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Cite This: J. Agric. Food Chem. 2019, 67, 7886−7897

Role of Gluten in Surface Chemistry: Nanometallic Bioconjugation of Hard, Medium, and Soft Wheat Protein Divya Mandial,§ Poonam Khullar,*,§ Vikas Gupta,∥ Harsh Kumar,⊥ Narpinder Singh,# Gurinder Kaur Ahluwalia,‡ and Mandeep Singh Bakshi*,†

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Department of Chemistry, Natural and Applied Sciences, University of Wisconsin - Green Bay, 2420 Nicolet Drive, Green Bay, Wisconsin 54311-7001, United States ‡ Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2V 2K7, Canada § Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India ∥ Department of Biotechnology, DAV College, Amritsar 143005, Punjab, India ⊥ Department of Chemistry, Dr. B. R. Ambedkar National Institute of Technology, Jalandhar 144011, Punjab, India # Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, Punjab, India S Supporting Information *

ABSTRACT: Hard, medium, and soft wheat proteins, based on gluten content, were studied for their important roles in nanometallic surface chemistry. In situ synthesis of Au nanoparticles (NPs) was followed to determine the surface adsorption behavior of wheat protein based on the gluten contents. A greater amount of gluten contents facilitated the nucleation to produce Au NPs. X-ray photoelectron spectroscopy (XPS) surface analysis clearly showed the surface adsorption of protein on nanometallic surfaces which was almost equally prevalent for the hard, medium, and soft wheat proteins. Wheat protein conjugated NPs were highly susceptible to phase transfer from aqueous to organic phase that was entirely related to the amount of gluten contents. The presence of higher gluten content in hard wheat protein readily enabled the hard wheat protein conjugated NPs to move across the aqueous−organic interface followed by medium and soft wheat protein conjugated NPs. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS page) analysis allowed us to determine molar masses of nanometallic surface adsorbed protein fractions. Only two protein fractions of high molar masses (74 and 85 kDa) from SDS solubilized hard, medium, and soft wheat proteins preferred to adsorb on nanometallic surfaces out of more than 15 protein fractions of pure wheat protein. This made the surface adsorption of wheat protein highly selective and closely related to gluten content. Cetyltrimethylammonium bromide (CTAB) solubilized wheat protein conjugated NPs demonstrated their strong antimicrobial activities against both Gram negative and Gram positive bacteria making them suitable for their applications in food industry. KEYWORDS: wheat protein, gluten contents, nanometallic surface adsorption, phase transfer, wheat protein conjugated nanomaterials

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INTRODUCTION Wheat is an important component of human diet, and wheat protein is extensively studied in food chemistry.1−3 On the basis of gluten contents, wheat protein is categorized into three categories of hard, medium, and soft protein.4−6 Hard wheat protein contains about 12−14% of gluten. Gluten consists of monomeric water-soluble gliadin, which shows viscous behavior, and polymeric water insoluble glutenin, which is elastic. Glutenin is a heterogeneous mixture of a number of different high and low molecular mass subunits connected through disulfide bonds with strong hydrophobic interactions and hence constitutes up to 47% of the total protein contents.4 The presence of glutenin makes gluten highly hydrophobic, and hence it cannot be easily solubilized in water but is readily soluble in aqueous micellar solution.7−10 Surfactant molecules in the aqueous phase interact with the hydrophobic domains of protein through their hydrocarbon chains while their polar head groups provide essential polarity for aqueous solubilization.11 Aqueous solubilized wheat protein is an excellent © 2019 American Chemical Society

candidate for nanometallic surface adsorption and its subsequent applications in materials chemistry. Fundamentally, it is interesting to observe how hard, medium, and soft proteins, based on gluten content, interact with the nanometallic surfaces, involve themselves in the crystal growth control, and provide essential colloidal stabilization. These aspects12−14 are the key for successful bioconjugation to produce hybrid nanomaterials that can perform diverse functions including biocompatibility.15,16 Bioconjugation of a protein on nanometallic surfaces is mainly driven by the amphiphilic and unfolding behavior of a protein.15,16 Predominantly hydrophobic protein demonstrates interesting surface adsorption that depends on the degree of unfolding.8−10 Greater unfolding promotes a greater deposition of Received: Revised: Accepted: Published: 7886

February 14, 2019 May 17, 2019 June 25, 2019 June 25, 2019 DOI: 10.1021/acs.jafc.9b01015 J. Agric. Food Chem. 2019, 67, 7886−7897

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Journal of Agricultural and Food Chemistry protein. If this process is studied under in situ reaction conditions that involve a simultaneous reduction of metal ions into metal atoms, then predominantly hydrophobic proteins prove to be in much better shape controlling, as well as colloidal stabilizing agents, than the hydrophilic proteins.17 These characteristic features of strongly hydrophobic proteins are similar to those of highly hydrophobic conventional surfactants which also act as excellent shape directing as well as stabilizing agents for the nanomaterials.18−20 However, nanomaterials stabilized by ionic surfactants are not suitable for biological applications due to their inherent toxicity. Bioconjugation achieved by using different proteins based on their functionalities provides the best compatibility of nanomaterials with biological systems and hence wheat protein conjugated nanoparticles (NPs) may also find several applications in food and pharmaceutical industries. The easy and low cost accessibility of wheat protein allows us to synthesize cost-effective bioconjugated nanomaterials for diverse applications. Hence, they require comprehensive study as they remain largely elusive so far. The focus and aim of this study is to demonstrate how gluten contents drive the surface adsorption of protein and bring about a clear distinction among the fundamental behaviors of hard, medium, and soft protein conjugation to nanometallic surfaces.

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Au 3 +(aq) + wheat protein(aq) + 3e− → Au o(s) + oxidizing products A NPs suspension thus obtained was cooled to room temperature and kept overnight. It was purified with pure water at least three times to remove unreacted protein. Purification was done by collecting the Au NPs at 8000−10000 rpm for 5 min after washing each time with distilled water. Characterization. All reactions were simultaneously monitored with the help of UV−visible (Shimadzu-Model No. 2450, double beam) measurements. The UV−visible instrument was equipped with a TCC 240A thermoelectrically temperature controlled Cell Holder that allowed us to measure the spectrum at a constant temperature of ±1 °C. Au NPs were characterized by transmission electron microscopic (TEM) analysis on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared by mounting a drop of a solution on a carbon-coated Cu grid which was allowed to dry in air. X-ray diffraction (XRD) patterns were characterized by using a Bruker-AXS D8-GADDS with Tsec = 480. Samples were prepared on glass slides by spotting a concentrated drop of aqueous suspension wgich was dried in a vacuum desiccator. The surface chemical composition of NPs was confirmed with the help of X-ray photoelectron spectroscopy (XPS) measurements. Each sample was analyzed by using a Kratos Axis Ultra X-ray photoelectron spectrometer. XPS can detect all elements except hydrogen and helium and can probe the surface of the sample to a depth of 7−10 nm. Survey scan analyses were carried out with an analysis area of 300 μm × 700 μm. SDS Page Gel Electrophoresis. SDS page analysis was performed to characterize the protein fractions of low and high molar masses of pure hard, medium, and soft wheat proteins. Similarly, SDS page analyses of wheat protein conjugated NPs samples were also performed to determine the nanometallic surface adsorbed fractions of hard, medium, and soft wheat protein. Analyses were done by taking the purified samples along with a 1× sample loading buffer. They were boiled in a water bath at 100 °C to remove the protein corona adsorbed on the NPs surface. The suspension thus obtained was further centrifuged to remove NPs, and the supernatant that contained adsorbed protein was subjected to SDS page analysis. A 10 μL portion of the sample was loaded in the well with 5% stacking gel which was solidified over 12% separating gel. The loaded gel was immersed with 1× Tris-Glycine SDS gel running buffer and was electrophoresed at 120 V and 10 mA. The gel was stained and destained to obtain clear visible bands. Antimicrobial Activity of Wheat Protein Conjugated NPs. The in vitro antibacterial activity of NPs was evaluated against Klebsiella pneumoniae (Gram negative bacteria) and Staphylococcus epidermidis (Gram positive bacteria) by using agar well diffusion assay, with nutrient agar as growth media and the determination of zone of inhibition measurement in millimeters. Bacterial suspensions of K. pneumoniae and S. epidermidis were prepared at a concentration of 0.5 McFarland Scale. Agar plates were inoculated with 50 μL of NPs (1 mM), oxytetracycline (0.5 mg/mL) (positive control), and distilled water (negative control) separately into three wells and incubated at 37 °C for 24 h. The diameter of the bacterial growth inhibition zone denoted the antibacterial activity. The minimum inhibitory concentrations (MICs) were determined by following the standard micro dilution method by using a 96 well plate.

EXPERIMENTAL SECTION

Materials. Chloroauric acid (HAuCl4), silver nitrate (AgNO3), sodium dodecyl sulfate (SDS), and cetyltrimethylammonium bromide (CTAB) were purchased from Aldrich. The hard, medium, and soft wheat proteins were extracted and characterized as mentioned elsewhere.4 They were characterized by SDS page analysis, and further details are mentioned in the Discussion section devoted to SDS page analysis. Physiochemical Characterization of Wheat Protein. Dynamic Light Scattering (DLS) Analysis. Wheat protein was solubilized in aqueous SDS and CTAB micellar solutions. Both solutions provided good aqueous medium for complete solubilization of wheat protein. Self-aggregation and ζ potential behaviors of aqueous solubilized wheat protein were determined by DLS measurements with temperature. Multiangle particle sizing and low angle ζ potential analyses were done by DLS and ELS (electrophoretic light scattering), respectively, using a minimum number of optical components in the apparatus (NICOMP Nano Particle Size Analyzer system, model: Z3000 ZLS). It was equipped with a Peltier thermoelectric element which regulated the temperature of the sample cell within ±0.2 °C with a lower limit of 0 °C and a upper limit of 90 °C. The Particle size analysis was calibrated with nanosphere size standards of 90 and 240 nm, while ζ potential was calibrated using ζ reference standards. The measurements were made using a quartz cuvette with a path length of 1 cm. The particle size analysis was recorded for both the Gaussian system and the NICOMP distribution over a varying temperature range of 20 °C − 70 °C. In Situ Synthesis of Wheat Protein Conjugated NPs. The synthesis of wheat protein conjugated Au NPs was carried out under in situ reaction conditions. First, the wheat protein was solubilized in 10 mM aqueous (SDS or CTAB) surfactant solution at room temperature that partially unfolds the protein and solubilizes it in aqueous phase. Then, aqueous mixtures (total 10 mL) of protein (0.1−0.4%) and HAuCl4 (0.25−1.0 mM) were taken in screw-capped glass bottles and kept in a water thermostat bath (Julabo F25) precisely at 70 ± 0.1 °C for 6 h under static conditions. Wheat protein acts as a weak reducing agent due to the presence of aqueous exposed reducing amino acids like cysteine and methionine. The following reaction is expected to occur in order to produce Au NPs.

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RESULTS AN DISCUSSION DLS and ζ Potential of Aqueous Wheat Protein. DLS and ζ potential studies are used to determine the selfaggregation and polarity of wheat protein with temperature, respectively. Figure 1 depicts the size and ζ potential variation of the aqueous solution of hard wheat protein. Three different size distribution histograms (Figure 1a, inset) are obtained, and their variation is shown in Figure 1a. The three prominent sizes, i.e., A, B, and C, are from the polymeric gluten 7887

DOI: 10.1021/acs.jafc.9b01015 J. Agric. Food Chem. 2019, 67, 7886−7897

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Figure 2. UV−visible scans with temperature (in °C) of a typical reduction reaction of Au(III) into Au(0) induced by SDS solubilized hard wheat protein (inset). Plots of variation of intensity due to surface plasmon resonance of Au NPs at 550 nm with temperature. Black arrows indicate the order of reduction potential of soft < medium < hard wheat protein. See details in the text.

CTAB) with temperature, but it eventually reverses its polarity through an isoelectric point at 60 °C. This is a unique example of a temperature induced isoelectric point in contrast to the usual method of a pH induced isoelectric point. A successive dehydration of protein macromolecules reduces size and hence increases the surface charge density that increases the respective ζ potential value initially (i.e., up to 60 °C).21,22 The higher respective ζ potential value attracts oppositely charged counter ions (i.e., Na+ for SDS and Br− for CTAB solubilized wheat protein) and hence reverses the polarity beyond 60 °C due to stronger ion−ion (surfactant ion− counterion) over ion−dipole (surfactant ion−water dipole) interactions. This is schematically shown in Figure 1c for CTAB solubilized wheat protein. The effect is highly prominent when the concentration of the surfactant used is greater than its critical micelle concentration (cmc); the cmc of SDS = 8 mM, (Figure S2). A greater amount of surfactant than its cmc induces a greater amount of hydration as well as a greater solubilization capacity due to the presence of the micellar state. The greater hydration is more responsive to temperature change, and that is why hard protein solubilized in 10 mM and 15 mM SDS showed clear and marked charge reversal while this is not the case when it is solubilized in 5 mM SDS (Figure S2). A comparison between all three, i.e., hard, medium, and soft wheat proteins indicates that a much prominent effect is induced by the soft wheat protein where a maximum increase in the negative ζ potential is observed with temperature, followed by the medium and hard wheat protein (Figure S3). A higher negative ζ potential value originates from the higher charge density.23,24 It is a maximum negative value for soft wheat protein because it contains the lowest amount of hydrophobic gluten content, such as glutenin, followed by medium and hard wheat protein. Thus, the higher glutenin with the maximum amount of hydrophobic content of hard wheat protein lowers its solubilization/hydration and hence lowers negative ζ potential.23,24 It is to be mentioned that this unique behavior of ζ potential variation with temperature of wheat protein is only depicted

Figure 1. Size distribution histograms of aqueous solution of SDS solubilized hard wheat protein, 1 mg/1 mL of 10 mM SDS solution showing three different kinds of aggregates (inset). (a) Shows their variation with temperature. (b) Plot of ζ potential of both SDS and CTAB solubilized wheat protein (1 mg/1 mL of 10 mM surfactant) with temperature. (c) Schematic representation of CTAB−wheat protein complex with positive ζ potential and its interactions with Br− counterions. Temperature induced dehydration causes the reversal of polarity. See details in the text.

(glutenin), monomeric gluten (gliadin), and structural proteins (globulin/albumin/amphiphilic proteins), respectively. Polymeric glutenin (“A”) provides the maximum size because of the large hydration sphere in comparison to the smaller “B” and “C”. All three components showed size fluctuations within the temperature range studied due to conformational changes with an overall decrease in the size induced by temperature dehydration. Similar behavior is observed for CTAB solubilized hard wheat protein (Supporting Information, Figure S1). A variation in the ζ potential (Figure 1b) provides interesting information for both aqueous SDS and CTAB solubilized wheat proteins. Initially, ζ potential becomes more negative (in aqueous SDS) and more positive (in aqueous 7888

DOI: 10.1021/acs.jafc.9b01015 J. Agric. Food Chem. 2019, 67, 7886−7897

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Figure 3. (a) TEM images of Au NPs synthesized in the presence of SDS solubilized hard wheat protein (1 mg/1 mL of 10 mM SDS). (b) TEM images of Au NPs synthesized in the presence of CTAB solubilized hard wheat protein (1 mg/1 mL of 10 mM CTAB). (c and d) XRD patterns of Au NPs from part a and part b. See details in the text.

hydrophobic protein.28,29 It mainly exists in the form of a cross-linked structure bound by the disulfide bonds. Aqueous solubilization breaks disulfide bonds and unfolds it. That makes it easier for the cysteine to induce the reduction of Au(III) into Au(0). Thus, the greater amount of gluten in hard wheat protein provides a greater reduction potential. The Au NPs thus produced are characterized by TEM measurements. A relative comparison of TEM images of Au NPs (Figure 3) suggests that SDS solubilized hard wheat protein produces mainly polyhedral Au NPs (Figure 3a), while polyhedral morphologies slowly convert into predominantly plate-like NPs from medium (Figure S8) to soft (Figure S9) wheat protein. Contrasting differences are observed when comparing the morphology of Au NPs produced by CTAB solubilized hard wheat protein. It produces clear faceted morphologies like triangular, hexagonal, and cubic geometries (Figure 3b), while CTAB solubilized medium and soft proteins are unable to produce such faceted morphologies (Figures S10 and S11). This indicates that the positively charged hard wheat protein proves to be an excellent shape controlling agent rather than the negatively charged hard wheat protein. XRD analysis further helps us to evaluate the crystal structures of Au NPs synthesized in the presence of SDS (Figure 3c) and CTAB (Figure 3d) solubilized hard wheat proteins. All peaks are indexed to the face centered cubic (fcc) geometry of Au. Au NPs synthesized with CTAB solubilized hard protein (Figure 3d) show prominent growth at the {111} crystal planes in

when it is solubilized in aqueous micellar solution. This typical behavior is not demonstrated by the wheat protein when it is solubilized in the basic medium without the presence of a surfactant (Figure S4). Although, DLS size distribution still demonstrates the presence of three prominent sizes (Figure S5) similar to that of Figure 1a, ζ potential (Figure S4) remains more or less close to zero over the temperature range studied because the isoelectric point of gluten is close to pH 6.2.25 Morphology Controlled Synthesis of Au NPs and Characterization. In this section, we demonstrate the shape directing and crystal growth effects26,27 of different fractions of wheat protein and their relation to gluten content. Figure 2 (inset) depicts the UV−visible scans of a typical reduction reaction of Au(III) into Au(0)10,11 by using SDS solubilized hard wheat protein with maximum gluten contents. Similar scans are obtained when SDS solubilized medium and soft wheat proteins are used (Figures S6 and S7, respectively). A variation in the absorbance due to surface plasmon resonance (SPR) of Au NPs at 550 nm versus temperature (Figure 2) demonstrates the order in reduction potential of soft < medium < hard wheat proteins. Hard wheat protein induces reduction of Au(III) into Au(0) to produce Au nucleating centers around 37 °C while medium starts it at 44 °C and soft wheat protein at 52 °C. Thus, the greater gluten content of hard wheat protein is responsible for the facilitated reduction of Au(III) into Au(0). In fact, gluten is a highly complex 7889

DOI: 10.1021/acs.jafc.9b01015 J. Agric. Food Chem. 2019, 67, 7886−7897

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Figure 4. XPS Analysis of (a) C-1s, (b) O-1s, (c) N-1s, (d) Br-3d and Au-4f, and (e) Br-3p of CTAB solubilized hard wheat protein conjugated Au NPs. (f) Schematic representation showing adsorption of Br ̅ ion on {100} crystal planes. That in turn facilitates the adsorption of CTAB solubilized protein during the crystal growth. See details in the text.

comparison to the {110} and {100} crystal planes,30,31 while this is not so for Au NPs synthesized with SDS solubilized hard wheat protein (Figure 3c). Prominent growth at the {111} crystal planes suggests the greater participation of the {111} planes in the nucleation process while the {110} and {100} crystal planes are predominantly passivated due to the

preferential surface adsorption of CTAB solubilized hard wheat protein. In contrast, this is not so for Au NPs synthesized with SDS solubilized hard wheat protein (Figure 3c) where almost equal amount of growth on different crystal planes of fcc geometry is observed. We will further explain 7890

DOI: 10.1021/acs.jafc.9b01015 J. Agric. Food Chem. 2019, 67, 7886−7897

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Table 1. XPS Analysis of CTAB Solubilized Hard (1), Medium (2), and Soft (3) Protein Conjugated Au NPs and SDS Solubilized Hard (4), Medium (5), and Soft (6) Protein Conjugated Au NPsa

a

All binding energies/eV are listed in parentheses in the same order in a column below the atomic % and area % entries.

facilitated by the protein−surfactant complex, the aqueous exposed protein domains are surfactant complexed, which contribute the most to the C-1s binding energy from the hydrocarbon chains followed by the C−OH groups of amino acid residues. The greater contribution of C-1s for CTAB solubilized protein can be related to the longer methylene chain of C16 in comparison to that of C12 of SDS. The next higher binding energy is contributed by O-1s which is much higher for the SDS solubilized proteins than for the CTAB solubilized proteins (Table 1). The higher amount in the former case is mainly contributed by the ketonic oxygens of the sulfate groups34 with binding energy close to 531 eV of SDS, apart from the −OH group of the tyrosine residues which constitute a major contribution to wheat protein (Figure 4b). The N-1s emission (Figure 4c) of CTAB solubilized protein coated NPs has two binding energies: one at 401.7 eV due to the alkylammonium functional group35−37 of CTAB and the other at 399.4 eV due to the uncharged amino functional groups38,39 of the protein structure. The contribution of the

these results from the XPS analysis of the surface adsorbed protein in the next section. Bioconjugation and Surface Adsorption of Wheat Protein. XPS analysis is a quantitative technique to determine the composition of the surface adsorbed species. Figure 4a−e shows the high resolution spectra of C-1s, O-1s, N-1s, Au-4f, and Br-3p of CTAB solubilized hard wheat protein coated Au NPs, respectively. Similar high resolution spectra of SDS solubilized hard wheat protein coated Au NPs are shown in Figure S12. The binding energies and atomic percent compositions are compared in Table 1. A perusal of Table 1 indicates that the maximum emission is demonstrated by C-1s which is much higher for the CTAB solubilized hard, medium, and soft proteins than for the respective SDS solubilized proteins. High resolution spectra for various C species (Figure 4a) indicate that C−C and C−H hydrocarbon functional groups are the major contributors followed by C−OH and C− O−C in both cases. 32,33 Since the wheat protein is predominantly hydrophobic and its aqueous solubilization is 7891

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surface when a capping layer is deposited by long chain amphiphilic species.40,44 Na-1s (Table 1) is only contributed by SDS molecules and hence its contribution is only displayed by the SDS solubilized protein coated NPs. High resolution scans of Br require considerable attention (Figure 4d,e). The Br− ion is a CTAB counterion and possesses a high affinity for Au surface adsorption. Several studies indicate that halide anions significantly influence the morphology of gold nanoparticles45,46 by preferentially adsorbing on the crystal planes of fcc geometry. This is further supported by density functional theory (DFT)-based calculations.47 Molecular dynamic simulations suggest that Br− ions in fact diffuse through water channels created in between distorted cylindrical CTAB micelles adsorbed on the Au surface.48 In the present study, aqueous solubilization of wheat protein happens when CTAB monomers form a complex with protein that provides a global positive charge to the protein. However, the reversal of positive to negative ζ potential at 60 °C (Figure 1b) is the consequence of the adsorption of Br− ions on the [CTAB−wheat protein complex] during temperature induced dehydration. Since, all in situ reactions are conducted at 70 °C (see experimental), the morphology control synthesis of faceted Au NPs is actually driven by the adsorption of Br− ions on the solid−liquid interface and is further stabilized by the CTAB solubilized protein during the crystal growth.30 This is shown as a schematic representation in Figure 4f. This causes two different XPS signals of Br− ions on CTAB solubilized wheat protein coated Au NPs. One is for the Br− ions which are adsorbed on the Au surface; this is depicted by the Br 3d region that exhibits two signals from spin−orbit-splitting corresponding to the Br 3d5/2 at 68.0 eV and the Br 3d3/2 at 69.0 eV (Figure 4d).49 The other one is due to the nonadsorbed free Br− counterions which are only electrostatically associated with the positively charged protein in the form of an electric double layer. This signal belongs to Br 3p3/2 at 181.1 eV and Br 3p1/2 at 187.7 eV (Figure 4e). The Br 3d signal overshadows the Au 4f signal (Figure 4d) because of the surface adsorption of Br− ions, and that is why the Br 3d signal contribution is much greater than that of the Au 4f signal (Table 1). Thus, the formation of fine faceted Au NPs in Figure 3b is the consequence of the preferential adsorption of Br− ions on the {100} and/or {110} crystal planes50−53 of Au NPs, because XRD patterns provide relatively minimum growth at these crystal planes and maximum growth is directed to the {111} crystal planes of fcc geometry. This causes a stronger surface passivation of the {100} and {110} crystal planes resulting in the formation of faceted Au NPs (Figure 3b). In contrast, SDS solubilized hard wheat protein lacks Br− ions and hence preferential surface passivation is not achieved, with the resultant growth randomly distributed over different crystal planes leading to the formation of polyhedral morphologies (Figure 3a). Thus, CTAB solubilized hard wheat protein proves to be a better shape controlling agent in comparison to SDS solubilized hard wheat protein. Phase Transfer and Interfacial Behavior of Bioconjugated NPs. It is possible to extract the wheat protein coated Au NPs from the aqueous to the organic phase by using water insoluble ionic liquids.54−57 Purified Au NPs suspension when mixed with 1-butyl-3-methylimidazolium hexafluorophosphate (50 mM) in ethyl acetate causes an instantaneous transfer of the Au NPs to the organic phase.11 Interestingly, the magnitude of the Au NP transfer depends on the gluten content of the wheat protein. Figure 5 shows the phase transfer

Figure 5. Images of sample tubes labeled 1−9 depicting the phase transfer of SDS solubilized hard (a, 1, 2, 3), medium (b, 4, 5, 6), and soft (c, 7, 8, 9) wheat protein (1 mg/1 mL of 10 mM SDS) conjugated Au NPs synthesized by using different concentrations of gold salt (0.25, 0.50, 1.0 mM) across the aqueous−organic interface. In parts a−c, the upper panel sample images are before the phase transfer and the lower panel represents the images after the phase transfer. See details in the text.

former emission is much higher than that of the latter because CTAB monomers are complexed with the hydrophobic domains of wheat protein and their tetralkylammonium head groups are surface exposed. In contrast, as expected, the N-1s high resolution scan of SDS solubilized protein coated NPs shows only emission due to amino functional groups of protein at 399.4 eV. This makes the overall binding energy contribution of N-1s for CTAB solubilized protein greater in comparison to that for SDS solubilized protein coated NPs. The lowest emission among all is exhibited by the oxidized Au species (Table 1) which are mainly located around 529.2 eV40 though many studies have also reported this value close to 530.1 eV.41−43 The low amount of the emission is the consequence of the thick surface coating of the wheat protein that reduces the intensity of the emission from the oxidized Au 7892

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Figure 6. (a) Schematic representation of proposed mechanism of phase transfer of wheat protein conjugated NPs under the effect of strong electrostatic interactions operating between the organic layer solubilized 1-butyl-3-methylimidazolium cations and negatively charged SDS solubilized protein conjugated NPs. (b) Similar schematic representation of interfacial adsorption tendency of electrostatically attracted CTAB solubilized wheat protein conjugated NPs with organic layer solubilized hexafluorophosphate anions. See details in the text.

of Au NPs prepared by using different concentrations of gold salt (i.e., 0.25/0.50/1.0 mM) in the presence of SDS solubilized hard, medium, and soft wheat proteins. All the samples of the hard wheat protein coated Au NPs demonstrated their complete transfer from the aqueous to the organic layer (Figure 5a). Samples prepared with medium wheat protein also showed complete transfer except for the one with 1.0 mM gold salt concentration (indicated by a black arrow in Figure 5b, where the pink color of the aqueous phase indicates its partial transfer). For soft wheat protein, two samples, i.e., the ones with 0.5 mM and 1.0 mM gold salt, showed partial phase transfers (Figure 5c). This phase transfer trend can be placed in the order of soft < medium < hard wheat protein. Phase transfer is driven by the strong electrostatic interactions operating between the organic layer solubilized 1-butyl-3-methylimidazolium cations and the negatively charged SDS solubilized protein coated NPs (Figure 6a).58 NPs which are fully coated with protein are transferred to the organic phase. Thus, all samples of hard wheat protein (i.e., 1, 2, and 3, Figure 5a) are fully coated, while this is not the case for sample 6 (Figure 5b) of medium wheat protein and for samples 8 and 9 (Figure 5c) of soft wheat protein. Since, the total amount of hard, medium, and soft wheat proteins used is constant, all samples are expected to

completely transfer if they are completely coated with their respective proteins. Thus, the hard wheat protein possesses a stronger ability to surface adsorb which systematically decreases from medium to soft wheat protein. This trend can be very well related to the amount of gluten content. The greater amount of gluten in hard wheat protein provides greater hydrophobicity which in turn makes it strongly amphiphilic and hence provides an effective driving force for the surface adsorption.30 Likewise, the decreasing amount of gluten from medium to soft wheat protein reduces their surface adsorptions and hence relatively lesser amounts of proteins are surface adsorbed. Interestingly, CTAB solubilized hard, medium, and soft wheat protein coated NPs do not show phase transfer and instead preferentially adsorb at the aqueous−organic interface (Figure S13). All samples of hard wheat protein coated NPs (samples 1, 2, and 3) show prominent, narrow, and concentrated interfacial adsorption, while samples 6 and 9 of medium of soft wheat proteins, respectively, do not show any visible interfacial adsorption. Samples 7 and 8 of soft protein show much broader and less concentrated bands with a low tendency to adsorb at the interface. The interfacial adsorption tendency of CTAB solubilized wheat protein coated NPs indicates their ability to simultaneously interact with the 7893

DOI: 10.1021/acs.jafc.9b01015 J. Agric. Food Chem. 2019, 67, 7886−7897

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Figure 7. SDS page scans of pure nonadsorbed SDS and CTAB solubilized hard, medium, and soft wheat proteins (1 mg/1 mL of 10 mM surfactant) as control depicting 3 major protein fractions with high, medium, and low molar masses. See details in the text.

and it is further related to the higher gluten content. Therefore, a greater interfacial adsorption is mainly depicted by the hard wheat protein coated NPs in comparison to those of medium and soft wheat protein coated NPs. SDS Page Analysis of Surface Adsorbed Protein. SDS page analysis helps us to characterize the protein fractions which are adsorbed on Au NP surfaces. Figure 7a shows the SDS page scans4,59 of pure nonadsorbed SDS and CTAB solubilized hard, medium, and soft wheat proteins as control. Each pure sample shows around 16 identical bands corresponding to different protein fractions. They only differ from one another in terms of their relative intensity. These protein fractions can be divided into three main categories of high (A), medium (B), and low (C) molecular mass glutenins based on the DLS size distribution shown in Figure 1a. The SDS page scans of protein fractions adsorbed on Au NPs during in situ reactions are shown in Figures S14 and S15 and are compared with the corresponding controls. For SDS solubilized hard, medium, and soft wheat protein adsorbed on nanometallic surfaces, only 74 and 85 kDa protein fractions of high molecular masses are present (Figure S14), while six protein fractions of low molecular masses (38−65 kDa) are conjugated to the Au NPs from CTAB solubilized protein (Figure S15). The surface adsorption of 74 and 85 kDa protein fractions is due to the direct consequence of the specific binding to nanometallic surfaces and is driven by the greater amphiphathic character of higher molar masses of protein fractions.10,11,15 The greater amphiphilic nature is usually induced by the higher molar masses of the proteins with plenty of polar and nonpolar amino acids residues which create distinct polar and nonpolar domains within a protein macromolecule.10,15 The larger polar and nonpolar domains provide higher respective surface potentials which in turn

Figure 8. Images of zone of inhibition against Klebsiella pneumonia (A) and zone of inhibition against Staphylococcus epidermidis (B). “−” represents negative control, “+” represents positive control, and “S” represents CTAB solubilized wheat protein conjugated NPs. See details in the text.

aqueous as well as organic phases rather than their strong preference for hexafluorophosphate anions in the organic phase (Figure 6b). It seems that NPs do not acquire enough positive potential to be attracted across the interface by the hexafluorophosphate anions in the organic phase. This is due to the already adsorbed Br− ions on the {100} lattice planes of Au NPs (as depicted in Figure 4d from XPS analysis). The surface adsorbed Br− ions are expected to reduce the positive potential contributed by the CTAB solubilized wheat protein and hence screen the electrostatic interactions with the hexafluorophosphate anions in the organic phase. This impedes the complete transfer of Au NPs across the interface and hence restricts them to interfacial adsorption. A stronger interfacial adsorption of Au NPs is the direct consequence of the stronger amphiphilic behavior of surface adsorbed protein 7894

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Journal of Agricultural and Food Chemistry

nanometallic surface adsorption of wheat protein is highly selective. In addition, CTAB solubilized wheat protein conjugated NPs show strong antimicrobial activity against both Gram positive and Gram negative bacteria which prompts its favorable applications in the food industry.

convert the macromolecule into a strongly amphiphilic entity with a strong ability to demonstrate solid−liquid interfacial adsorption.15 Whereas the adsorption of protein fractions of 38−65 kDa from CTAB solubilized wheat proteins is considered to be the consequence of indirect surface adsorption in view of the preferential surface adsorption of Br− ions on the {100} crystal planes of Au NPs as stated in the XPS analysis of Figure 4d,e. Thus, the surface adsorption of CTAB stabilized protein fractions of 38−65 kDa is prompted by the already adsorbed Br− ions due to the electrostatic interactions on the nanometallic surfaces rather than specific adsorption. In this way, a clear and prominent aqueous−organic interfacial transfer of SDS solubilized wheat protein conjugated NPs is driven by the surface adsorbed high molar masses of 74 and 85 kDa protein fractions of gluten in comparison to the low molar masses (38−65 kDa) of surface adsorbed protein fractions of CTAB solubilized wheat protein which prefer to adsorb at the aqueous−organic interface. Antimicrobial Activity of Wheat Protein Conjugated NPs. Both SDS and CTAB solubilized hard wheat protein conjugated Au NPs are used for antimicrobial activities against K. pneumoniae (Gram negative bacteria) and S. epidermidis (Gram positive bacteria). Interestingly, only CTAB solubilized wheat protein conjugated NPs show antimicrobial activity against both strains unlike SDS solubilized wheat protein conjugated NPs. Figure 8 shows the bacterial growth inhibition zone for CTAB solubilized hard wheat protein conjugated Au NPs with minimum inhibition concentrations (MIC) against Gram negative and Gram positive bacteria of 6.1 and 49.1 μg/ mL, respectively. This value is lower against Gram negative bacteria than against Gram positive bacteria and can be attributed to the nature of the bacterial cell wall, which is quite resistant to antibiotics in Gram negative bacteria due to the presence of an outer cell membrane in comparison to Gram positive bacteria in which the outer cell membrane is absent. However, both strains interact effectively with positively charged NPs through their predominantly negatively charged cell membranes;60 this is why CTAB solubilized hard wheat protein conjugated Au NPs demonstrate MIC unlike SDS solubilized NPs. The antimicrobial activities of wheat protein conjugated NPs are mainly related to the overall global charge rather than the gluten content. The electropositive nature of nanomaterials drives stronger electrostatic interactions with the predominantly negatively charged bacterial cell wall and hence only the electropositive nature of the bioconjugated nanomaterials induces the antimicrobial activity. From the above results, we can conclude that wheat protein possesses high affinity for nanometallic surface adsorption which is mainly governed by the gluten content. Higher gluten contents facilitate not only the in situ reduction of Au(III) into Au(0) but also the selective adsorption of wheat protein fractions on nanometallic surfaces. Wheat protein conjugated NPs can also be transferred into the organic phase for their applications in nonaqueous phases. The amounts of wheat protein conjugated NPs that transfer across the aqueous− organic interface are further related to the gluten contents. SDS page analysis indicates the presence of more than 15 protein fractions in each hard, medium, and soft wheat protein. Out of 15, only two protein fractions of high molar masses (74 and 85 kDa) preferentially adsorb on the NP surface during the growth process of Au NPs from nucleating centers in the case of SDS solubilized wheat protein. It shows that the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01015. UV−visible spectra, TEM images, DLS plots, and SDS page scans (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (M. S. Bakshi): [email protected]. *E-mail: (P. Khullar): [email protected]. ORCID

Harsh Kumar: 0000-0003-3874-4614 Mandeep Singh Bakshi: 0000-0003-1251-9590 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS These studies were partially supported by the financial assistance from UWGB, NAS, Green Bay, and DST under nanomission research project [ref no: SR/NM/NS-1057/ 2015(G)], New Delhi. Dr. Gurinder Kaur thankfully acknowledges the financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA. P.K. acknowledges the TEM studies done by the SAIF Lab, Nehu, Shillong.

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REFERENCES

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