Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins Ashkan Madadlou, Juliane Floury, and Didier Dupont*
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STLO, UMR 1253, INRA, Agrocampus Ouest, 35000 Rennes, France ABSTRACT: Chemical modification of whey proteins allows manipulation of their characteristics, such as surface charge and hydrophobicity. Herein, we report the influence of hydrophobization accomplished by a preacetylation stage and a subsequent combined acetylation−heating process on some characteristics of whey proteins. Hydrophobization extensively (≥90%) acetylated the available free amino groups of whey proteins. The produced protein particles were nanoscaled (75 nm) and had a significantly low isoelectric point (3.70). Hydrophobization increased the β-sheet content of whey proteins and significantly decreased the solvent exposure of tyrosine residues. It also conferred a less compact tertiary structure to the proteins and decreased the extent of disulfide-bond formation by heating. The mobility of α-lactalbumin in nonreducing electrophoresis gel increased as a consequence of hydrophobization. Then, the ability of whey proteins to catalyze hydroquinone autoxidation was examined, and it was found that the activity decreased as a result of hydrophobization. The catalytic activity of the proteins was associated with the free-amino-group content, which determined the extent of cation−π attractive interactions; ζ-potential, which determined the extent of anion−π repulsive interactions; and π-stacking between hydrophobic residues and the electron cloud of the quinone ring. KEYWORDS: protein modification, hydrophobization, oxidation, catalysis wine, broccoli, coffee, and tea.15 The oxidative conversion of HQ has been widely used for assessing the photocatalytic activity of various inorganic compounds.16−19 In contrast, little, if any, information is available about HQ oxidation catalyzed by food proteins. Histamine, an amine-bearing biomolecule, has been declared a catalyst of HQ autoxidation.20 It was anticipated by the authors of the present communication that proteins and peptides could also serve as oxidative-reaction catalysts. The effect of acetylation was then assessed on the catalytic activity of whey proteins. Acetylation has been shown to influence the biological activity of whey-protein-derived peptides21 and was expected to alter the catalytic efficiency of oxidation of the proteins. Therefore, the objective of the present study was to develop and introduce a whey-protein hydrophobization process by using acetic anhydride and heat treatment and assess the activity of the proteins in catalyzing HQ autoxidation.
1. INTRODUCTION Whey proteins are readily available and inexpensive ingredients that are used for diverse purposes, ranging from emulsification and gel formation to protein delivery. Enzymatic hydrolysis to smaller molecules and heat-induced polymerization to larger aggregates are conventional methods that are carried out in the food industry to enhance traditional functional properties of whey proteins and introduce new characteristics.1 Hydrophobization of proteins by chemical substances is an old technique that is gaining interest. Acylation with fatty acids was carried out to synthesize hydrophobic-whey-protein isolate. However, some of the chemicals used (pyridine,2 for example) show hazardous impacts on human health following inhalation and skin contact,3 posing dangers to workers. Alternatively, hydrophobization with food-grade ingredients can be safer and may expand the application of whey proteins. Acetic anhydride is a food-grade acetylating agent4 that is permitted for direct addition to food for human consumption.5 It has been utilized for acetylation of different proteins, including mucan-bean,6 chickpea,7 potato,8 and oat9 proteins, as well as casein micelles.10 Lakkis and Villota11 hydrophobized whey proteins with acetic anhydride in a saturated sodium acetate solution, and Zhao et al.12 assessed the influence of acetylation on the conformation of whey proteins. Like acetylation, heat treatment enhances the surface hydrophobicity of whey proteins.13 In the current study, we introduce a method consisting of a preacetylation stage and a subsequent combined acetylation−heating process to form hydrophobized whey-protein nanoparticles. Hydroquinone (HQ) is a nonvolatile chemical compound used in cosmetics, pesticides, dyes, and plastics, as well as in photostabilization and petroleum refining.14 It may also occur as a glucose conjugate (arbutin) in wheat products, pears, red © XXXX American Chemical Society
2. MATERIALS AND METHODS 2.1. Materials. Whey-protein isolate (WPI) was a kind gift from Lactalis Ingredients. According to the information provided by the manufacturer, the WPI had 90% protein, 5.1% water, 3.0% lactose, and 1.97% ash contents. Reversed-phase high-performance liquid chromatography (RP-HPLC) indicated that the α-lactalbumin and βlactoglobulin contents were 16 and 56% of the total protein in the WPI sample. Distilled water was used throughout the study, and all chemicals used were analytical-grade. 2.2. WPI Hydrophobization. After a series of preliminary tests to minimize the amount of added acetic anhydride, the following Received: May 4, 2018 Revised: September 3, 2018 Accepted: October 25, 2018
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DOI: 10.1021/acs.jafc.8b02362 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
mode with a resolution of 4 cm−1 and 256 scans for each sample (in duplicate) using Opus software (Bruker). Water was used as the background reference and the spectral contribution of atmospheric water vapor and CO2 was subtracted from the spectra. Then, the spectra were derivatized (polynomial order 2) by the Savitzky−Golay algorithm with 13-point smoothing, further smoothed (smoothingpoints number = 9), and normalized.22 The derivatized spectra were Fourier self-deconvoluted using Opus software, and the peaks were fitted with the Gaussian function in wavenumber ranges between 1700 and 1600 cm−1 (amide I) and 1570 and 1510 cm−1 (amide II). The percentages of secondary structures were calculated23 according to deconvoluted amide I band. 2.7. Intrinsic-Fluorescence Spectroscopy. The peak emission wavelengths (λem) of the WPI samples were measured using a cuvette spectrofluorometer (FLX-Xenius, SAFAS Monaco). Samples were diluted 100-fold with distilled water and excited at different wavelengths of 280, 290, and 295 nm. The latter wavelength results in selective stimulation of tryptophan.24 Emission spectra were recorded at 300−400 nm with the entrance and exit slits set at 8 nm. 2.8. Gel Electrophoresis. WPI samples were analyzed for protein profiles by sodium dodecyl sulfate−polyacrylamide-gel electrophoresis (SDS-PAGE) following the method of Ménard et al.25 Samples were diluted with NuPAGE LDS sample buffer (Invitrogen) and then treated with either DL-dithiothreitol (for reducing conditions) or deionized water (for nonreducing conditions). Specimens (20 μL) were loaded into 4−12% polyacrylamide NuPAGE Novex Bis-Tris precast gels (Invitrogen) and Mark 12 Unstained Standard (Invitrogen) was used as the molecular weight (Mw) marker. Later, electrophoresis gels were fixed in a mixed solution of ethanol, acetic acid, and distilled water (30, 10, and 60%, v/v, respectively). Then, the gels were rinsed with distilled water and stained with Bio-Safe Coomassie stain (Bio-Rad Laboratories). Subsequently, the gels were destained in distilled water. Image analyses of gels were carried out using Image Scanner III (GE Healthcare Europe GmbH). 2.9. Hydroquinone Oxidation. WPI solution or water at pH 8.2 (50 μL) was mixed with 950 μL of a hydroquinone solution (6 mg/ mL, pH 8.1) and stored at 20 °C. Yellowish-brown-colored products that had a maximum absorption wavelength (λmax) of 345 nm were formed over time. Yellow-product formation as a consequence of complexation of a veterinary drug with a derivative of 1,4benzoquinone has already been measured at 345 nm.26 The absorbance of samples was read against a corresponding blank (either water or matching WPI solution). 2.10. Statistical Analysis. Samples were fabricated at least three times, and the experiments were performed in triplicate. The results were analyzed by the general-linear-model univariate procedure with SPSS software version 16 (IBM) using Duncan’s test at a significance level of p < 0.05.
procedure was adopted to hydrophobize whey proteins. WPI powder was dissolved in distilled water (65 mg/mL) and stirred for 2 h at 20 °C; sodium azide was added (100 ppm) as an antimicrobial agent. After storage for 18 h at 5 °C to warrant complete hydration, the WPI solution (9.5 mL) was titrated successively with acetic anhydride (two injections, each 50 μL) and 7 M NaOH (five 50 μL injections and three 10 μL injections). The ratio of acetic anhydride to whey protein was ≈0.18 g/g. This was followed by titration of the WPI solution with 1 M NaOH (2−3 injections immediately and 3−4 injections over time, each 10 μL) during the next 30 min to keep pH above 8.0. Immediately after the last alkaline titration, the WPI solution (pH ≈8.70) was heat-treated at 80 °C for 20 min while being stirred at 800 rpm, after which the pH dropped to 7.41 ± 0.03. Subsequently, the pH of the acetylated heat-treated WPI solution was increased to 8.25 ± 0.1 by addition of 1 M NaOH (35 μL). The pH value did not change during further storage. Native (nonacetylated and nonheated) and heat-treated (nonacetylated) WPI samples were also prepared. For this purpose, the stock WPI solution (9.5 mL) was titrated with 1 M HCl (100 μL), 1 M NaOH (200 μL), and distilled water (200 μL) to a final pH of 8.40 ± 0.1. The native WPI sample was stored at 20 °C, whereas the other sample was heat-treated at 80 °C. The sample pH value was adjusted to ≈8.2 by 0.5 or 1 M NaOH (10 μL). 2.3. Acetylation Extent. The extent of WPI acetylation was determined by measuring the free-amino-group content according to the method used by Miedzianka et al.8 with some modifications. First, the WPI solution was diluted 75-fold with distilled water, and 1 mL of the diluted protein solution was mixed with 1 mL of 4% NaHCO3 and 1 mL of 0.1% 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution. The mixture was heated at 50 °C for 2 h and cooled down rapidly. Then, 1 mL of 5% sodium dodecyl sulfate (SDS) solution and 0.5 mL of 1 M HCl were added to the mixture, and the absorbance was read at 390 nm using a UV−visible spectrophotometer (model UVmc2, SAFAS Monaco) against a blank consisting of reagents and water. The absorbance of a native (nonacetylated and nonheated) WPI solution was set equal to 100% free amino groups. The free-amino-group content of the heat-treated but not acetylated WPI solution was also measured for comparison. 2.4. Particle Size and Electric Charge. The sizes and ζpotentials of the WPI samples (native, heat-treated, and acetylated− heated) were measured by the dynamic-light-scattering (DLS) technique using a Zetasizer Nano ZS (Malvern Instruments Ltd.). A laser wavelength of 633 nm at a backscattering angle of 173° was applied for size measurements at 20 °C. 2.5. 8-Anilino-1-naphthalenesulfonic Acid (ANS)-Binding Affinity. The affinity of WPI samples (native, heat-treated and acetylated−heated) to bind to ANS was measured following the method of Alizadeh-Pasdar and Li-Chan.13 WPI samples at different pH values (above and below their corresponding pI values) were diluted to several extents (37.5-, 50-, 75-, and 150-fold) with distilled water, and 30 μL of ANS solution (5 mM) in water was added to 1.5 mL of diluted WPI sample. The mixture was vortexed, and after storage for 10 min in the dark, ANS was excited at 390 nm. The emission intensity was read using a cuvette spectrofluorometer (FLXXenius, SAFAS Monaco) at 470 nm, and the excitation- and emissionslit widths were both 8 nm. The emission intensity of each WPI dilution without ANS was also measured and subtracted from the intensity of the corresponding sample to obtain a net fluorescence intensity. The initial slope (S0) of the net-fluorescence-intensityversus-protein-concentration plot was calculated by linear-regression analysis with Microsoft Excel. The fluorescence intensity of a sample including water and ANS (without protein) was considered as protein concentration 0. 2.6. Fourier-Transform-Infrared (FTIR) Analysis. To assess changes in protein secondary-structure, attenuated-total-reflectance (ATR) FTIR spectra of the WPI-solution samples were acquired at 20 °C using a TENSOR 27 FTIR spectrometer (Bruker) equipped with a single-reflection germanium crystal ATR accessory (MIRacle, PIKE Technologies) and a liquid-nitrogen-cooled mercury−cadmium− telluride detector (Bruker). Spectra were collected in absorbance
3. RESULTS AND DISCUSSION 3.1. Acetylation Extent. Preliminary experiments showed that storage of the WPI solution with added acetic anhydride at either 20 or 40 °C before heating and titration with 1 M NaOH from time to time to keep the pH above 8.0 resulted in a constant pH of 8.32 ± 0.04 after 9 h. Nonetheless, the pH of the solution decreased by a value of ≈0.5 as a consequence of subsequent heat treatment. In contrast, slight heating (≤0.15 value) decreased the pH of the WPI solution without added acetic anhydride. The reduction of the pH of the preacetylated sample after heat treatment indicated that acetic anhydride was not fully consumed at lower temperatures (20 and 40 °C) even after prolonged storage and reaching a constant pH value. Heating was obviously required to cause consumption of all acetylating agent and achieve the highest possible extent of free-amino-group modification. Acetylation of proteins preferentially modifies the ε-amino group of lysine9 in a nucleophilic substitution reaction. Table 1 reports that the relative B
DOI: 10.1021/acs.jafc.8b02362 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 1. Free Amino Acid Contents, Particle Characteristics, and Maximum Emission Wavelengths of Whey-Protein-Isolate (WPI) Samplesa WPI samples
free amino groups (%)b
native heat-treated hydrophobized native with added HQd heat-treated with added HQ hydrophobized with added HQ
100a 87.7 ± 4.8b 8.6 ± 2.6c
z-average diameter (nm) NA 45.70 74.56 NA 46.96 75.84
± 1.73b ± 4.02a ± 4.15b ± 4.14a
PDIc 0.790 0.216 0.263 0.750 0.236 0.260
± ± ± ± ± ±
emission peak (nm)
0.043a 0.011b 0.030b 0.030a 0.025b 0.020b
329 334 336 328 330 331
± ± ± ± ± ±
1 0 0 0 0 1
a
Means with different letters within a column differ significantly (p < 0.05). bThe free-amino-group content of native WPI was set to 100%. Polydispersity index. dHydroquinone.
c
Figure 1. ζ-Potential values and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding affinities of WPI samples at different pHs.
percentage of free-amino-group contents significantly decreased (>90%) as a consequence of hydrophobization (i.e., preacetylation and subsequent acetylation−heating). Miedzianka et al.8 obtained a maximum extent (99%) of potatoprotein acetylation by the lowest concentration (0.4 mL per gram of protein preparation) of acetic anhydride and higher concentrations of acetic anhydride did not influence the acetylation degree. Likewise, Zhao et al.9 found that a low ratio of acetic anhydride to oat proteins (0.2 g/g) resulted in a remarkably high (90%) N-acetylation degree and further increasing the acetic anhydride ratio did not significantly
increase the acetylation extent. Bean-protein concentrate was acetylated as well at a level of 87% using acetic anhydride at a fixed ratio of 0.5 g/g protein.6 Because of preliminary tests and the above-mentioned literature, a low ratio of acetic anhydride to whey proteins (≈0.18 g/g) was employed in the current study, which brought about a remarkable level of free-aminogroup acetylation. The heat-treated WPI solution had a lower free-amino-group content than the native counterpart (Table 1). Whey proteins are readily denatured during thermal processing and undergo conformational changes to an unfolded state. This is followed C
DOI: 10.1021/acs.jafc.8b02362 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
other fluorescent dyes, such as PRODAN (N,N-dimethyl-6propionyl-2-naphthylamine). Heat-treated WPI at pH ≈7.8 (i.e., pI + 3) had significantly higher (approximately 2.4-fold) ANS binding (i.e., surface hydrophobicity) than native WPI (Figure 1). In agreement with our result, Alizadeh-Pasdar and Li-Chan,13 using the ANS method, observed that the surface hydrophobicity of WPI and β-lg increased after heating at pH 7.0 and 9.0. Stănciuc et al.36 attributed the increase of surface hydrophobicity of heat-treated β-lg to protein unfolding. Exposure of previously hidden hydrophobic patches upon protein unfolding provided more sites for ANS binding. Far from our initial expectation (i.e., increasing surface hydrophobicity by acetylation),11 hydrophobized WPI had lower ANS-binding than both native and heat-treated samples at pH ≥ 6.7 (Figure 1). Zhao et al.,9 using ANS, observed that oat protein acetylated at anhydride-to-protein ratios between 0.1 and 0.4 g/g had lower surface hydrophobicity than native protein. The negatively charged sulfonate group of ANS can bind to the cationic side chains of lysine and arginine residues in proteins, leading to fluorescence enhancement.37 Acetylation, which is proceeded by the consumption of free amino groups, diminished ion pairing between ANS and whey proteins, leading to a reduction of ANS binding. Furthermore, the smaller surface-area-to-mass ratio of hydrophobized WPI nanoparticles at a given protein concentration in comparison with those of the other samples could limit the availability of fluorophore-binding sites and might contribute to the lower surface hydrophobicity of WPI at pH 7.8 as a consequence of acetylation. Decreasing the pH reduced electrostatic repulsion between whey proteins and the ligand (i.e., pHs above the corresponding pI) or caused electrostatic interactions between ANS and whey proteins (i.e., at pHs below the corresponding pI), increasing the surface hydrophobicity of all WPI samples. The ANS-binding affinity of a hydrophobized sample at pH 2.7 (pH = pI − 1) and that of a heated sample at pH 3.8 (pH = pI − 1) were comparable. Because the heat-treated WPI sample had a higher (positively charged) ζ-potential than the hydrophobized sample at pH values below their corresponding pIs (Figure 1), it was concluded that the hydrophobic interactions of acetyl moieties with ANS were of sufficient magnitude to compensate for the lower extent of electrostatic interactions between ANS and positively charged amino groups at a pH below the pI. Indeed, van der Waals interactions are required to stabilize ion pairings between proteins and ligands. Furthermore, it has been observed that the binding of the dimeric analogous of ANS (i.e., 4,4′-bis-1anilinonaphthalene-8-sulfonate, Bis-ANS) to proteins is dominated by hydrophobic interactions.35 3.4. Influence of Acetylation on Protein Structure. The FTIR spectra of WPI samples are illustrated in Figure 2. The amide I band (1600−1700 cm−1)23 mainly (≈80%) arises from CO stretching of peptide linkages, weakly coupled with C−N stretching and N−H bending.38 It is the main amide vibration studied for assessing protein secondary structure. The band was derivatized and deconvoluted, and the resulting peaks were assigned to different secondary structures, including random-coil and helical (α-helix and 310-helix) structures (1645−1666 cm−1), β-sheets (1620−1643 and 1689−1697 cm−1), and β-turns (1666−1687 cm−1).22 The effects of heating and acetylation−heating on the proportions of different secondary structures are reported in Table 2. The total β-sheet content of whey proteins decreased as a result of heat treatment, coinciding with an increased amount of β-
by formation of protein aggregates of varying molecular sizes depending on protein composition and heating conditions.27 Formation of the aggregates, which confine some amino groups within their compact matrix, and reaction of proteins with small amounts of innate lactose present in the WPI powder have been given as possible reasons for the slight decreases in the levels of free amino groups after heating.28 3.2. Influence of Acetylation on Protein-Particle Size and Charge. The intensity-based particle-size distributions indicated that native WPI was highly polydisperse (Table 1), and the quality of the calculated value of the z-average diameter, which is the intensity-weighted mean harmonic size,29 was poor. However, the number-based size distribution showed a monomodal (only one peak) pattern. This indicated that the contribution from other populations to hydrodynamic size was substantially small (
