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Design of novel edible hydrocolloids by structural interplays between wheat gluten proteins and soy protein isolates
Food Hydrocolloids 100 (2020) 105395
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Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd
Design of novel edible hydrocolloids by structural interplays between wheat gluten proteins and soy protein isolates Jian He a, b, c, d, Ren Wang a, b, c, d, Wei Feng a, b, c, d, Zhengxing Chen a, b, c, d, **, Tao Wang a, b, c, d, * a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, 214122, China d School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Wheat gluten proteins Soy protein isolates Water solubility Structural interaction
Wheat gluten proteins (WPs) are nutritious protein with a large yield, but they are rarely industrially attractive due to their poor water solubility. In this experiment, a pH-cycle was used by co-dissolving WPs and soy protein isolates (SPIs) with WP/SPI ratios (W/S) from 1:0.1 to 1:2 at pH 12, and further readjusting the pH to neutral, which improved the water solubility of WPs from 4.5% to 72.4% while maintaining the intact primary structure of proteins. The morphological analysis, fluorescence and far-UV spectra illustrated that the WPs interacted with SPIs to form combined particulate architectures by the co-folding of proteins during neutralization. The com bined structures are significantly resistant to renaturation due to the unfolded protein backbones with high structural tenacity. The more charged surface provided complexed proteins with greater affinity to water mol ecules and protected the protein molecules from aggregation. Moreover, the amino acid compositions closely met the amino acid requirements for adults. The results indicate that the protein complexation induced by adjusting the pH may be a facile and applicative approach to modify insoluble proteins into edible hydrocolloids, and has great potential for enhancing nutritional values of proteins.
1. Introduction The growing world population greatly improves the demands for high nutrient value-added food sources in the past few decades. Efforts has focused on the substitute of expensive animal proteins using the cheap and nutritious plant proteins (Elmalimadi et al., 2017). Wheat gluten, the products of wheat deep processing with a high protein con tent over 70%, is an abundant and modestly priced protein source available in large amounts (Day, Augustin, Batey, & Wrigley, 2006). As the major constituent of wheat gluten, the wheat gluten proteins (WPs) are generally composed of two protein fractions: insoluble glutenin and alcohol-soluble gliadin. The two fractions easily aggregate when hy drated due to strong hydrogen bonds and hydrophobic interactions (Wang, Jin, & Xu, 2015), giving WPs great viscoelasticity, making it the key to the unique ability of wheat gluten to enhance structure of prod ucts (Day et al., 2006; Wang, Gan, Zhou, Cheng, & Nirasawa, 2017). This intermolecular aggregation, as well as the large molecular size and the
wealth contents of nonpolar amino acids such as proline, leucine and glutamine (Mejri, Rog�e, BenSouissi, Michels, & Mathlouthi. 2005), are considered as the reason of limited solubility of WPs in aqueous solvent around neutral pH. Although the techniques for the industrialization of WPs seem to be mature in traditional industry, e.g. baking, meat and animal feed (Bert, Thewissen, Kristof, & Delcour, 2007; Hand, Cren welge, & Terrell, 2010), lack of desirable solubility, emulsifying and foaming lead to extremely limited applications of WPs in high value-added fields. The threatened gluten surplus also starves for methods of converting WPs into precious protein sources with preferable functionalities. Among the functional properties of proteins, the aqueous solubility is generally a prerequisite for extensive application of proteins in liquid foods or medicines, therefore substantial efforts have focused on the solubilization of WPs during the last decades (Apichartsrangkoon, Led ward, Bell, & Brennan, 1998; Kong, Zhou, & Qian, 2007; Wang et al., 2017). Deamidation and enzymic hydrolysis successfully improve the
* Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. ** Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. E-mail addresses: [email protected] (Z. Chen), [email protected] (T. Wang). https://doi.org/10.1016/j.foodhyd.2019.105395 Received 5 June 2019; Received in revised form 19 September 2019; Accepted 24 September 2019 Available online 25 September 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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solubility of WPs to some extent by removing the amino groups from glutamine and asparagine residues, or partial hydrolyzing the peptide chains to enhance the charge density (Wang et al., 2017; Yong, Yama guchi, & Matsumura, 2006). However, the unavoidable damage to the primary structures results in the undesired loss of functional properties and the generation of unwelcome bitter peptides (Kristinsson & Rasco, 2000), far from the current demands for all-natural plant products. Physical methods are also effective attempts that almost caused no in fluence on the primary structures of protein molecules. However, the excessive costs of equipment and energy restrict their widespread applications. Upon proper alkali treatment, the proteins are subjected to be unfolded, and the disulfide bonds of cystine are broken without peptide hydrolysis (Day et al., 2006). However, once the alkalinity is reduced, the proteins refold and aggregate rapidly due to the absence of elec trostatic repulsions, in turn leading to precipitation (Fabian & Ju, 2011). In this context, maintaining the exposure of charged groups and pre venting protein refolding during acidification are the key to enhance the aqueous solubility of proteins. Recently, we improved the aqueous sol ubility of rice proteins to more than 90% by forming composites with casein, which was achieved by simply dissolving both proteins at pH 12 and readjusting to pH 7 (pH-cycle) (Wang, Yue, Xu, Wang, & Chen, 2018). We envisaged that, abiding this technique, the structural unfolding route may probably be extended to WPs, and their water solubilization can be eventually enabled. Soy protein isolates (SPIs) are superior plant proteins with moderate price, high nutritional properties and great consumer acceptability. In present study, we chose SPIs as the foreign proteins to verify the feasi bility of the pH-cycle on the solubilization of WPs. Specifically, we firstly attempted to co-assemble WPs with SPIs into structural composites to enhance the water solubility of WPs. Secondly, the structural alterations of proteins were investigated to learn the mechanism underlying the structural interaction between WPs and SPIs. Finally, the amino acid compositions of the protein composites were measured in order to evaluate their performance on nutritional properties. The development of soluble hydrocolloids might provide a possible solubilization tech nique for hydrophobic plant proteins and enlarge the application domain of WPs.
20 � C before usage. Complexation of WPs and SPIs. Wheat gluten powder (265.7 mg) was added into 20 mL distilled water, and was fully dissolved by dropping 1 M NaOH in the mixtures to pH 12. By centrifugation at 4000g for 10 min, insoluble substances were separated to obtain WPs solution with a final protein concentration of 10 mg/mL. The WP-SPI mixtures of various WP/SPI ratios (W/S, w/w) were obtained by adding various amounts of SPIs to aforementioned WPs suspensions (1%, w/v). The protein solutions were stirred by a magnetic stirring bar at room tem perature for 1.5 h, slowly neutralized to pH 7 using 0.05 M HCl, and then dialyzed for 24 h to remove salts. The dialyzed mixtures were subse quently centrifuged at 4000 g for 10 min. Then, the supernatants were stored at 4 � C as the stock solutions for further analysis. The precipitates were freeze-dried with the aforementioned freeze-drier into powder with moisture contents ranging from 3.9% to 4.5%, and then subjected to SDSGAGE and amino acid composition analysis. 2.2.2. Nitrogen solubility index (NSI) NSI was measured following a previous described method (Bera & Mukherjee, 2010). After preparation of the stock solutions (section 2.2.1), the recollected residues were subjected to nitrogen measure ments using a Kjeldahl method (N � 5.7) (Wang et al., 2017). Before the measurement, an SDS-PAGE analysis had verified that only a single subunit of SPIs with a dim stripe remained in precipitates (Fig. S1). Therefore, we ignored the trace amounts of residual subunits and calculated the NSI of WPs using the following equation (1): NSI ¼
PWP
Presidue � 100% PWP
(1)
where PWP is the weight of initial WPs, and Presidue is the weight (g) of residual proteins. 2.2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) SDSPAGE analysis were performed following the report of Laemmli (Laemmli, 1970). The Tris–HCl buffer (0.125 M) containing SDS (2%, w/v), 2- glycerol (10%, v/v) and mercaptoethanol (2%, v/v) was applied to solubilize the WP-SPI composites. The solutions were diluted with the mentioned Tris-HCl buffer to a protein concentration of 20 μg/μL, and then heated in boiling water for 5 min. By centrifuging the solutions at 10 000 g for 10 s, 10 μL of the supernatants were collected for electrophoresis. Coomassie Brilliant Blue-R250 (0.05%) was used for the staining of gels.
2. Materials and methods 2.1. Materials Wheat gluten powder were purchased from Anhui Ante Food Co., Ltd (Suzhou, Anhui, China). The content of moisture, ash, starch and protein were 8.31%, 0.54%, 12.73% and 75.27% (N � 5.7). Defatted soy flakes with a protein content of 58.3% (N � 6.25, wet basis) were purchased from Sinograin Oils Co., Ltd. (Peking, China). All other chemicals and reagents were analytical grade.
2.2.4. Morphological microscopy Field emission scanning electron microscopy (FE-SEM). The observation was conducted by stock solutions diluted to 0.02% (w/v) with distilled water. The solutions were spread onto silicon slices (5 � 5 mm) and dried in a dark cabinet overnight at room temperature. The slices were subsequently coated with a film of gold (10 nm) under vacuum and then subjected to morphological microscopy by a SU-8220 FE-SEM (Hitachi, Japan). Atomic force microscopy (AFM). The WP-SPI composites solutions were diluted to 0.002% (w/v) with distilled water. A 2.5 μL drop of each diluted solution was dripped on a mica substrate (10 � 10 mm), and the substrate was placed in a dark cabinet at room temperature overnight to evaporate the solvent. AFM analysis was performed using a Multimode VIII microscope (Bruker Corporation, Billerica, MA, USA) equipped with a nanoprobe cantilever tip (Bruker Nanoprobe, Camarillo, CA, USA).
2.2. Methods 2.2.1. Sample preparation Extraction of SPIs. Samples of SPIs with a protein content of 90.5% (N � 6.25, wet basis) were extracted using a previously described approach (Wang, Xu, Chen, & Wang, 2018). In detail, soy flakes (300 g) were ground to pass through an 80-mesh sieve and then mixed with 3 L of water. The mixture was adjusted to pH 9 using 1 M NaOH and stirred by a magnetic stirring bar for 1 h. After the centrifugation at 7000g for 10 min, the supernatant was collected and adjusted to pH 4.5 using 1 M HCl. The slurry with precipitated protein was then centrifuge at 7000 g for 10 min. The precipitate was then collected and re-dispersed in 5 times of water. The crude proteins were pre-frozen at 80 � C and then freeze-dried in accordance to the procedures at 55 � C for 30 h with a Beta 2–8 LD plus freeze-drier (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The obtained SPIs were stored at
2.2.5. Spectrum acquisition Fluorescence spectrum. The protein concentrations of stock solutions were adjusted to 0.1% (w/v) with distilled water, and the intrinsic fluorescence spectra of protein composites were measured by a F-7000 spectrofluorometer (Horiba, Kyoto, Japan) at room temperature. The 2
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emission was excited at 295 nm and recorded from 310 to 410 nm with an excitation and emission slit of 5 nm. The theoretical fluorescence spectra were obtained by adding up the fluorescence spectra of indi vidual WPs and SPIs with corresponding concentrations in the protein composite solutions. The experimental fluorescence spectra were the fluorescence spectra of complexed proteins (Shigemitsu et al., 2018). To determine the contributions of hydrogen bindings, hydrophobic re actions and electrostatic interactions in the formation of protein com posites, WPs and SPIs were dissolved in 18 mL distilled water at pH 12 following the method mentioned in section 2.2.1 (complexation of WPs and SPIs), and 2 mL of thiourea, SDS, and NaCl solutions (0.1 M) were then separately added into the protein solutions. The final concentration of thiourea, SDS, and NaCl was 10 mM. The fluorescence spectra were collected at pH 7 with the same method as other samples. Circular dichroism (CD). A MOS-450 spectrometer (BioLogic Science Instruments, Ltd., Claix, France) was applied for the collection of CD spectra of protein composites. The protein concentrations of stock so lutions were adjusted to 0.05% (w/v) and 0.1% (w/v) for scanning of CD spectra at the wavelengths ranging from 190 to 250 nm and 250–320 nm which were corresponding to the far-UV region and near-UV region, respectively. The determination was conducted using distilled water as a blank solution. Data were generated as an average of 3 scans at 20 � C.
2.2.8. Statistical analysis All preparations were performed in triplicates. Data were analyzed by one-way analysis of variance (ANOVA) using Origin 9.0 (OriginLab Co., USA). The means comparison was determined by Duncan’s test (p < 0.05). 3. Results and discussion 3.1. NSIs of WPs Soluble protein composites were prepared by co-dissolving WPs and SPIs at pH 12 and recollecting at pH 7. Although the focus of this investigation was WPs, the amount of SPIs was increased to twice that of WPs for deeply learning of the protein-protein interaction. As shown in Fig. 2B, only a dim band of the basic subunit (19 kDa) from 11S of SPIs was observed in the SDS-PAGE profile of residuals, indicating that almost all the subunits of SPIs were dissolved in the supernatant and further collected after interact with WPs. Thus, the amounts of insoluble subunits were neglected, and the NSIs of WPs were approximately calculated as the percentage of WPs dissolved in the solution against the initial WPs used in the reaction. As listed in Fig. 1, the NSI of WPs was improved to nearly 50% by adding SPIs at a W/S ratio of 1:0.1, while that of control was just 4.5%. The NSI of WPs was finally increased up to 72.4% at a W/S ratio of 1:2. It is worth noting that the prepared protein composites can be completely dissolved in water (Fig. 2J). However, our attempt on reducing the W/S ratio to 0.05 was failed as WPs almost completely precipitated. The SDS-PAGE profiles of complexed proteins (Fig. 2A) exhibited distinct bands of all subunits in WPs and SPIs, and the bands of SPIs were brightened along with the addition of SPIs, demon strating that the intact primary structures of proteins were retained. We further tried to remix the residue with SPIs and recollected them after a pH-cycle, it was observed that a part of the residual WPs were assembled with SPIs without any hydrolyzation of peptide chains (Fig. S1), which provided a path to further improve the recovery rate of WPs.
2.2.6. Surface properties of proteins Surface hydrophobicity (H0). The index of H0 was determined by a previously described method (Qiu, Sun, Cui, & Zhao, 2013) with 1-Ani lino-8-naphthalenesulfonate (ANS) as fluorescence probe. A series of protein composite solutions with protein concentrations ranging from 0.0125% to 0.1% (w/v) were obtained by diluting the stock solutions with distilled water, and then each dilution (4 mL) was mixed with 10 μL of 8 mM ANS solution. The fluorescence intensities of samples were measured by the mentioned spectrofluorometer at excitation and emission wavelengths of 390 nm and 484 nm, respectively. H0 was defined as the slope of the fluorescence intensity against protein concentration. Zeta-potential. The zeta-potential analysis was performed following the method described by Wang (Wang et al., 2018) with a Zetasizer Nano instrument (Malvern Instruments Ltd., Malvern, UK). After dilu tion to 0.1% (w/v) with distilled water, all protein samples were analyzed at 25 � C with refractive indices of 1.450 and 1.330 for proteins and dispersion medium, respectively. WPs and SPIs were subjected to the same pH-cycle treatment, and the supernatants after centrifugation were used for the measurement.
3.2. Morphologies of the WP–SPI composites The FE-SEM observation was conducted to visually reveal the in teractions between WPs and SPIs in water. The images showed that WPs strongly aggregated to form massive particles with densely packed structures (Fig. 2C), while the SPIs showed a laminar microstructure
2.2.7. Amino acid composition The amino acid composition (g/100 g protein) was detected by an acid hydrolyzation method using an amino acid analyzer (835-50, Hitachi, Japan) equipped with a PicoTag column at 254 nm. The freezedried proteins were hydrolyzed in sealed tubes with 6 M HCl at 110 � C for 24 h. Then, the obtained hydrolyzed solutions were neutralized by 4 M NaOH, and centrifuged at 10,000 g for 10 min. The collected su pernatants were subjected to amino acid analysis at 38 � C. The data (expressed as g per 100 g protein) did not include Tryptophan (Trp) because of the easy degradation of Trp. The essential amino acid index (EAAI) was the geometric mean of the content of the essential amino acids in measured proteins to their corresponding contents in the whole ~ as, Gomez, Frias, Baeza, & Vidal-Valverde, 2010). The egg protein (Pen values were calculated following equation (2): sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Thrp Valp n Lysp EAAI ¼ 100 � (2) � � ⋅⋅⋅ � Lyss Thrs Vals where the subscript ‘p’ means the protein sample, ‘s’ means the whole egg protein, and ‘n’ means the number of amino acids.
Fig. 1. Nitrogen solubility index (NSI) of WPs. Control was wheat gluten powder with the same pH-cycle treatment. W/S means the WPs/SPIs ratio. Asterisks (*) and (**) above bars respectively represent significant differences (p < 0.05) and very significant differences (p < 0.01) relative to the control. 3
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Fig. 2. SDS-PAGE analysis and FE-SEM images of WP-SPI composites. (A) and (B) SDS-PAGE profiles of preprared WP-SPI composites and residual proteins. Lanes 1–3 are markers, WPs, and SPIs, respectively; lanes 4–8 are protein composites or residual proteins with W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2, respectively; (C), (D), (E), (F), (G), (H)and (I) are FE-SEM images of WPs, SPIs and protein composites prepared by W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2, respectively; Scale bars ¼ 1 μm. (J) Pictures of redissolved protein composites. No.1 is control, 2–6 are protein composites correspoding to lanes 4–8 in SDS-PAGE, respectively. The concentration of each sample was 10 mg/mL.
tiled on the silicon slice (Fig. 2D). After incorporating with a handful of SPIs, the WPs decreased their self-aggregation but formed cobblestonelike particles with smooth surfaces and diameters between 100 and 250 nm (Fig. 2E). Similar shapes and sizes were observed for all of the samples till the W/S ratio was increased to 1:1.5 (Fig. 2F–H). However, when the amount of SPIs that could be incorporated into WPs reached the limit (W/S ¼ 1:2), the excess of SPIs aggregated into membrane and simultaneously integrated with the WP-SPI composites, leading to the generation of distinctive film structures which were observed in Fig. 2I. AFM images showed similar morphologies of protein composites to those observed by FE-SEM. With moderate addition of SPIs (W/ S ¼ 1:0.1–1:1.5), the protein composites appeared as uniformly dispersed nanoparticles which had diameters around 100 nm (Fig. 3A–D) and thicknesses of several nanometers (insets at the right of Fig. 3) at a low concentration (0.002%, w/w). The stretched structures might contribute to easier hydration due to larger contact surface. When the W/S ratio reached 1:2, the stable dispersion of protein composites in water was destroyed as the particles aggregated into clusters by the interactions with excess of SPIs (Fig. 3E), which was in accordance with the FE-SEM results. Morphological transition of proteins indicated that the agglomerate structures of WPs were transformed into new hydratable structures due to the incorporation of SPIs, leading to the improvement of protein solubility. Therefore, to understand the formation of the co-assembled structures, the process of protein structural alternation was investi gated subsequently.
and SPIs, which could distinguish this structural reaction from the simple mix of two proteins. The fluorescence profiles showed that both WPs and SPIs had a distinct peak intensity (Fmax) around 340 nm that was the mainly emission of Trp (Fig. S2). Numerous studies have verified that the interactions of proteins with foreign substances noticeably quench the intrinsic fluorescence of proteins, and the quenching was strengthened with the increase of addition amount (Rawel, Meidtner, & Kroll, 2005; Xiao et al., 2008; Zhang, Wright, & Zhong, 2013). There fore, the change of the emission can be applied as an evidence for the structural transition of proteins. In this scenario, the theoretical (sum of the fluorescence spectra of WPs and SPIs with corresponding concen trations) and experimental fluorescence spectra (the fluorescence spectra of complexed proteins) were compared. Taking the solution with a W/S ratio of 1:1 as an example (Fig. 4A), the Fmax of experimental fluorescence spectra were much lower than those of the theoretical ones owing to the photoinduced electron transferred from the electron-rich aromatic amino groups to the electron-deficient groups (Shiki et al., 2014), which indicated that the two proteins combined with each other to form novel composites. The fluorescence quenching was observed at all W/S ratios. The differences between the theoretical and experimental Fmax values that expressed the degree of quenching increased along with the increasing of SPIs (Fig. 4B), confirming that more SPIs participated in the formation of protein composites. The far-UV CD spectra showed that all protein composites had wide negative bands between 200 and 240 nm at pH 7, but the ellipticity was reduced with the increase of SPI proportion (Fig. 4D), which was a strong indication of complexed sec ondary structures. A previous research (Yang et al., 2007) had proved that the folding degree of proteins could be characterized by ellipticity at the wavelength of 222 nm, the ellipticities of protein composites at 222 nm were therefore plotted against the W/S ratios to show protein refolding explicitly. As shown in Fig. 4C, incrementing addition of SPIs significantly weakened the ellipticity, indicating that the WP-SPI com posites had more stable and unfolding structures. Since the interactions between the two proteins were confirmed, it was necessary to illuminate the molecular forces involved in the for mation of protein composites. Therefore, thiourea, SDS and NaCl were
3.3. Molecular binding Successful researches had been reported that the addition of grain constituents, e.g. fibers, improved the dough properties of wheat flour as a result of interactions between polysaccharides structure and wheat proteins (Wang, Rosell, & Barber, 2002). Although the morphology analysis has showed the formation of novel protein composites, protein mixtures neutralized to pH 7 were subjected to fluorescence and far-UV CD spectra to demonstrate the protein-protein interactions between WPs 4
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Fig. 3. AFM images of the WP-SPI composites; (A), (B), (C), (D)and (E) are protein composites prepared with W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2, respectively; Scale bars ¼ 200 nm. The insets at the right of each image are height profiles of the structures marked with white lines, in the direction from left to right.
solely added as blocking agents in the reaction mixtures with a final concentration of 10 mM at the beginning of the reaction, and the fluo rescence intensities of neutralized protein solutions were then compared to explore the contributions of hydrogen bindings, hydrophobic re actions and electrostatic interactions, respectively (Lennart & Bertil, 2005). Contrary to the previously mentioned fluorescence quenching caused by the interactions, growing fluorescence intensities (Fig. 4E) suggested that the interactions between WPs and SPIs were prevented by the blocking agents, verifying that the interactions were simultaneously driven by hydrogen bindings, hydrophobic reactions and electrostatic interactions. However, inconsistent with the results obtained in our previous studies on rice proteins that the main force involved in for mation of protein composites was hydrogen bonding (Wang, Xu, Chen,
Zhou, & Wang, 2018), the reaction mixture supplemented with SDS exhibited the maximum fluorescence intensity, implying the primary role of hydrophobic reactions playing in the co-assembly of proteins. However, an opposite phenomenon compared to fluorescence was observed, showing that the neutralized reaction mixture with SDS addition showed the highest recovery rate (Fig. S3). This abnormal result indicated that the addition of SDS inhibited the aggregation of WPs by blocking the hydrophobic interactions. This subjected WPs to be inclined to interact with the SPIs during neutralization, thereby pro moting the formation of the soluble protein composites. Therefore, the formation process of the combined structures was further characterized in this study.
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Fig. 4. Characterization of the molecular structures of WP-SPI composites. (A) Theo retical and experimental emission spectra of composites prepared with a W/S of 1:1 at pH 7; (B) The difference values between the peak intensity (Fmax) of theoretical and experimental emission spectra of each pro tein composites prepared at W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2. (C) The elliptic extrema at 222 nm corresponding to CD spectra of each protein composites. (D) Far-UV CD spectra of WPs, SPIs and WP-SPI protein composites at pH 7; WPs and SPIs were treated in the same way as the protein composite but without any addition of other proteins; (E) Fluorescence emission spectra of protein composites (W/S ¼ 1:1) mixed with 10 mM NaCl, thiourea, or SDS during complexation at pH 7. Different letters close to error bars indicate significant differences (p < 0.05).
3.4. Inhibition of structural folding of proteins
promoted the co-existed proteins structured into unfolded conformations. The ellipticity at 275 nm of each sample was a typical signal of Tyr (Kelly & Price, 1997), which was plotted against pH to intuitively show the process of protein folding (Javid et al., 2013). For co-solvated pro teins with a W/S ratio of 1:0.1, negligible change was observed when pH was above 10 (Fig. 5B). Keeping titration of HCl until pH dropped below 10, the ellipticity was weakened sharply as a result of protein refolding, a hint that the interactions of WPs with SPIs possibly began at pH 10. Although similar phenomenon was observed on samples with W/S ratios of 1:0.5 and 1:1, the incorporation of more SPIs eventually reduced the refolding degree of proteins. And for samples with ratios of 1:1.5 and 1:2, the ellipticity approximately remained unchanged along with the reduction of pH, except for tiny changes in the pH range of 8-7. The CD result reiterated that the SPIs merged into WPs conformations by the
Each aromatic amino acid residue exhibits CD signals in the near-UV region in the asymmetric microenvironment, which is a reflection of the tertiary structures of proteins (Kelly & Price, 2000). Therefore, CD spectrum was used as a sensitive spectroscopic probe to characterize the structural refolding of protein during neutralization. As showed in Fig. S4, the near-UV region of co-existent WPs and SPIs at pH 12 and 11 had negligible ellipticities that were strengthened subsequently along with the HCl addition, suggesting that the renatured proteins approached more intricate tertiary structures. Therefore, the formation of protein composites was mainly induced by the large synergistic conformational changes during protein refolding resultant from neutralization. Besides, growing proportion of SPIs weakened the ellipticity at pH 7 (Fig. 5A), proving that the incorporation of SPIs
Fig. 5. (A) Near-UV CD spectra of protein composites at pH 7; WPs were treated in the same way as the preparation of protein composite but without SPIs. (B) Plotting of the ellipticity at 275 nm against pH. 6
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surface that could provide intermolecular repulsions among adjacent proteins. The zeta-potential analysis, along with the hydrophobicity, again revealed the unfolding structures of protein composites, and explained the increasingly soluble nature stemming from the partici pation of SPIs. Substantial evidences jointly corroborated that both of WPs and SPIs unfolded at pH 12 and interacted with the exposed charged groups during neutralization to form nanoparticles that were uniformly dispersed in water. The nanoparticles, with relatively unfolded struc tures, showed good stability that was similar to the protein structures at pH 12, thus greatly improving the solubility of the proteins. In this study, the protein composites show over 72% solubility at a W/S ratio of 1:2, which was similar to that of deamidated samples (Wang et al., 2017). Considering the intact primary structures of protein composites, the nutritional values may be greatly maintained, and the amino acid compositions were further measured.
co-folding of proteins, and more tenacious backbones of the combined structures formed by the interactions was the reason of weaker folding and less self-aggregation of co-solved proteins. To adequately under stand the dispersibility of protein composites, the interfacial properties analysis was further performed. 3.5. Interfacial properties of proteins affected by interaction As shown in Fig. 6B, the hydrophobicity of the complexed proteins increased along with the reduction of pH, which was another evidence of protein refolding. In this regard, the decrease of hydrophobicity was actually caused by the formation of hydrophobic pockets which were available to ANS probes due to protein refolding (Hawe, Sutter, & Jis koot, 2008). Correspondingly, the zeta-potential showed slight im provements with decreasing of pH (Fig. 6D), suggesting that a part of charged groups that exposed at pH 12 were buried along with the packing of unfolded structures. From this view, the hydrophobic groups were packed inside the co-assembled structures, inducing the formation of protein composites, in good agreement with the fluorescence quenching listed in Fig. 4E. Furthermore, based on the experimental phenomenon, it could be speculated that the embedding of hydrophobic groups played a driving role in the co-assembly of the two proteins, which was different from the interaction between fibers and WPs (Wang et al., 2002). On the other hand, the increasing level of added SPIs obviously reduced the hydrophobicity of the protein composites, and all of the complexed samples had lower hydrophobicity than individual WPs (Fig. 6A). The SPIs showed much higher zeta-potential intensity (32.3 mV) than WPs (14.3 mV), and the zeta-potential showed a sharper increase as more SPIs was incorporated into WPs (Fig. 6C). Although it was lower than that of SPIs, all of the potential values of the complexed proteins were higher than that of WPs, evidencing the more charged
3.6. Amino acid analysis WPs and SPIs are considered as good sources of amino acids for humans, which widely exist in human diet (Friedman, 1996), especially for WPs. The Food and Agriculture Organization and the World Health Organization (FAO/WHO) provide a recommendation about the dietary proteins that human need nine essential amino acids, i.e. His, Ile, Leu, Lys, Met þ Cys, Phe þ Tyr, Thr, Trp, and Val to maintain normal body function. The recommended levels of each amino acid are 19, 28, 66, 58, 25, 63, 34, 11, and 35 mg/g protein for pre-school children, and are 16, 13, 19, 16, 17, 19, 9, 5, and 13 mg/g protein for adults, respectively. As shown in Table 1, all of complexed proteins showed compromised amino acid compositions with EAAI values ranging from 58.97 to 70.22 while the SPIs and WPs showed EAAI values of 70.39 and 55.95, respectively. By interacting with SPIs, the WPs made up for the deficiency of Arg and
Fig. 6. Surface properties of protein composites; (A) The hydrophobicity of WPs, SPIs and the protein composites at pH 7; (B) The hydrophobicity of the protein composites prepared at W/S ration of 1:1 at pH 7–12; (C) The zeta-potential of WPs, SPIs and the protein composites at pH 7. (D) The zeta-potential of the protein composites prepared at W/S ration of 1:1 at pH ranging from 7 to 12. Different letters above bars indicate significant differences (p < 0.05). 7
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Table 1 Amino acid analysis of WP-SPI composites. a Amino acid
SPI
Essential amino acids His 2.65 � 0.42ab Thr 3.10 � 0.21a Tyr 3.16 � 0.33a Cys-s 0.18 � 0.01a Val 4.89 � 0.11ab Met 1.11 � 0.18a Phe 6.02 � 0.31b Ile 4.48 � 0.33ab Leu 8.18 � 0.32b Lys 6.02 � 0.37e Nonessential amino acids Asp 12.17 � 0.20e Glu 23.92 � 0.28a Ser 4.16 � 0.13a Gly 4.27 � 0.23a Arg 6.72 � 0.30c Ala 3.76 � 0.08a Pro 5.23 � 0.08a EAAI 70.39 � 1.83c a
WP
1:0.1
1:0.5
1:1
1:1.5
1:2
2.02 � 0.27a 2.44 � 0.21a 2.73 � 0.31a 0.82 � 0.11c 5.28 � 0.13b 1.78 � 0.28b 3.83 � 0.36a 4.20 � 0.17a 7.10 � 0.10a 1.56 � 0.32a
2.44 � 0.20ab 2.69 � 0.40a 2.51 � 0.17a 0.28 � 0.23ab 5.14 � 0.13b 1.12 � 0.41ab 5.27 � 0.31b 4.07 � 0.20a 7.08 � 0.37a 2.78 � 0.34b
2.44 � 0.33ab 2.67 � 0.40a 2.72 � 0.03a 0.36 � 0.14ab 4.99 � 0.06ab 1.48 � 0.18ab 5.68 � 0.25b 4.49 � 0.13ab 7.48 � 0.41a 3.44 � 0.42bc
2.24 � 0.06ab 2.47 � 0.11a 2.81 � 0.28a 0.44 � 0.08ab 4.65 � 0.31a 1.36 � 0.14ab 5.73 � 0.11b 4.31 � 0.10ab 7.20 � 0.16a 3.29 � 0.42bc
2.51 � 0.13ab 2.88 � 0.37a 3.12 � 0.34a 0.51 � 0.25abc 5.10 � 0.07b 1.26 � 0.11ab 5.72 � 0.38b 4.74 � 0.27b 7.45 � 0.08a 4.01 � 0.36cd
2.73 � 0.30b 2.93 � 0.06a 2.97 � 0.36a 0.62 � 0.03bc 5.23 � 0.21b 1.27 � 0.35ab 5.68 � 0.25b 4.49 � 0.16ab 7.64 � 0.20ab 4.28 � 0.26d
5.18 � 0.28a 38.44 � 0.30g 4.07 � 0.17a 4.32 � 0.08a 3.83 � 0.35a 3.01 � 0.38a 9.38 � 0.03e 55.95 � 0.99a
7.24 � 0.06b 33.92 � 0.30g 4.01 � 0.34a 4.15 � 0.17a 4.90 � 0.03b 3.22 � 0.31a 9.19 � 0.07e 58.97 � 4.14ab
9.41 � 0.18c 31.25 � 0.20d 3.64 � 0.34a 3.82 � 0.10a 5.45 � 0.22b 3.37 � 0.39a 7.33 � 0.38d 64.91 � 0.68bc
9.76 � 0.27c 32.48 � 0.41e 3.75 � 0.17a 3.95 � 0.17a 5.20 � 0.16b 3.44 � 0.30a 6.93 � 0.04c 61.70 � 2.30ab
9.42 � 0.28c 29.63 � 0.11c 3.65 � 0.33a 4.03 � 0.17a 6.28 � 0.27c 3.52 � 0.38a 6.18 � 0.11b 68.12 � 3.32bc
10.34 � 0.25d 27.62 � 0.41b 4.01 � 0.10a 4.19 � 0.41a 6.24 � 0.34c 3.76 � 0.40a 6.01 � 0.04b 70.22 � 2.88c
Values are expressed as the percentage weight of the amino acid against protein weight. Different superscript letters indicate significant differences (p < 0.05).
Appendix A. Supplementary data
Lys, and compensated for the lack of Pro in SPIs at the same time. However, the contents of Lys and Thr in protein composites were still too low to meet the requirements for pre-school children, which hindered the application to a certain extent. Nonetheless, the soluble protein composites have great potential becoming a superior protein source for adults. The amino acid compositions of residual proteins collected during the preparation of protein composites were also determined (Table S1). The residual proteins with different W/S ratios showed similar amino acid compositions, which was another indication that the amounts of residual SPIs in the precipitates were very small. However, the glutamic (Glu) contents of precipitates were lower than that of WPs, implying that some glutamic -enriched peptide chains were more likely to assemble with SPIs.
Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105395. References Apichartsrangkoon, A., Ledward, D. A., Bell, A. E., & Brennan, J. G. (1998). Physicochemical properties of high pressure treated wheat gluten. Food Chemistry, 63 (2), 215–220. Bera, M. B., & Mukherjee, R. K. (2010). Solubility, emulsifying, and foaming properties of rice bran protein concentrates. Journal of Food Science, 54(1), 142–145. Bert, L., Thewissen, B. G., Kristof, B., & Delcour, J. A. (2007). Impact of redox agents on the extractability of gluten proteins during bread making. Journal of Agricultural and Food Chemistry, 55(13), 5320–5325. Day, L., Augustin, M. A., Batey, I. L., & Wrigley, C. W. (2006). Wheat-gluten uses and industry needs. Trends in Food Science & Technology, 17(2), 82–90. � � Zu� � za, M. G., Lukovi�c, N. D., Elmalimadi, M. B., Stefanovi�c, A. B., Sekuljica, N.Z., Jovanovi�c, J. R., et al. (2017). The synergistic effect of heat treatment on alcalaseassisted hydrolysis of wheat gluten proteins: Functional and antioxidant properties. Journal of Food Processing and Preservation, 41. https://doi.org/10.1111/jfpp.13207. Fabian, C., & Ju, Y. H. (2011). A review on rice bran protein: Its properties and extraction methods. Critical Reviews in Food Science and Nutrition, 51(9), 816–827. Friedman, M. (1996). Nutritional value of proteins from different food sources. A review. Journal of Agricultural and Food Chemistry, 44(1), 6–29. Hand, L. W., Crenwelge, C. H., & Terrell, R. N. (2010). Effects of wheat gluten, soy isolate and flavorings on properties of restructured beef steaks. Journal of Food Science, 46 (4), 1004–1006. Hawe, A., Sutter, M., & Jiskoot, W. (2008). Extrinsic fluorescent dyes as tools for protein characterization. Pharmaceutical Research, 25(7), 1487–1499. Javid, N., Roy, S., Zelzer, M., Yang, Z. M., Sefcik, J., & Ulijn, R. V. (2013). Cooperative self-assembly of peptide gelators and proteins. Biomacromolecules, 14(12), 4368–4376. Kelly, S. M., & Price, N. C. (1997). The application of circular dichroism to studies of protein folding and unfolding. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1338(2), 161–185. Kelly, S. M., & Price, N. C. (2000). The use of circular dichroism in the investigation of protein structure and function. Current Protein & Peptide Science, 1(4), 349–384. Kong, X., Zhou, H., & Qian, H. (2007). Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates. Food Chemistry, 102(3), 759–763. Kristinsson, H. G., & Rasco, B. A. (2000). Fish protein hydrolysates: Production, biochemical, and functional properties. Critical Reviews in Food Science and Nutrition, 40(1), 43–81. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. Lennart, N., & Bertil, H. (2005). Molecular origin of time-dependent fluorescence shifts in proteins. Proceedings of the National Academy of Sciences of the United States of America, 102(39), 13867–13872. Mejri, M., Rog�e, B., BenSouissi, A., Michels, F., & Mathlouthi, M. (2005). Effects of some additives on wheat gluten solubility: A structural approach. Food Chemistry, 92(1), 7–15. Pe~ nas, E., Gomez, R., Frias, J., Baeza, M. L., & Vidal-Valverde, C. (2010). High hydrostatic pressure effects on immunoreactivity and nutritional quality of soybean products. Food Chemistry, 125(2), 423–429.
4. Conclusion In summary, soluble protein composites with hydrophilic particulate nanostructures can be prepared by co-dissolving WPs and SPIs at pH 12 and neutralizing the solutions to pH 7 (a pH-cycle). The interactions between the two proteins inhibited the structural refolding and sus tained the charged surface of proteins under neutral condition. With W/ S ratios ranging from 1:0.1 to 1:2, the solubility of WPs was improved from <5% to >72% without negligible damages on the primary struc tures of proteins. The amino acid compositions of the composites met the requirement of FAO/WHO for the daily intake for adults. This facile technique relying on a simple pH-cycle procedure is believed to have feasibility for solubilization of various insoluble plant proteins, and for the preparation of tailor-made protein ingredients. Declaration of competing interest Authors declare no conflicts of interest. Acknowledgements The research was financially supported by the National Natural Science Foundation of China (No. 31778198; 31901602), and National first-class discipline program of Food Science and Technology (JUFSTR20180203), the Foundation of Scientific Development of Wuxi, China (No. 0302B010504180011PB), the Scientific Plan for Jiangsu Province, China (BE2018309).
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Qiu, C., Sun, W., Cui, C., & Zhao, M. (2013). Effect of citric acid deamidation on in vitro digestibility and antioxidant properties of wheat gluten. Food Chemistry, 141(3), 2772–2778. Rawel, H. A., Meidtner, K., & Kroll, J. (2005). Binding of selected phenolic compounds to proteins. Journal of Agricultural and Food Chemistry, 53(10), 4228–4235. Shigemitsu, H., Fujisaku, T., Tanaka, W., Kubota, R., Minami, S., Urayama, K., et al. (2018). An adaptive supramolecular hydrogel comprising self-sorting double nanofibre networks. Nature Nanotechnology, 13(3), 165–172. Shiki, Y., Kazunori, I., Mitsuaki, Y., Takashi, K., Akihide, K., Shinobu, U., et al. (2014). Photocontrol over self-assembled nanostructures of π-π stacked dyes supported by the parallel conformer of diarylethene. Angewandte Chemie, 126(10), 2640–2644. Wang, Y., Gan, J., Zhou, Y., Cheng, Y., & Nirasawa, S. (2017). Improving solubility and emulsifying property of wheat gluten by deamidation with four different acids: Effect of replacement of folded conformation by extended structure. Food Hydrocolloids, 72, 105–114. Wang, P., Jin, Z., & Xu, X. (2015). Physicochemical alterations of wheat gluten proteins upon dough formation and frozen storage - a review from gluten, glutenin and gliadin perspectives. Trends in Food Science & Technology, 46(2), 189–198. Wang, J., Rosell, C. M., & Barber, C. B. D. (2002). Effect of the addition of different fibres on wheat dough performance and bread quality. Food Chemistry, 79(2), 221–226.
Wang, T., Xu, P., Chen, Z., & Wang, R. (2018). Mechanism of structural interplay between rice proteins and soy protein isolates to design novel protein hydrocolloids. Food Hydrocolloids, 84, 361–367. Wang, T., Xu, P., Chen, Z., Zhou, X., & Wang, R. (2018). Alteration of the structure of rice proteins by their interaction with soy protein isolates to design novel protein composites. Food & Function, 9, 10.1039.C1038FO00661J-. Wang, T., Yue, M., Xu, P., Wang, R., & Chen, Z. (2018). Toward water-solvation of rice proteins via backbone hybridization by casein. Food Chemistry, 258(30), 278–283. Xiao, J., Suzuki, M., Jiang, X., Chen, X., Yamamoto, K., Ren, F., et al. (2008). Influence of B-ring hydroxylation on interactions of flavonols with bovine serum albumin. Journal of Agricultural and Food Chemistry, 56(7), 2350–2356. Yang, F., Zhou, B., Zhang, P., Zhao, Y. F., Chen, J., & Liang, Y. (2007). Binding of ferulic acid to cytochrome c enhances stability of the protein at physiological pH and inhibits cytochrome c-induced apoptosis. Chemico-Biological Interactions, 170(3), 231–243. Yong, Y. H., Yamaguchi, S., & Matsumura, Y. (2006). Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of wheat gluten. Journal of Agricultural and Food Chemistry, 54(16), 6034–6040. Zhang, Y., Wright, E., & Zhong, Q. (2013). Effects of pH on the molecular binding between β-lactoglobulin and bixin. Journal of Agricultural and Food Chemistry, 61(4), 947–954.
9
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd
Design of novel edible hydrocolloids by structural interplays between wheat gluten proteins and soy protein isolates Jian He a, b, c, d, Ren Wang a, b, c, d, Wei Feng a, b, c, d, Zhengxing Chen a, b, c, d, **, Tao Wang a, b, c, d, * a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, 214122, China d School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Wheat gluten proteins Soy protein isolates Water solubility Structural interaction
Wheat gluten proteins (WPs) are nutritious protein with a large yield, but they are rarely industrially attractive due to their poor water solubility. In this experiment, a pH-cycle was used by co-dissolving WPs and soy protein isolates (SPIs) with WP/SPI ratios (W/S) from 1:0.1 to 1:2 at pH 12, and further readjusting the pH to neutral, which improved the water solubility of WPs from 4.5% to 72.4% while maintaining the intact primary structure of proteins. The morphological analysis, fluorescence and far-UV spectra illustrated that the WPs interacted with SPIs to form combined particulate architectures by the co-folding of proteins during neutralization. The com bined structures are significantly resistant to renaturation due to the unfolded protein backbones with high structural tenacity. The more charged surface provided complexed proteins with greater affinity to water mol ecules and protected the protein molecules from aggregation. Moreover, the amino acid compositions closely met the amino acid requirements for adults. The results indicate that the protein complexation induced by adjusting the pH may be a facile and applicative approach to modify insoluble proteins into edible hydrocolloids, and has great potential for enhancing nutritional values of proteins.
1. Introduction The growing world population greatly improves the demands for high nutrient value-added food sources in the past few decades. Efforts has focused on the substitute of expensive animal proteins using the cheap and nutritious plant proteins (Elmalimadi et al., 2017). Wheat gluten, the products of wheat deep processing with a high protein con tent over 70%, is an abundant and modestly priced protein source available in large amounts (Day, Augustin, Batey, & Wrigley, 2006). As the major constituent of wheat gluten, the wheat gluten proteins (WPs) are generally composed of two protein fractions: insoluble glutenin and alcohol-soluble gliadin. The two fractions easily aggregate when hy drated due to strong hydrogen bonds and hydrophobic interactions (Wang, Jin, & Xu, 2015), giving WPs great viscoelasticity, making it the key to the unique ability of wheat gluten to enhance structure of prod ucts (Day et al., 2006; Wang, Gan, Zhou, Cheng, & Nirasawa, 2017). This intermolecular aggregation, as well as the large molecular size and the
wealth contents of nonpolar amino acids such as proline, leucine and glutamine (Mejri, Rog�e, BenSouissi, Michels, & Mathlouthi. 2005), are considered as the reason of limited solubility of WPs in aqueous solvent around neutral pH. Although the techniques for the industrialization of WPs seem to be mature in traditional industry, e.g. baking, meat and animal feed (Bert, Thewissen, Kristof, & Delcour, 2007; Hand, Cren welge, & Terrell, 2010), lack of desirable solubility, emulsifying and foaming lead to extremely limited applications of WPs in high value-added fields. The threatened gluten surplus also starves for methods of converting WPs into precious protein sources with preferable functionalities. Among the functional properties of proteins, the aqueous solubility is generally a prerequisite for extensive application of proteins in liquid foods or medicines, therefore substantial efforts have focused on the solubilization of WPs during the last decades (Apichartsrangkoon, Led ward, Bell, & Brennan, 1998; Kong, Zhou, & Qian, 2007; Wang et al., 2017). Deamidation and enzymic hydrolysis successfully improve the
* Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. ** Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. E-mail addresses: [email protected] (Z. Chen), [email protected] (T. Wang). https://doi.org/10.1016/j.foodhyd.2019.105395 Received 5 June 2019; Received in revised form 19 September 2019; Accepted 24 September 2019 Available online 25 September 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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solubility of WPs to some extent by removing the amino groups from glutamine and asparagine residues, or partial hydrolyzing the peptide chains to enhance the charge density (Wang et al., 2017; Yong, Yama guchi, & Matsumura, 2006). However, the unavoidable damage to the primary structures results in the undesired loss of functional properties and the generation of unwelcome bitter peptides (Kristinsson & Rasco, 2000), far from the current demands for all-natural plant products. Physical methods are also effective attempts that almost caused no in fluence on the primary structures of protein molecules. However, the excessive costs of equipment and energy restrict their widespread applications. Upon proper alkali treatment, the proteins are subjected to be unfolded, and the disulfide bonds of cystine are broken without peptide hydrolysis (Day et al., 2006). However, once the alkalinity is reduced, the proteins refold and aggregate rapidly due to the absence of elec trostatic repulsions, in turn leading to precipitation (Fabian & Ju, 2011). In this context, maintaining the exposure of charged groups and pre venting protein refolding during acidification are the key to enhance the aqueous solubility of proteins. Recently, we improved the aqueous sol ubility of rice proteins to more than 90% by forming composites with casein, which was achieved by simply dissolving both proteins at pH 12 and readjusting to pH 7 (pH-cycle) (Wang, Yue, Xu, Wang, & Chen, 2018). We envisaged that, abiding this technique, the structural unfolding route may probably be extended to WPs, and their water solubilization can be eventually enabled. Soy protein isolates (SPIs) are superior plant proteins with moderate price, high nutritional properties and great consumer acceptability. In present study, we chose SPIs as the foreign proteins to verify the feasi bility of the pH-cycle on the solubilization of WPs. Specifically, we firstly attempted to co-assemble WPs with SPIs into structural composites to enhance the water solubility of WPs. Secondly, the structural alterations of proteins were investigated to learn the mechanism underlying the structural interaction between WPs and SPIs. Finally, the amino acid compositions of the protein composites were measured in order to evaluate their performance on nutritional properties. The development of soluble hydrocolloids might provide a possible solubilization tech nique for hydrophobic plant proteins and enlarge the application domain of WPs.
20 � C before usage. Complexation of WPs and SPIs. Wheat gluten powder (265.7 mg) was added into 20 mL distilled water, and was fully dissolved by dropping 1 M NaOH in the mixtures to pH 12. By centrifugation at 4000g for 10 min, insoluble substances were separated to obtain WPs solution with a final protein concentration of 10 mg/mL. The WP-SPI mixtures of various WP/SPI ratios (W/S, w/w) were obtained by adding various amounts of SPIs to aforementioned WPs suspensions (1%, w/v). The protein solutions were stirred by a magnetic stirring bar at room tem perature for 1.5 h, slowly neutralized to pH 7 using 0.05 M HCl, and then dialyzed for 24 h to remove salts. The dialyzed mixtures were subse quently centrifuged at 4000 g for 10 min. Then, the supernatants were stored at 4 � C as the stock solutions for further analysis. The precipitates were freeze-dried with the aforementioned freeze-drier into powder with moisture contents ranging from 3.9% to 4.5%, and then subjected to SDSGAGE and amino acid composition analysis. 2.2.2. Nitrogen solubility index (NSI) NSI was measured following a previous described method (Bera & Mukherjee, 2010). After preparation of the stock solutions (section 2.2.1), the recollected residues were subjected to nitrogen measure ments using a Kjeldahl method (N � 5.7) (Wang et al., 2017). Before the measurement, an SDS-PAGE analysis had verified that only a single subunit of SPIs with a dim stripe remained in precipitates (Fig. S1). Therefore, we ignored the trace amounts of residual subunits and calculated the NSI of WPs using the following equation (1): NSI ¼
PWP
Presidue � 100% PWP
(1)
where PWP is the weight of initial WPs, and Presidue is the weight (g) of residual proteins. 2.2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) SDSPAGE analysis were performed following the report of Laemmli (Laemmli, 1970). The Tris–HCl buffer (0.125 M) containing SDS (2%, w/v), 2- glycerol (10%, v/v) and mercaptoethanol (2%, v/v) was applied to solubilize the WP-SPI composites. The solutions were diluted with the mentioned Tris-HCl buffer to a protein concentration of 20 μg/μL, and then heated in boiling water for 5 min. By centrifuging the solutions at 10 000 g for 10 s, 10 μL of the supernatants were collected for electrophoresis. Coomassie Brilliant Blue-R250 (0.05%) was used for the staining of gels.
2. Materials and methods 2.1. Materials Wheat gluten powder were purchased from Anhui Ante Food Co., Ltd (Suzhou, Anhui, China). The content of moisture, ash, starch and protein were 8.31%, 0.54%, 12.73% and 75.27% (N � 5.7). Defatted soy flakes with a protein content of 58.3% (N � 6.25, wet basis) were purchased from Sinograin Oils Co., Ltd. (Peking, China). All other chemicals and reagents were analytical grade.
2.2.4. Morphological microscopy Field emission scanning electron microscopy (FE-SEM). The observation was conducted by stock solutions diluted to 0.02% (w/v) with distilled water. The solutions were spread onto silicon slices (5 � 5 mm) and dried in a dark cabinet overnight at room temperature. The slices were subsequently coated with a film of gold (10 nm) under vacuum and then subjected to morphological microscopy by a SU-8220 FE-SEM (Hitachi, Japan). Atomic force microscopy (AFM). The WP-SPI composites solutions were diluted to 0.002% (w/v) with distilled water. A 2.5 μL drop of each diluted solution was dripped on a mica substrate (10 � 10 mm), and the substrate was placed in a dark cabinet at room temperature overnight to evaporate the solvent. AFM analysis was performed using a Multimode VIII microscope (Bruker Corporation, Billerica, MA, USA) equipped with a nanoprobe cantilever tip (Bruker Nanoprobe, Camarillo, CA, USA).
2.2. Methods 2.2.1. Sample preparation Extraction of SPIs. Samples of SPIs with a protein content of 90.5% (N � 6.25, wet basis) were extracted using a previously described approach (Wang, Xu, Chen, & Wang, 2018). In detail, soy flakes (300 g) were ground to pass through an 80-mesh sieve and then mixed with 3 L of water. The mixture was adjusted to pH 9 using 1 M NaOH and stirred by a magnetic stirring bar for 1 h. After the centrifugation at 7000g for 10 min, the supernatant was collected and adjusted to pH 4.5 using 1 M HCl. The slurry with precipitated protein was then centrifuge at 7000 g for 10 min. The precipitate was then collected and re-dispersed in 5 times of water. The crude proteins were pre-frozen at 80 � C and then freeze-dried in accordance to the procedures at 55 � C for 30 h with a Beta 2–8 LD plus freeze-drier (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The obtained SPIs were stored at
2.2.5. Spectrum acquisition Fluorescence spectrum. The protein concentrations of stock solutions were adjusted to 0.1% (w/v) with distilled water, and the intrinsic fluorescence spectra of protein composites were measured by a F-7000 spectrofluorometer (Horiba, Kyoto, Japan) at room temperature. The 2
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emission was excited at 295 nm and recorded from 310 to 410 nm with an excitation and emission slit of 5 nm. The theoretical fluorescence spectra were obtained by adding up the fluorescence spectra of indi vidual WPs and SPIs with corresponding concentrations in the protein composite solutions. The experimental fluorescence spectra were the fluorescence spectra of complexed proteins (Shigemitsu et al., 2018). To determine the contributions of hydrogen bindings, hydrophobic re actions and electrostatic interactions in the formation of protein com posites, WPs and SPIs were dissolved in 18 mL distilled water at pH 12 following the method mentioned in section 2.2.1 (complexation of WPs and SPIs), and 2 mL of thiourea, SDS, and NaCl solutions (0.1 M) were then separately added into the protein solutions. The final concentration of thiourea, SDS, and NaCl was 10 mM. The fluorescence spectra were collected at pH 7 with the same method as other samples. Circular dichroism (CD). A MOS-450 spectrometer (BioLogic Science Instruments, Ltd., Claix, France) was applied for the collection of CD spectra of protein composites. The protein concentrations of stock so lutions were adjusted to 0.05% (w/v) and 0.1% (w/v) for scanning of CD spectra at the wavelengths ranging from 190 to 250 nm and 250–320 nm which were corresponding to the far-UV region and near-UV region, respectively. The determination was conducted using distilled water as a blank solution. Data were generated as an average of 3 scans at 20 � C.
2.2.8. Statistical analysis All preparations were performed in triplicates. Data were analyzed by one-way analysis of variance (ANOVA) using Origin 9.0 (OriginLab Co., USA). The means comparison was determined by Duncan’s test (p < 0.05). 3. Results and discussion 3.1. NSIs of WPs Soluble protein composites were prepared by co-dissolving WPs and SPIs at pH 12 and recollecting at pH 7. Although the focus of this investigation was WPs, the amount of SPIs was increased to twice that of WPs for deeply learning of the protein-protein interaction. As shown in Fig. 2B, only a dim band of the basic subunit (19 kDa) from 11S of SPIs was observed in the SDS-PAGE profile of residuals, indicating that almost all the subunits of SPIs were dissolved in the supernatant and further collected after interact with WPs. Thus, the amounts of insoluble subunits were neglected, and the NSIs of WPs were approximately calculated as the percentage of WPs dissolved in the solution against the initial WPs used in the reaction. As listed in Fig. 1, the NSI of WPs was improved to nearly 50% by adding SPIs at a W/S ratio of 1:0.1, while that of control was just 4.5%. The NSI of WPs was finally increased up to 72.4% at a W/S ratio of 1:2. It is worth noting that the prepared protein composites can be completely dissolved in water (Fig. 2J). However, our attempt on reducing the W/S ratio to 0.05 was failed as WPs almost completely precipitated. The SDS-PAGE profiles of complexed proteins (Fig. 2A) exhibited distinct bands of all subunits in WPs and SPIs, and the bands of SPIs were brightened along with the addition of SPIs, demon strating that the intact primary structures of proteins were retained. We further tried to remix the residue with SPIs and recollected them after a pH-cycle, it was observed that a part of the residual WPs were assembled with SPIs without any hydrolyzation of peptide chains (Fig. S1), which provided a path to further improve the recovery rate of WPs.
2.2.6. Surface properties of proteins Surface hydrophobicity (H0). The index of H0 was determined by a previously described method (Qiu, Sun, Cui, & Zhao, 2013) with 1-Ani lino-8-naphthalenesulfonate (ANS) as fluorescence probe. A series of protein composite solutions with protein concentrations ranging from 0.0125% to 0.1% (w/v) were obtained by diluting the stock solutions with distilled water, and then each dilution (4 mL) was mixed with 10 μL of 8 mM ANS solution. The fluorescence intensities of samples were measured by the mentioned spectrofluorometer at excitation and emission wavelengths of 390 nm and 484 nm, respectively. H0 was defined as the slope of the fluorescence intensity against protein concentration. Zeta-potential. The zeta-potential analysis was performed following the method described by Wang (Wang et al., 2018) with a Zetasizer Nano instrument (Malvern Instruments Ltd., Malvern, UK). After dilu tion to 0.1% (w/v) with distilled water, all protein samples were analyzed at 25 � C with refractive indices of 1.450 and 1.330 for proteins and dispersion medium, respectively. WPs and SPIs were subjected to the same pH-cycle treatment, and the supernatants after centrifugation were used for the measurement.
3.2. Morphologies of the WP–SPI composites The FE-SEM observation was conducted to visually reveal the in teractions between WPs and SPIs in water. The images showed that WPs strongly aggregated to form massive particles with densely packed structures (Fig. 2C), while the SPIs showed a laminar microstructure
2.2.7. Amino acid composition The amino acid composition (g/100 g protein) was detected by an acid hydrolyzation method using an amino acid analyzer (835-50, Hitachi, Japan) equipped with a PicoTag column at 254 nm. The freezedried proteins were hydrolyzed in sealed tubes with 6 M HCl at 110 � C for 24 h. Then, the obtained hydrolyzed solutions were neutralized by 4 M NaOH, and centrifuged at 10,000 g for 10 min. The collected su pernatants were subjected to amino acid analysis at 38 � C. The data (expressed as g per 100 g protein) did not include Tryptophan (Trp) because of the easy degradation of Trp. The essential amino acid index (EAAI) was the geometric mean of the content of the essential amino acids in measured proteins to their corresponding contents in the whole ~ as, Gomez, Frias, Baeza, & Vidal-Valverde, 2010). The egg protein (Pen values were calculated following equation (2): sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Thrp Valp n Lysp EAAI ¼ 100 � (2) � � ⋅⋅⋅ � Lyss Thrs Vals where the subscript ‘p’ means the protein sample, ‘s’ means the whole egg protein, and ‘n’ means the number of amino acids.
Fig. 1. Nitrogen solubility index (NSI) of WPs. Control was wheat gluten powder with the same pH-cycle treatment. W/S means the WPs/SPIs ratio. Asterisks (*) and (**) above bars respectively represent significant differences (p < 0.05) and very significant differences (p < 0.01) relative to the control. 3
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Fig. 2. SDS-PAGE analysis and FE-SEM images of WP-SPI composites. (A) and (B) SDS-PAGE profiles of preprared WP-SPI composites and residual proteins. Lanes 1–3 are markers, WPs, and SPIs, respectively; lanes 4–8 are protein composites or residual proteins with W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2, respectively; (C), (D), (E), (F), (G), (H)and (I) are FE-SEM images of WPs, SPIs and protein composites prepared by W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2, respectively; Scale bars ¼ 1 μm. (J) Pictures of redissolved protein composites. No.1 is control, 2–6 are protein composites correspoding to lanes 4–8 in SDS-PAGE, respectively. The concentration of each sample was 10 mg/mL.
tiled on the silicon slice (Fig. 2D). After incorporating with a handful of SPIs, the WPs decreased their self-aggregation but formed cobblestonelike particles with smooth surfaces and diameters between 100 and 250 nm (Fig. 2E). Similar shapes and sizes were observed for all of the samples till the W/S ratio was increased to 1:1.5 (Fig. 2F–H). However, when the amount of SPIs that could be incorporated into WPs reached the limit (W/S ¼ 1:2), the excess of SPIs aggregated into membrane and simultaneously integrated with the WP-SPI composites, leading to the generation of distinctive film structures which were observed in Fig. 2I. AFM images showed similar morphologies of protein composites to those observed by FE-SEM. With moderate addition of SPIs (W/ S ¼ 1:0.1–1:1.5), the protein composites appeared as uniformly dispersed nanoparticles which had diameters around 100 nm (Fig. 3A–D) and thicknesses of several nanometers (insets at the right of Fig. 3) at a low concentration (0.002%, w/w). The stretched structures might contribute to easier hydration due to larger contact surface. When the W/S ratio reached 1:2, the stable dispersion of protein composites in water was destroyed as the particles aggregated into clusters by the interactions with excess of SPIs (Fig. 3E), which was in accordance with the FE-SEM results. Morphological transition of proteins indicated that the agglomerate structures of WPs were transformed into new hydratable structures due to the incorporation of SPIs, leading to the improvement of protein solubility. Therefore, to understand the formation of the co-assembled structures, the process of protein structural alternation was investi gated subsequently.
and SPIs, which could distinguish this structural reaction from the simple mix of two proteins. The fluorescence profiles showed that both WPs and SPIs had a distinct peak intensity (Fmax) around 340 nm that was the mainly emission of Trp (Fig. S2). Numerous studies have verified that the interactions of proteins with foreign substances noticeably quench the intrinsic fluorescence of proteins, and the quenching was strengthened with the increase of addition amount (Rawel, Meidtner, & Kroll, 2005; Xiao et al., 2008; Zhang, Wright, & Zhong, 2013). There fore, the change of the emission can be applied as an evidence for the structural transition of proteins. In this scenario, the theoretical (sum of the fluorescence spectra of WPs and SPIs with corresponding concen trations) and experimental fluorescence spectra (the fluorescence spectra of complexed proteins) were compared. Taking the solution with a W/S ratio of 1:1 as an example (Fig. 4A), the Fmax of experimental fluorescence spectra were much lower than those of the theoretical ones owing to the photoinduced electron transferred from the electron-rich aromatic amino groups to the electron-deficient groups (Shiki et al., 2014), which indicated that the two proteins combined with each other to form novel composites. The fluorescence quenching was observed at all W/S ratios. The differences between the theoretical and experimental Fmax values that expressed the degree of quenching increased along with the increasing of SPIs (Fig. 4B), confirming that more SPIs participated in the formation of protein composites. The far-UV CD spectra showed that all protein composites had wide negative bands between 200 and 240 nm at pH 7, but the ellipticity was reduced with the increase of SPI proportion (Fig. 4D), which was a strong indication of complexed sec ondary structures. A previous research (Yang et al., 2007) had proved that the folding degree of proteins could be characterized by ellipticity at the wavelength of 222 nm, the ellipticities of protein composites at 222 nm were therefore plotted against the W/S ratios to show protein refolding explicitly. As shown in Fig. 4C, incrementing addition of SPIs significantly weakened the ellipticity, indicating that the WP-SPI com posites had more stable and unfolding structures. Since the interactions between the two proteins were confirmed, it was necessary to illuminate the molecular forces involved in the for mation of protein composites. Therefore, thiourea, SDS and NaCl were
3.3. Molecular binding Successful researches had been reported that the addition of grain constituents, e.g. fibers, improved the dough properties of wheat flour as a result of interactions between polysaccharides structure and wheat proteins (Wang, Rosell, & Barber, 2002). Although the morphology analysis has showed the formation of novel protein composites, protein mixtures neutralized to pH 7 were subjected to fluorescence and far-UV CD spectra to demonstrate the protein-protein interactions between WPs 4
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Fig. 3. AFM images of the WP-SPI composites; (A), (B), (C), (D)and (E) are protein composites prepared with W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2, respectively; Scale bars ¼ 200 nm. The insets at the right of each image are height profiles of the structures marked with white lines, in the direction from left to right.
solely added as blocking agents in the reaction mixtures with a final concentration of 10 mM at the beginning of the reaction, and the fluo rescence intensities of neutralized protein solutions were then compared to explore the contributions of hydrogen bindings, hydrophobic re actions and electrostatic interactions, respectively (Lennart & Bertil, 2005). Contrary to the previously mentioned fluorescence quenching caused by the interactions, growing fluorescence intensities (Fig. 4E) suggested that the interactions between WPs and SPIs were prevented by the blocking agents, verifying that the interactions were simultaneously driven by hydrogen bindings, hydrophobic reactions and electrostatic interactions. However, inconsistent with the results obtained in our previous studies on rice proteins that the main force involved in for mation of protein composites was hydrogen bonding (Wang, Xu, Chen,
Zhou, & Wang, 2018), the reaction mixture supplemented with SDS exhibited the maximum fluorescence intensity, implying the primary role of hydrophobic reactions playing in the co-assembly of proteins. However, an opposite phenomenon compared to fluorescence was observed, showing that the neutralized reaction mixture with SDS addition showed the highest recovery rate (Fig. S3). This abnormal result indicated that the addition of SDS inhibited the aggregation of WPs by blocking the hydrophobic interactions. This subjected WPs to be inclined to interact with the SPIs during neutralization, thereby pro moting the formation of the soluble protein composites. Therefore, the formation process of the combined structures was further characterized in this study.
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Fig. 4. Characterization of the molecular structures of WP-SPI composites. (A) Theo retical and experimental emission spectra of composites prepared with a W/S of 1:1 at pH 7; (B) The difference values between the peak intensity (Fmax) of theoretical and experimental emission spectra of each pro tein composites prepared at W/S ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2. (C) The elliptic extrema at 222 nm corresponding to CD spectra of each protein composites. (D) Far-UV CD spectra of WPs, SPIs and WP-SPI protein composites at pH 7; WPs and SPIs were treated in the same way as the protein composite but without any addition of other proteins; (E) Fluorescence emission spectra of protein composites (W/S ¼ 1:1) mixed with 10 mM NaCl, thiourea, or SDS during complexation at pH 7. Different letters close to error bars indicate significant differences (p < 0.05).
3.4. Inhibition of structural folding of proteins
promoted the co-existed proteins structured into unfolded conformations. The ellipticity at 275 nm of each sample was a typical signal of Tyr (Kelly & Price, 1997), which was plotted against pH to intuitively show the process of protein folding (Javid et al., 2013). For co-solvated pro teins with a W/S ratio of 1:0.1, negligible change was observed when pH was above 10 (Fig. 5B). Keeping titration of HCl until pH dropped below 10, the ellipticity was weakened sharply as a result of protein refolding, a hint that the interactions of WPs with SPIs possibly began at pH 10. Although similar phenomenon was observed on samples with W/S ratios of 1:0.5 and 1:1, the incorporation of more SPIs eventually reduced the refolding degree of proteins. And for samples with ratios of 1:1.5 and 1:2, the ellipticity approximately remained unchanged along with the reduction of pH, except for tiny changes in the pH range of 8-7. The CD result reiterated that the SPIs merged into WPs conformations by the
Each aromatic amino acid residue exhibits CD signals in the near-UV region in the asymmetric microenvironment, which is a reflection of the tertiary structures of proteins (Kelly & Price, 2000). Therefore, CD spectrum was used as a sensitive spectroscopic probe to characterize the structural refolding of protein during neutralization. As showed in Fig. S4, the near-UV region of co-existent WPs and SPIs at pH 12 and 11 had negligible ellipticities that were strengthened subsequently along with the HCl addition, suggesting that the renatured proteins approached more intricate tertiary structures. Therefore, the formation of protein composites was mainly induced by the large synergistic conformational changes during protein refolding resultant from neutralization. Besides, growing proportion of SPIs weakened the ellipticity at pH 7 (Fig. 5A), proving that the incorporation of SPIs
Fig. 5. (A) Near-UV CD spectra of protein composites at pH 7; WPs were treated in the same way as the preparation of protein composite but without SPIs. (B) Plotting of the ellipticity at 275 nm against pH. 6
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surface that could provide intermolecular repulsions among adjacent proteins. The zeta-potential analysis, along with the hydrophobicity, again revealed the unfolding structures of protein composites, and explained the increasingly soluble nature stemming from the partici pation of SPIs. Substantial evidences jointly corroborated that both of WPs and SPIs unfolded at pH 12 and interacted with the exposed charged groups during neutralization to form nanoparticles that were uniformly dispersed in water. The nanoparticles, with relatively unfolded struc tures, showed good stability that was similar to the protein structures at pH 12, thus greatly improving the solubility of the proteins. In this study, the protein composites show over 72% solubility at a W/S ratio of 1:2, which was similar to that of deamidated samples (Wang et al., 2017). Considering the intact primary structures of protein composites, the nutritional values may be greatly maintained, and the amino acid compositions were further measured.
co-folding of proteins, and more tenacious backbones of the combined structures formed by the interactions was the reason of weaker folding and less self-aggregation of co-solved proteins. To adequately under stand the dispersibility of protein composites, the interfacial properties analysis was further performed. 3.5. Interfacial properties of proteins affected by interaction As shown in Fig. 6B, the hydrophobicity of the complexed proteins increased along with the reduction of pH, which was another evidence of protein refolding. In this regard, the decrease of hydrophobicity was actually caused by the formation of hydrophobic pockets which were available to ANS probes due to protein refolding (Hawe, Sutter, & Jis koot, 2008). Correspondingly, the zeta-potential showed slight im provements with decreasing of pH (Fig. 6D), suggesting that a part of charged groups that exposed at pH 12 were buried along with the packing of unfolded structures. From this view, the hydrophobic groups were packed inside the co-assembled structures, inducing the formation of protein composites, in good agreement with the fluorescence quenching listed in Fig. 4E. Furthermore, based on the experimental phenomenon, it could be speculated that the embedding of hydrophobic groups played a driving role in the co-assembly of the two proteins, which was different from the interaction between fibers and WPs (Wang et al., 2002). On the other hand, the increasing level of added SPIs obviously reduced the hydrophobicity of the protein composites, and all of the complexed samples had lower hydrophobicity than individual WPs (Fig. 6A). The SPIs showed much higher zeta-potential intensity (32.3 mV) than WPs (14.3 mV), and the zeta-potential showed a sharper increase as more SPIs was incorporated into WPs (Fig. 6C). Although it was lower than that of SPIs, all of the potential values of the complexed proteins were higher than that of WPs, evidencing the more charged
3.6. Amino acid analysis WPs and SPIs are considered as good sources of amino acids for humans, which widely exist in human diet (Friedman, 1996), especially for WPs. The Food and Agriculture Organization and the World Health Organization (FAO/WHO) provide a recommendation about the dietary proteins that human need nine essential amino acids, i.e. His, Ile, Leu, Lys, Met þ Cys, Phe þ Tyr, Thr, Trp, and Val to maintain normal body function. The recommended levels of each amino acid are 19, 28, 66, 58, 25, 63, 34, 11, and 35 mg/g protein for pre-school children, and are 16, 13, 19, 16, 17, 19, 9, 5, and 13 mg/g protein for adults, respectively. As shown in Table 1, all of complexed proteins showed compromised amino acid compositions with EAAI values ranging from 58.97 to 70.22 while the SPIs and WPs showed EAAI values of 70.39 and 55.95, respectively. By interacting with SPIs, the WPs made up for the deficiency of Arg and
Fig. 6. Surface properties of protein composites; (A) The hydrophobicity of WPs, SPIs and the protein composites at pH 7; (B) The hydrophobicity of the protein composites prepared at W/S ration of 1:1 at pH 7–12; (C) The zeta-potential of WPs, SPIs and the protein composites at pH 7. (D) The zeta-potential of the protein composites prepared at W/S ration of 1:1 at pH ranging from 7 to 12. Different letters above bars indicate significant differences (p < 0.05). 7
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Table 1 Amino acid analysis of WP-SPI composites. a Amino acid
SPI
Essential amino acids His 2.65 � 0.42ab Thr 3.10 � 0.21a Tyr 3.16 � 0.33a Cys-s 0.18 � 0.01a Val 4.89 � 0.11ab Met 1.11 � 0.18a Phe 6.02 � 0.31b Ile 4.48 � 0.33ab Leu 8.18 � 0.32b Lys 6.02 � 0.37e Nonessential amino acids Asp 12.17 � 0.20e Glu 23.92 � 0.28a Ser 4.16 � 0.13a Gly 4.27 � 0.23a Arg 6.72 � 0.30c Ala 3.76 � 0.08a Pro 5.23 � 0.08a EAAI 70.39 � 1.83c a
WP
1:0.1
1:0.5
1:1
1:1.5
1:2
2.02 � 0.27a 2.44 � 0.21a 2.73 � 0.31a 0.82 � 0.11c 5.28 � 0.13b 1.78 � 0.28b 3.83 � 0.36a 4.20 � 0.17a 7.10 � 0.10a 1.56 � 0.32a
2.44 � 0.20ab 2.69 � 0.40a 2.51 � 0.17a 0.28 � 0.23ab 5.14 � 0.13b 1.12 � 0.41ab 5.27 � 0.31b 4.07 � 0.20a 7.08 � 0.37a 2.78 � 0.34b
2.44 � 0.33ab 2.67 � 0.40a 2.72 � 0.03a 0.36 � 0.14ab 4.99 � 0.06ab 1.48 � 0.18ab 5.68 � 0.25b 4.49 � 0.13ab 7.48 � 0.41a 3.44 � 0.42bc
2.24 � 0.06ab 2.47 � 0.11a 2.81 � 0.28a 0.44 � 0.08ab 4.65 � 0.31a 1.36 � 0.14ab 5.73 � 0.11b 4.31 � 0.10ab 7.20 � 0.16a 3.29 � 0.42bc
2.51 � 0.13ab 2.88 � 0.37a 3.12 � 0.34a 0.51 � 0.25abc 5.10 � 0.07b 1.26 � 0.11ab 5.72 � 0.38b 4.74 � 0.27b 7.45 � 0.08a 4.01 � 0.36cd
2.73 � 0.30b 2.93 � 0.06a 2.97 � 0.36a 0.62 � 0.03bc 5.23 � 0.21b 1.27 � 0.35ab 5.68 � 0.25b 4.49 � 0.16ab 7.64 � 0.20ab 4.28 � 0.26d
5.18 � 0.28a 38.44 � 0.30g 4.07 � 0.17a 4.32 � 0.08a 3.83 � 0.35a 3.01 � 0.38a 9.38 � 0.03e 55.95 � 0.99a
7.24 � 0.06b 33.92 � 0.30g 4.01 � 0.34a 4.15 � 0.17a 4.90 � 0.03b 3.22 � 0.31a 9.19 � 0.07e 58.97 � 4.14ab
9.41 � 0.18c 31.25 � 0.20d 3.64 � 0.34a 3.82 � 0.10a 5.45 � 0.22b 3.37 � 0.39a 7.33 � 0.38d 64.91 � 0.68bc
9.76 � 0.27c 32.48 � 0.41e 3.75 � 0.17a 3.95 � 0.17a 5.20 � 0.16b 3.44 � 0.30a 6.93 � 0.04c 61.70 � 2.30ab
9.42 � 0.28c 29.63 � 0.11c 3.65 � 0.33a 4.03 � 0.17a 6.28 � 0.27c 3.52 � 0.38a 6.18 � 0.11b 68.12 � 3.32bc
10.34 � 0.25d 27.62 � 0.41b 4.01 � 0.10a 4.19 � 0.41a 6.24 � 0.34c 3.76 � 0.40a 6.01 � 0.04b 70.22 � 2.88c
Values are expressed as the percentage weight of the amino acid against protein weight. Different superscript letters indicate significant differences (p < 0.05).
Appendix A. Supplementary data
Lys, and compensated for the lack of Pro in SPIs at the same time. However, the contents of Lys and Thr in protein composites were still too low to meet the requirements for pre-school children, which hindered the application to a certain extent. Nonetheless, the soluble protein composites have great potential becoming a superior protein source for adults. The amino acid compositions of residual proteins collected during the preparation of protein composites were also determined (Table S1). The residual proteins with different W/S ratios showed similar amino acid compositions, which was another indication that the amounts of residual SPIs in the precipitates were very small. However, the glutamic (Glu) contents of precipitates were lower than that of WPs, implying that some glutamic -enriched peptide chains were more likely to assemble with SPIs.
Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105395. References Apichartsrangkoon, A., Ledward, D. A., Bell, A. E., & Brennan, J. G. (1998). Physicochemical properties of high pressure treated wheat gluten. Food Chemistry, 63 (2), 215–220. Bera, M. B., & Mukherjee, R. K. (2010). Solubility, emulsifying, and foaming properties of rice bran protein concentrates. Journal of Food Science, 54(1), 142–145. Bert, L., Thewissen, B. G., Kristof, B., & Delcour, J. A. (2007). Impact of redox agents on the extractability of gluten proteins during bread making. Journal of Agricultural and Food Chemistry, 55(13), 5320–5325. Day, L., Augustin, M. A., Batey, I. L., & Wrigley, C. W. (2006). Wheat-gluten uses and industry needs. Trends in Food Science & Technology, 17(2), 82–90. � � Zu� � za, M. G., Lukovi�c, N. D., Elmalimadi, M. B., Stefanovi�c, A. B., Sekuljica, N.Z., Jovanovi�c, J. R., et al. (2017). The synergistic effect of heat treatment on alcalaseassisted hydrolysis of wheat gluten proteins: Functional and antioxidant properties. Journal of Food Processing and Preservation, 41. https://doi.org/10.1111/jfpp.13207. Fabian, C., & Ju, Y. H. (2011). A review on rice bran protein: Its properties and extraction methods. Critical Reviews in Food Science and Nutrition, 51(9), 816–827. Friedman, M. (1996). Nutritional value of proteins from different food sources. A review. Journal of Agricultural and Food Chemistry, 44(1), 6–29. Hand, L. W., Crenwelge, C. H., & Terrell, R. N. (2010). Effects of wheat gluten, soy isolate and flavorings on properties of restructured beef steaks. Journal of Food Science, 46 (4), 1004–1006. Hawe, A., Sutter, M., & Jiskoot, W. (2008). Extrinsic fluorescent dyes as tools for protein characterization. Pharmaceutical Research, 25(7), 1487–1499. Javid, N., Roy, S., Zelzer, M., Yang, Z. M., Sefcik, J., & Ulijn, R. V. (2013). Cooperative self-assembly of peptide gelators and proteins. Biomacromolecules, 14(12), 4368–4376. Kelly, S. M., & Price, N. C. (1997). The application of circular dichroism to studies of protein folding and unfolding. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1338(2), 161–185. Kelly, S. M., & Price, N. C. (2000). The use of circular dichroism in the investigation of protein structure and function. Current Protein & Peptide Science, 1(4), 349–384. Kong, X., Zhou, H., & Qian, H. (2007). Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates. Food Chemistry, 102(3), 759–763. Kristinsson, H. G., & Rasco, B. A. (2000). Fish protein hydrolysates: Production, biochemical, and functional properties. Critical Reviews in Food Science and Nutrition, 40(1), 43–81. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. Lennart, N., & Bertil, H. (2005). Molecular origin of time-dependent fluorescence shifts in proteins. Proceedings of the National Academy of Sciences of the United States of America, 102(39), 13867–13872. Mejri, M., Rog�e, B., BenSouissi, A., Michels, F., & Mathlouthi, M. (2005). Effects of some additives on wheat gluten solubility: A structural approach. Food Chemistry, 92(1), 7–15. Pe~ nas, E., Gomez, R., Frias, J., Baeza, M. L., & Vidal-Valverde, C. (2010). High hydrostatic pressure effects on immunoreactivity and nutritional quality of soybean products. Food Chemistry, 125(2), 423–429.
4. Conclusion In summary, soluble protein composites with hydrophilic particulate nanostructures can be prepared by co-dissolving WPs and SPIs at pH 12 and neutralizing the solutions to pH 7 (a pH-cycle). The interactions between the two proteins inhibited the structural refolding and sus tained the charged surface of proteins under neutral condition. With W/ S ratios ranging from 1:0.1 to 1:2, the solubility of WPs was improved from <5% to >72% without negligible damages on the primary struc tures of proteins. The amino acid compositions of the composites met the requirement of FAO/WHO for the daily intake for adults. This facile technique relying on a simple pH-cycle procedure is believed to have feasibility for solubilization of various insoluble plant proteins, and for the preparation of tailor-made protein ingredients. Declaration of competing interest Authors declare no conflicts of interest. Acknowledgements The research was financially supported by the National Natural Science Foundation of China (No. 31778198; 31901602), and National first-class discipline program of Food Science and Technology (JUFSTR20180203), the Foundation of Scientific Development of Wuxi, China (No. 0302B010504180011PB), the Scientific Plan for Jiangsu Province, China (BE2018309).
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Qiu, C., Sun, W., Cui, C., & Zhao, M. (2013). Effect of citric acid deamidation on in vitro digestibility and antioxidant properties of wheat gluten. Food Chemistry, 141(3), 2772–2778. Rawel, H. A., Meidtner, K., & Kroll, J. (2005). Binding of selected phenolic compounds to proteins. Journal of Agricultural and Food Chemistry, 53(10), 4228–4235. Shigemitsu, H., Fujisaku, T., Tanaka, W., Kubota, R., Minami, S., Urayama, K., et al. (2018). An adaptive supramolecular hydrogel comprising self-sorting double nanofibre networks. Nature Nanotechnology, 13(3), 165–172. Shiki, Y., Kazunori, I., Mitsuaki, Y., Takashi, K., Akihide, K., Shinobu, U., et al. (2014). Photocontrol over self-assembled nanostructures of π-π stacked dyes supported by the parallel conformer of diarylethene. Angewandte Chemie, 126(10), 2640–2644. Wang, Y., Gan, J., Zhou, Y., Cheng, Y., & Nirasawa, S. (2017). Improving solubility and emulsifying property of wheat gluten by deamidation with four different acids: Effect of replacement of folded conformation by extended structure. Food Hydrocolloids, 72, 105–114. Wang, P., Jin, Z., & Xu, X. (2015). Physicochemical alterations of wheat gluten proteins upon dough formation and frozen storage - a review from gluten, glutenin and gliadin perspectives. Trends in Food Science & Technology, 46(2), 189–198. Wang, J., Rosell, C. M., & Barber, C. B. D. (2002). Effect of the addition of different fibres on wheat dough performance and bread quality. Food Chemistry, 79(2), 221–226.
Wang, T., Xu, P., Chen, Z., & Wang, R. (2018). Mechanism of structural interplay between rice proteins and soy protein isolates to design novel protein hydrocolloids. Food Hydrocolloids, 84, 361–367. Wang, T., Xu, P., Chen, Z., Zhou, X., & Wang, R. (2018). Alteration of the structure of rice proteins by their interaction with soy protein isolates to design novel protein composites. Food & Function, 9, 10.1039.C1038FO00661J-. Wang, T., Yue, M., Xu, P., Wang, R., & Chen, Z. (2018). Toward water-solvation of rice proteins via backbone hybridization by casein. Food Chemistry, 258(30), 278–283. Xiao, J., Suzuki, M., Jiang, X., Chen, X., Yamamoto, K., Ren, F., et al. (2008). Influence of B-ring hydroxylation on interactions of flavonols with bovine serum albumin. Journal of Agricultural and Food Chemistry, 56(7), 2350–2356. Yang, F., Zhou, B., Zhang, P., Zhao, Y. F., Chen, J., & Liang, Y. (2007). Binding of ferulic acid to cytochrome c enhances stability of the protein at physiological pH and inhibits cytochrome c-induced apoptosis. Chemico-Biological Interactions, 170(3), 231–243. Yong, Y. H., Yamaguchi, S., & Matsumura, Y. (2006). Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of wheat gluten. Journal of Agricultural and Food Chemistry, 54(16), 6034–6040. Zhang, Y., Wright, E., & Zhong, Q. (2013). Effects of pH on the molecular binding between β-lactoglobulin and bixin. Journal of Agricultural and Food Chemistry, 61(4), 947–954.
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