−717. (5) Aouada, F. A.; de Moura, M. R.; Orts, W. J.; Mattoso, L. H. Preparation and characterization of novel micro- and nanocomposite hydrogels containing cellulosic fibrils. J. Agric. Food Chem. 2011, 59, 9433−9442. (6) Yano, H.; Sugiyama, J.; Nakagaito, A. N.; Nogi, M.; Matsuura, T.; Hikita, M.; Handa, K. Optically transparent composites reinforced with networks of bacterial nanofibers. Adv. Mater. 2005, 17, 153−155. (7) Päak̈ kö, M.; Ankerfors, M.; Kosonen, H.; Nykänen, A.; Ahola, S.; Ö sterberg, M.; Ruokolainen, J.; Laine, J.; Larsson, P. T.; Ikkala, O.; Lindstrom, T. Enzymatic hydrolysis combined with mechanical shearing and high-pressure homogenization for nanoscale cellulose fibrils and strong gels. Biomacromolecules 2007, 8, 1934−1941. (8) Zeng, J. B.; He, Y. S.; Li, S. L.; Wang, Y. Z. Chitin whiskers: an overview. Biomacromolecules 2012, 13, 1−11. (9) Lee, S. Y.; Chun, S. J.; Kang, I. A.; Park, J. Y. Preparation of cellulose nanofibrils by high-pressure homogenizer and cellulose-based composite films. J. Ind. Eng. Chem. 2009, 15, 50−55. (10) Liu, D. G.; Chang, P. R.; Chen, M. D.; Wu, Q. L. Chitosan colloidal suspension composed of mechanically disassembled nanofibers. J. Colloid Interface Sci. 2011, 354, 637−643. (11) Koide, S. S. Chitin-chitosan: properties, benefits and risks. Nutr. Res. (N.Y.) 1998, 18, 1091−1101. (12) Nagahama, H.; Nwe, N.; Jayakumar, R.; Koiwa, S.; Furuike, T.; Tamura, H. Novel biodegradable chitin membranes for tissue engineering applications. Carbohyd. Polym. 2008, 73, 295−302. (13) Liu, D. G.; Wu, Q. L.; Chang, P. R.; Gao, G. Z. Self-assembled liquid crystal film from mechanically defibrillated chitosan nanofibers. Carbohyd. Polym. 2011, 84, 686−689. 4441



Article

(35) Le, X. T.; Turgeon, S. L. Rheological and structural study of electrostatic cross-linked xanthan gum hydrogels induced by βlactoglobulin. Soft Matter 2013, 9, 3063−3073. (36) Renkema, J. M. S.; Knabben, J. H. M.; Van Vliet, T. Gel formation by β-conglycinin and glycinin and their mixtures. Food Hydrocolloids 2001, 15, 407−414. (37) Marchessault, R. H.; Morehead, F. F.; Walter, N. M. Liquid crystal systems from fibrillar polysaccharides. Nature 1959, 184, 632− 633. (38) Shin, M. K.; Spinks, G. M.; Shin, S. R.; Kim, S. I.; Kim, S. J. Nanocomposite hydrogel with high toughness for bioactuators. Adv. Mater. 2009, 21, 1712−1715. (39) Zhou, C. J.; Wu, Q. L.; Yue, Y. Y.; Zhang, Q. G. Application of rod-shaped cellulose nanocrystals in polyacrylamide hydrogels. J. Colloid Interface Sci. 2011, 353, 116−123. (40) Wu, Y. T.; Zhou, Z.; Fan, Q. Q.; Chen, L.; Zhu, M. F. Facile insitu fabrication of novel organic nanoparticle hydrogels with excellent mechanical properties. J. Mater. Chem. 2009, 19, 7340−7346.

4442


Chitin Microfibers Reinforce Soy Protein Gels ... - ACS Publications

Recommend Documents