Article pubs.acs.org/JAFC
Quercetagetin-Loaded Zein−Propylene Glycol Alginate Ternary Composite Particles Induced by Calcium Ions: Structure Characterization and Formation Mechanism Cuixia Sun, Yang Wei, Ruirui Li, Lei Dai, and Yanxiang Gao* Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China ABSTRACT: The complexation of zein and propylene glycol alginate (PGA) was confirmed to improve the entrapment efficiency and loading capacity of quercetagetin (Q) in our previous study. The present work focused on the influence and induction mechanism of calcium ions on structures of Q-loaded zein−PGA ternary composite particles. The incorporation of Ca2+ resulted in the formation of aggregates with a large dimension between zein particles, led to obvious conformational, secondary, and tertiary structural changes of zein, and caused the disappearance of crystalline structure of zein. PGA exhibited a fine filamentous network structure and became much thicker and stronger in the presence of Ca2+. The presence of Q promoted the affinity and binding capacity of Ca2+ to zein and PGA. An interwoven network structure with enhanced firmness and density was observed in Q-loaded zein−PGA composite particles, leading to improved thermal stability. Three potential mechanisms were proposed to explain the structural characteristics induced by Ca2+, including particle−particle collision for zein particles, chain−chain association for PGA molecules, and simultaneous cross-linking coupled with aggregating for Q-loaded zein−PGA composite particles. KEYWORDS: zein, propylene glycol alginate, quercetagetin, calcium induction, structure, morphology
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albumin,15 and soy protein isolate.16 Zein, as a typical prolamin, contains a high proportion of nonpolar amino acid residues, which makes up >50% of its total amino acid content.17 Due to its inherent hydrophobicity, zein can be easily fabricated into spherical colloidal nanoparticles by the antisolvent precipitation method, which makes it an ideal delivery system for drugs and micronutrients in the food, pharmaceutical, and biotechnological industries.18 To the best of our knowledge, little information is available about the influence of calcium ions on the structural modification of alcohol-soluble proteins. We hypothesize that the incorporation of Ca2+ will modify the physicochemical properties of zein, which may be adapted to the requirement of processes and broaden its application fields. According to the aforementioned descriptions, it is worth mentioning that calcium ions are commonly utilized to modify the properties of individual water-soluble proteins or hydrophilic polysaccharides. Any application of calcium ions to the biopolymer complexes is still limited. Zein−PGA binary complexes were formed through the noncovalent interaction forces according to our previous paper.10 Therefore, we are extremely fortunate to have access to exploit the structural and morphological characteristics of zein−PGA binary complex in the presence of Ca2+. Quercetagetin (Q), as a typical alcohol-soluble flavonol compound, exhibited a stronger antioxidant activity than quercetin due to a similar structure but with an additional 6-
INTRODUCTION Calcium ions have a number of diverse functions in biological systems, from biomineralization in bones, teeth, and shells to a complex role as an intracellular messenger.1 In recent years, calcium ions are particularly applied to fabricate hydrogel beads as delivery systems of bioactive compounds based on foodgrade hydrophilic polysaccharides, such as starch,2 pectin,3 carrageenan,4 and especially alginate5 due to the formation of cationic bridges between the guluronic-rich entities along the biopolymer backbone.6 Propylene glycol alginate (PGA), chemically modified alginate, is the reaction derivative between propylene oxide and alginic acid, which is composed of 1,4linked D-mannuronic acid (31−65%) and L-guluronic acid (69− 35%).7 Compared to nonesterified alginates, PGA possesses unique surface-active properties and is much less sensitive to low-pH conditions attributed to the existence of propylene glycol groups. PGA has been approved for utilization as a stabilizer and emulsifier as well as thickener in a variety of food products.8 Previous studies uncovered the gelling potential of PGA in the presence of calcium ions.9 However, investigations referring to the effect of calcium ions on the structure and morphology of PGA are still limited. According to our previous study, PGA showed a great solubility in aqueous ethanol solution.10 It may be interesting to explore the characteristics of PGA induced by Ca2+ under the condition of aqueous ethanol solution. In addition to hydrophilic polysaccharides, calcium ions are also usually used to modify the functional and structural properties of water-soluble proteins, such as carp muscle protein,11 bovine plasma protein,12 wheat germ protein hydrolysates,13 whey protein concentrate,14 bovine serum © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
February 27, 2017 April 27, 2017 May 1, 2017 May 1, 2017 DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry OH group attached to the flavone backbone.19 Such a structure with many hydroxyls made Q show a strong affinity to proteins,20 which induced a significant effect on the tertiary structure of zein.21 The combination of zein and PGA was confirmed to improve the entrapment efficiency and loading capacity of Q in our previous study.10 As a result, it was proposed that the addition of calcium ions may lead to unique distinct changes in the physicochemical and structural properties of Q-loaded zein−PGA composite particles. It should be noted that PGA at low and intermediate degrees of esterification was able to form gels with relative strength.9 However, in the present work gelling of PGA was not the goal. Therefore, to avoid the occurrence of gel formation, PGA with a high degree of esterification (87.9%) was selected because it showed poor gelling ability due to the lower fraction of protonizable carboxyl groups. The object of the present work was to explore the influence of calcium ions at different concentrations on the structural and morphological characteristics of individual zein and PGA, zein− PGA binary complexes, and Q-loaded zein−PGA ternary composite particles. Besides, the potential mechanism was proposed to explain these characteristic changes of individual biopolymers and their complexes. Fourier-transform infrared and X-ray diffraction spectroscopies were applied to determine the possible interaction among zein, PGA, and Q after inducement by calcium ions. Far-/near-UV circular dichroism spectroscopy was utilized to probe the secondary and tertiary structural changes of zein. Field emission scanning electron microscopy was introduced to observe the micromorphology of samples in the presence of calcium ions. Findings from this work will provide a theoretical basis for the application of calcium ion induction on the characteristic modification of individual or complex biopolymers, especially for alcoholsoluble proteins, and may bring new insight into the development of potential carriers for bioactive compounds.
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parts of the dispersions were frozen and dried for 48 h with an Alpha 1-2 D Plus freeze-drying apparatus (Marin Christ, Germany) to obtain dry particles for solid state characterization analysis. The samples of zein, zein−Q, PGA, PGA−Q, and zein−PGA complex colloidal dispersions with various levels of Ca2+ were obtained by the aforementioned process. At the same time, all samples in the absence of Ca2+ were prepared by the aforementioned method and used as the reference. In this work, samples of zein, zein−Q, PGA, PGA−Q, zein−PGA, and Q-loaded zein−PGA composite particles in the presence of different Ca2+ concentrations (1.5625, 3.1250, 6.2500, and 12.5000 mM) were divided into six types and termed (i) zein-1, zein-3, zein-6, and zein-12; (ii) zein−Q-1, zein−Q-3, zein−Q-6, and zein−Q-12; (iii) PGA-1, PGA-3, PGA-6, and PGA-12; (iv) PGA−Q-1, PGA−Q-3, PGA−Q-6, and PGA−Q-12; (v) zein−PGA-1, zein−PGA-3, zein− PGA-6, and zein−PGA-12; and (vi) zein−PGA−Q-1, zein−PGA−Q3, zein−PGA−Q-6, and zein−PGA−Q-12, respectively. Particle Size and Zeta-Potential. Particle size and zeta-potential of colloidal dispersions in the presence and absence of Ca2+ were determined using a combined dynamic light scattering (DLS) and particle electrophoresis instrument (Zetasizer Nano-ZS90, Malvern Instruments Ltd., Worcestershire, UK). The particle size data were reported as a cumulative mean diameter (size, nm), which was calculated by the intensity weighted using the Stokes−Einstein equation. Zeta-potential of samples was determined by measuring the direction and velocity of particle movement in a well-defined electric field, and the data were obtained by the instrument using the Smoluchowski model. All measurements were carried out at room temperature (25 °C), and each sample was analyzed in triplicate. Fluorescence Spectroscopy. Fluorometric experiments were performed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). The excitation wavelength was set at 280 nm, and the emission spectra were collected between 290 and 450 nm with a scanning speed of 100 nm/min. Intrinsic fluorescence of the protein was measured at a constant concentration of 0.2 mg/mL. All data were collected at room temperature, and each individual emission spectrum was the average result of three runs. Circular Dichroism Spectroscopy. Both far-UV (190−250 nm) and near-UV (250−320 nm) CD spectra were recorded using a CD spectropolarimeter (Pistar π-180, Applied Photophysics Ltd., Surrey, UK).10 The protein concentration was 0.2 mg/mL, the path lengths were 0.1 and 1.0 cm for far-UV and near-UV regions, respectively, and constant nitrogen flush was applied during data acquisition. Ellipticity was recorded at a speed of 100 nm/min, 0.2 nm resolution, 20 accumulations, and 2.0 nm bandwidth. Fourier-Transform Infrared Spectroscopy (FTIR). FTIR was used to prove the possible interactions in the system including zein, Q, and PGA in the presence of Ca2+. Infrared spectra of samples were recorded at room temperature on a Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, UK). Freeze-dried powders were analyzed as KBr pellets. Briefly, 2.0 mg of sample was mixed with 198.0 mg of pure KBr powder. The mixture was ground into fine powder, pressed into a pellet, and measured by FTIR. The spectra were acquired at the spectral width ranging from 4000 to 400 cm−1 with a 4 cm−1 resolution and an accumulation of 64 scans. Pure KBr powder was used as a baseline. X-ray Diffraction (XRD). The effect of Ca2+ on the crystalline structure of the samples was analyzed on a wide-angle X-ray diffractometer (Bruker D8, Germany). The instrument was equipped with a copper anode that produces Cu Kα radiation and was operated at 40 kV of accelerating voltage and 40 mA of tube current. The 2θ angle was set from 5° to 50° in continuous mode using a step size of 0.02° and a step time of 5 s. Thermogravimetric Analysis. Dynamic thermogravimetric analysis of samples was carried out with a thermogravimetric analyzer model STA 449 F3 Jupiter (NETZSCH) according to a modified method.23 The measurements were performed on samples of about 10 mg, placed in ceramic crucibles, heated from 25 to 600 °C at a heating rate of 10 °C/min, under a nitrogen atmosphere with a nominal gas flow rate of 25 mL/min.
EXPERIMENTAL PROCEDURES
Materials. Zein with a protein content of 91.3% (w/w) and calcium chloride (≥99.9%) were purchased from Sigma-Aldrich Chemical Co. (USA). Propylene glycol alginate with an esterification degree of 87.9% was kindly provided by Hanjun Sugar Industry Co. Ltd. (Shanghai, China). Quercetagetin with a purity of 91% (w/w) was extracted from marigold (Tagetes erecta L.) flower according to the method described in our previous study.19,22 Absolute ethanol (99.99%), solid sodium hydroxide, and liquid hydrochloric acid (36%, w/w) were acquired from Eshowbokoo Biological Technology Co., Ltd. (Beijing, China). Preparation of Q-Loaded Zein−PGA Composite Particles in the Presence of Calcium Ions. Q-loaded zein−PGA composite particles were prepared by the antisolvent coprecipitation method coupled with calcium ion induction. Briefly, zein (1.0 g) and PGA (0.2 g) were dissolved in 100 mL of aqueous ethanol solution (70%, v/v) with stirring at 700 rpm on a magnetic for 60 min until completely soluble. Q powder (0.1 g) was added to glass beakers containing zein− PGA aqueous ethanol solution. The mixtures were bath-sonicated for 10 min, and the resulting Q-loaded zein−PGA complex suspensions were centrifuged at 765g for 10 min to remove the undissolved Q. The final Q-loaded zein−PGA complex stock solution (40 mL) was injected in 2 min to the beaker containing 120 mL of aqueous solutions with 1.5625, 3.1250, 6.2500, or 12.5000 mM CaCl2. The pH of final dispersions was adjusted to 4.0 by 0.1 M NaOH or HCl. To acquire aqueous dispersions, approximately three-fourths of ethanol was removed under reduced pressure (−0.1 MPa) by rotary evaporation at 45 °C for 35 min. Finally, Q-loaded zein−PGA composite colloidal dispersions in the presence of Ca2+ were stored in the refrigerator at 4 °C for further analysis in the form of liquid, and B
DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Effect of Ca2+ addition at different concentrations on particle size and zeta-potential of colloidal dispersions for zein (A), zein−Q (B), PGA (C), PGA−Q (D), zein−PGA (E), and zein−PGA−Q (F), respectively.
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Field Emission Scanning Electron Microscopy (FE-SEM). The microstructure of the freeze-dried samples was observed by a field emission scanning electron microscope (FE-SEM, SU8010, Hitachi) at an accelerating voltage of 5.0 kV. Prior to the observation, the surfaces of samples were sputter-coated with a gold layer to avoid charging under the electron beam. Statistical Analysis. All results are reported as means and standard deviations for at least three replicate samples, and statistical differences were evaluated using SPSS 18.0 for Windows (SPSS Inc., Chicago, IL, USA). Differences between pairs of means were compared using a Tukey test. Least significant differences (P < 0.05) were accepted among the treatments.
RESULTS AND DISCUSSION Particle Size and Zeta-Potential. As shown in Figure 1, at a designed pH 4.0, the size and zeta-potential of particles were dependent on the calcium concentration. For the sample of zein (Figure 1A) or zein−Q (Figure 1B), the incorporation of Ca2+ resulted in an increased size and a decreased zeta-potential of particles, especially the higher the calcium level, the larger the size and the lower the surface positive charge of particles. For instance, when the Ca2+ concentration was increased from 1.5625 to 12.5000 mM, the particle dimension was increased from 142.3 ± 2.0 to 260.5 ± 7.4 nm and from 156.1 ± 4.9 to 415.8 ± 27.6 nm for the samples of zein and zein−Q, respectively. The result may be expected from the reduced zetaC
DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Effect of Ca2+ addition on the conformation of zein. Panels A, B, C, and D represent samples of zein, zein−Q, zein−PGA, and zein−PGA− Q, respectively.
potential, from 17.9 ± 0.6 to 12.7 ± 1.1 mV and from 22.1 ± 0.4 mV to 13.8 ± 0.3 mV, respectively. Both positively and negatively charged patches are deemed accessible on a given protein surface.24 One possible explanation for the reduced positive net charge was that Ca2+ interacted with the negatively charged patches on the surface of zein, and the neutralization caused the decreased electrostatic inter-repulsion among zein particles and induced the generation of particle aggregates with a large dimension. A similar result was reported by Zhang et al.,25 who found that the addition of Ca2+ led to the increased size of soy protein isolate particles. Donato et al.26 also found that aggregates were formed between bovine serum albumin in the presence of CaCl2. In addition, the findings also indicated that the presence of Q made zein particles much more efficient in facilitating the aggregation, especially at a higher Ca2+ concentration. However, for the samples in the presence of PGA, the particle size was obviously reduced with the increase of Ca2+ level, which supposed that protein aggregation may be delayed by the addition of PGA. When the Ca2+ concentration was increased to 12.5000 mM, the particle sizes of samples of PGA (Figure 1C), PGA−Q (Figure 1D), zein−PGA (Figure 1E), and zein−PGA−Q (Figure 1F) were significantly decreased, from 1638.5 ± 12.0 to 498.2 ± 22.9 nm, from 1618.2 ± 3.9 to 912.0 ± 72.6 nm, from 774.1 ± 15.3 to 564.8 ± 11.4 nm, and from 759.3 ± 26.4 to 540.2 ± 39.8 nm, respectively. The result indicated that particles occupied negative charges due to the presence of PGA, which was screened by Ca2+, leading to the occurrence of cross-linking among PGA molecular chains and resulting in the formation of a more compact structure with a reduced size of particles. A similar result was obtained by Fang et al.,27 who suggested that
the binding of Ca2+ to alginate chains decreased the intramolecular repulsion and resulted in a more compact conformation of alginate gel. Donato et al.28 reported the formation of cross-links between low-methoxyl pectin chains via calcium ions. Jodra and Mijangos29 also revealed that the particle size of alginate gel was decreased as the metal concentration in the solution was increased. Fluorescence Property. The effect of Ca2+ at different levels on the conformation of zein is shown in Figure 2. For the samples of zein (Figure 2A) or zein−PGA (Figure 2C), the presence of Ca2+ led to an increase of fluorescence intensity, indicating the conformational change induced by Ca2+. The result may be ascribed to the unfolding of hydrophobic zones in zein molecules and exposure of more aromatic amino acids conserved in protein chains. Another possible explanation may be that the incorporation of Ca2+ changed the local environmental polarity of fluorophore because previous studies30 suggested that the fluorescence intensity of proteins was increased due to the decreased environmental polarity. Borgogna et al.31 revealed that the addition of calcium ions caused a modification of the electronic environment of the chromophores, resulting in a variation of the conformation. The findings were also consistent with those of Zhang et al.,25 who reported that calcium incorporation caused the enhanced fluorescence intensity of soy protein isolate (SPI). As shown in Figure 2B,D, the presence of Q resulted in decreased fluorescence intensity, which was also gradually reduced with the increase of Ca2+ concentration, indicating the occurrence of fluorescence quenching dominated by Q incorporation. The findings may be ascribed to diverse molecular interactions such as molecular rearrangements, energy transfer, ground state D
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Figure 3. Effect of Ca2+ addition on the secondary structure (A−D) and tertiary structure (E−H) of zein.
complex formation, and collisional quenching,32 which agreed
tertiary structural changes of zein as shown in Figure 3. Zein, as exhibited in Figure 3A, showed a positive peak at 193 nm and two negative peaks at 209 and 223 nm with a zero crossing around 202 nm, which was consistent with our previous
10,33
with our previous investigations. Far-/Near-UV CD Analysis. Far- and near-UV CD spectroscopies were performed to evaluate the secondary and E
DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. FTIR spectra of samples in the presence of Ca2+ at different concentrations. Panels A, B, C, D, E, and F represent samples of zein, zein−Q, PGA, PGA−Q, zein−PGA, and zein−PGA−Q, respectively.
investigation,33 implying a characteristic α-helix-rich secondary structure.34 After the addition of Ca2+, a significant decrease was observed in both positive and absolute negative ellipticities of CD spectra for all samples, indicating the loss of the α-helical structure of zein.35 At the same time, an obvious red shift occurred from 202 to 210 nm for the sample of zein at the Ca2+ concentration of 12.5000 mM. The result revealed a characteristic of the second structural change of zein, namely, the reduced content of α-helical structure consequently accompanied by a great increase in the amount of β-sheets. The findings also implied that the aggregation among zein molecules occurred at a high Ca2+ concentration because increased β-sheets were commonly found in aggregated proteins,36 which could be used to further confirm the result in Figure 1. Different secondary structural contents of zein at different concentrations of Ca2+ are calculated by SELCON3. It could be observed that the α-helix content of zein was significantly (P < 0.05) decreased from 32.9 to 15.2% after the incorporation of 12.5000 mM Ca 2+ and subsequently
accompanied by a great (P < 0.05) increase in the amount of β-sheets from 22.2 to 34.1%. Zhang et al.25 also reported a similar result when 5.0 mM calcium was added: the content of α-helix of SPI decreased to 12.6% and β-sheet increased to 29.9%. It can be found from Figure 3C,D that the changed extent of secondary structure for the samples in the presence of PGA was much smaller compared to the sample of zein or zein−Q. For example, after adding Ca2+ at a concentration of 12.5000 mM, the α-helix content of zein was reduced to 28.1 and 22.2% for the sample of zein−PGA and zein−PGA−Q, respectively. The result suggested that the presence of PGA effectively prevented secondary structural change of zein induced by Ca2+, which may be due to the fact that a kinetically competitive association between zein and PGA with Ca2+ took place. On the basis of the result, PGA seemed to show a preference to have a specific affinity to Ca2+ ascribed to its carboxy groups.37 A competition between whey proteins and low methoxyl pectins for the binding of calcium and water was proposed by Beaulieu,38 whereas zein−PGA binary composite F
DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. XRD patterns of samples in the presence of Ca2+ at different concentrations. Panels A, B, C, D, E, and F represent samples of zein, zein−Q, PGA, PGA−Q, zein−PGA, and zein−PGA−Q, respectively.
high incorporated level of Ca2+. According to the paper of Kelly et al.,39 the CD spectra in the region of 250−280 nm are influenced by the number of each type of aromatic amino acid and their spatial disposition in the protein. On the basis of the principle, the finding in the present work revealed that calcium addition resulted in the rearrangement of protein molecular chain, altered the microenvironment of the aromatic amino acids, led to restructuring of secondary structure of protein, and finally induced the different tertiary structures of zein.34 In conclusion, the higher the calcium concentration, the larger the dimension of zein particles (Figure 1A) along with a more
particles were still considered compatible because no phase separation was noted in the medium containing these two biopolymers. Compared to the spectrum of zein (Figure 3E), the incorporation of Ca2+ induced the formation of new peaks between the wavelengths of 250 and 280 nm. For example, new peaks appeared at wavelengths of 257, 267, 263, and 255 nm after the incorporation of Ca2+ at concentrations of 1.5625, 3.1250, 6.25, and 12.5000 mM, indicating that the tertiary structural change of zein took place after calcium addition. It should be noted that samples in the presence of Q (Figure 3F,H) exhibited a remarkable increase in the ellipticities at a G
DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 6. Effect of Ca2+ addition on the thermal behavior of samples. Panels A, B, C, D, E, and F represent samples of zein, zein−Q, PGA, PGA−Q, zein−PGA, and zein−PGA−Q, respectively.
obvious decrease of α-helix content and a subsequent increase of β-sheet of zein (Figure 3A). FTIR Analysis. FTIR was performed to unravel the type of interactions that may take place in such complex systems containing zein, PGA, Q, and calcium. As shown in Figure 4A, the FTIR spectrum of zein exhibited a broad peak band at 3311.2 cm−1, which was attributed to the O−H stretching vibration of hydroxyl-bound water.40 With increasing concentration of Ca2+ incorporation, a significant blue shift occurred. For example, the wavenumber greatly shifted to 3466.8 cm−1 (Figure 4A), 3399.7 cm−1 (Figure 4B), 3397.9 cm−1 (Figure 4E), and 3390.3 cm−1 (Figure 4F) after 12.5000 mM Ca2+ addition, indicating the formation of strong intermolecular hydrogen bonds due to the participation of hydroxyl groups.41 In addition, the band of 2800−3000 cm−1 was attributed to C−
H stretching vibrations.42 For the examples of zein−Q (Figure 4B) and PGA (Figure 4C), the peak band at 2936.2 and 2959.1 cm−1 disappeared after 6.25000 and 12.5000 mM Ca2+ addition, respectively, implying the elimination of the C−H antisymmetric stretch mode induced by calcium ions. The range of peak band from 1500 to 1700 cm−1 represented amide I and amide II groups. Amide I (1600−1700 cm−1) was mainly due to the stretching vibrations of C−O and C−N groups,43 whereas amide II (1400−1500 cm−1) was ascribed to the bending vibration of N−H groups and stretching vibrations of C−N groups.44 It can be found that a more obvious red shift to a lower wavenumber at the range of amide I and II groups was observed after Ca2+ addition for the examples in the presence of Q compared to the samples in the absence of Q, suggesting that such a unique structure with six hydroxyls of Q H
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Figure 7. FE-SEM images of samples in the presence of Ca2+ at various concentrations.
may promote a stronger binding of Ca2+ to protein via noncovalent interactions. It can be noted that the peak band at 1517.5 cm−1 almost disappeared for the sample of zein−Q-12, which may be related to the vanishing of C−C vibration of an aromatic ring after calcium ion induction.45 The results at the
same time revealed the existence of hydrophobic effects, and the interaction may be due to the fact that Ca2+ addition caused a decrease of electrostatic repulsive forces among particles, resulting in the formation of protein aggregates mainly consisting of β-sheets and consequently led to the changes of I
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represented by molecules of water bound to the polymeric chain through hydrogen bonds. As shown in Figure 6, the loss of water was continuous in a wide interval of temperature for all samples. In the samples containing calcium ions, the mass loss before the decomposition was significantly higher than that of original samples. This may be related to the fact that a certain number of molecular chains of biopolymers before interacting with each other could interact with additional molecules of water via hydrogen bonds, resulting in the increased quantity of water absorbed. A similar result was obtained by Russo et al.49 The decomposition process started at a lower temperature for the samples in the absence of calcium ions, and the onset decomposition temperature for samples induced by Ca2+ was shifted to a higher temperature, especially at a high level of Ca2+ incorporation, implying that cross-linked samples experienced a delay in the degradation. The pronouncedly enhanced thermal stability may be expected from the formation of protein aggregates and more compact structure of biopolymer complex after the addition of Ca2+ as discussed in Figure 1. Micromorphology. The corresponding microscopic structural observations of samples are displayed in Figure 7. It can be clearly noted that the morphology formed in the presence of different Ca2+ concentrations is distinguished from those in the absence of Ca2+. As shown in Figure. 7A, both zein particles and zein−Q complex particles were typically spherical and exhibited a uniform size distribution as reported in our previous study.10 The addition of Ca2+ induced the cross-link occurrence among zein particles due to the decreased repulsive forces, resulting in the formation of protein aggregates with a large size, which further confirmed the size analysis in Figure 1A,B. With increasing calcium concentration, the aggregates became more densely cross-linked and overlapped, indicating that the presence of Ca2+ at a high concentration resulted in the formation of the combined aggregates. The micromorphology of PGA is shown in Figure 7B. Native PGA exhibited a fine filamentous network structure. However, PGA fortified with Ca2+ was observed to show a branch-like pattern, which became much thicker and stronger with increasing levels of Ca2+. This observation strongly indicated the existence of cross-linking and association among PGA molecular chains and greater crosslinking at higher Ca2+ concentrations, leading to tighter biopolymer packing. A similar structural changing trend was found for the sample of PGA−Q as shown in Figure 7B, but the difference was still observed that the presence of both Q and Ca2+ induced a more severely cross-linked structure between PGA molecular chains. The profiles for the zein−PGA binary complexes differed dramatically from that of zein or PGA alone. As shown in Figure 7C, a fruit tree-like microstructure was presented for the zein−PGA complex; PGA was regarded as the branches, and the surface was closely adsorbed by zein particles, which agreed with our previous study.10 The presence of Ca2+ induced a more compact and denser structure. With the increase of Ca2+ concentration, the “branches” became closer to each other and cross-linking occurred until an interwoven network structure was formed. The finding may be interpreted as PGA forming a basic strong branch-like network structure, and large zein aggregates were adsorbed and interpenetrated into such a matrix, resulting in the final zein−PGA complex structure with enhanced firmness and density. For the samples of zein−PGA− Q (Figure 7C), phase separation was even observed after 12.5000 mM Ca2+ addition, which may confirm our hypothesis
secondary and tertiary structures of zein, which can be confirmed by the CD analysis as shown in Figure 3. The band of 1800−1650 cm−1 was assigned to a significant carbonyl region.46 For the samples of PGA (Figure 4C) and PGA-Q ((Figure 4D) after the addition of 12.5000 mM Ca2+, the peak bands at 1738.3 and 1739.4 cm−1 pronouncedly shifted to lower wavenumbers of 1728.2 and 1725.6 cm−1, respectively. The result implied that Ca2+ incorporation resulted in the chain−chain association of PGA molecules due to the attendance of its carboxy groups, which could be applied to explain the decreased dimension of particles in the presence of PGA as shown in Figure 1. A similar opinion was pointed out by Borgogna et al.31 in the investigation into alginate gel induced by calcium ion. X-ray Diffraction Analysis. The X-ray diffraction patterns of samples were recorded as shown in Figure 5. Variations in the peak intensity and shapes are clearly detected for the samples prior to and after the addition of Ca2+. The diffraction patterns (Figure 5A) showed two relatively sharp peaks of zein at diffraction angles of 9.1° and 19.2°. However, the addition of Ca2+ brought about a considerable decrease of peak intensity, indicating that the crystal structure of zein was disrupted by Ca2+ and led to low crystallinity. Kandori et al.23 also pointed out a reduced crystallinity of hydroxyapatite nanoparticles due to the addition of calcium ions. As shown in Figure 5C, PGA exhibited three peaks at diffraction angles of 6.9°, 20.2°, and 37.5°, which gradually disappeared with increasing concentration of Ca2+, whereas a new flat peak was observed at a diffraction angle around 30°. The result suggested that the binding of Ca2+ to PGA molecule not only changed the native structure of PGA but also induced the formation of a distinctive new feature due to the chain−chain interactions as confirmed by the FTIR analysis (Figure 4). Compared to the individual pattern of zein and PGA, there were characteristic new broad peaks at diffraction angles of 30° appearing on the patterns of the zein−PGA binary complexes (Figure 5E), indicating the formation of the amorphous complex with the intermolecular interaction occurred between zein and PGA.10 However, all of these characteristic peaks were almost lost by the addition of calcium ions, especially at a high level of Ca2+ incorporation. The finding strongly indicated that the presence of Ca2+ in zein−PGA showed an overlapped efficient induction combined protein aggregation and chain association of PGA molecules,47 resulting in the generation of a new biopolymer with poor crystallinity, which provided additional evidence of the interaction among zein, PGA, and calcium ions and demonstrated the structural changes of the complex due to the hydrogen bonding and hydrophobic effects. It is worth noting that the patterns of samples in the presence of Q exhibited a similar changing trend in peak intensity and shapes as shown in panels B, D, and F of Figure 5, but they occupied a much lower crystallinity than the samples in the absence of Q, indicating that Q may play an important role in synergistically prompting the induction activity of Ca2+ in biopolymers. Thermogravimetric Analysis. Figure 6 shows the mass loss of the samples in the presence and absence of calcium ions. The thermogravimetric curves can be divided into three regions: the first from room temperature to 100 °C, the second between 100 and 200 °C, and the third, above 200 °C, where decomposition occurs.48 The mass loss in the first region may be due to the elimination of free water. Above 100 °C, the decreased mass of samples may involve the bound water J
DOI: 10.1021/acs.jafc.7b00921 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
PGA molecular chains, which was already applied to explain the gelation of alginate in the presence of divalent cations.50 According to our previous study,10 Q-loaded zein−PGA composite particles presented a tree-like pattern, but it was not obviously observed in the presence of Ca2+. The result revealed a new formation mechanism of simultaneous cross-linking between PGA molecule chains and aggregation between zein particles. That is, Ca2+ was first bound to PGA, which resulted in a basic strong branch-like network structure, and at the same time, aggregation took place between zein particles due to Ca2+ induction; then the aggregates were closely adsorbed on the surface of such a branch until the formation of an interwoven network as shown in Figure 7C,D, indicating that Q-loaded zein−PGA composites showed the combined features of zein particles and PGA molecules, which seemed to be a densely colorful tree hung with different fruits. Overall, the incorporation of Ca2+ resulted in significant structural changes of individual zein and PGA, as well as zein−PGA binary complexes, in the absence and presence of Q, which was dependent on the different concentrations of Ca2+.
that Q may synergistically prompt the induction capacity of Ca2+ in biopolymers as described in Figure 5. Above all, microscopic observations suggested that calcium ions established specific interactions with both zein and PGA, which can be enhanced in the presence of Q. Potential Mechanism. On the basis of the cumulative evidence obtained from the various techniques, especially through the FE-SEM observation on the microstructure of samples, three potential mechanisms are proposed in this work to explain the effect of Ca2+ on the physical and structural characteristics of zein, PGA, and zein−PGA composite particles in the presence of Q as illustrated in Figure 8. For the sample of
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AUTHOR INFORMATION
Corresponding Author
*(Y.G.) E-mail:
[email protected]. ORCID
Yanxiang Gao: 0000-0003-2331-5956 Funding
Financial support from the National Natural Science Foundation of China (No. 31371835) is gratefully acknowledged. Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 8. Potential mechanisms and structures of the individual zein, PGA, and zein−PGA binary complexes in the presence of Q after the addition of Ca2+.
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