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Effect of malondialdehyde modification on the binding of aroma compounds to soy protein isolates
Food Research International 105 (2018) 150–158
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Effect of malondialdehyde modification on the binding of aroma compounds to soy protein isolates Juan Wanga, Mouming Zhaoa, Chaoying Qiub, Weizheng Suna, a b
T
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School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soy protein isolation MDA modification Flavor binding Aldehydes trans-s-Undecenal SPME
The interactions of soy protein isolate (SPI) and flavor compounds (hexanal, trans-2-hexenal, 1-octen-3-ol, trans2-octenal, nonanal, and trans-2-nonenal) were investigated. The influence of SPI structure modified by malondialdehyde (MDA) and flavor compound structure on the interactions were determined by using headspace solid-phase microextraction (SPME) and gas chromatography (GC) combined with mass spectrometry (MS). The binding of native SPI to the flavor compounds decreased in the order trans-2-nonenal > nonanal > trans-2octenal > trans-2-hexenal > hexanal > 1-octen-3-ol. It might be attributed to that aldehydes are more hydrophobic than alcohols. The former is more conducive to hydrophobic binding with the SPI. Furthermore, the aldehydes, in particular trans-s-undecenal, could also react covalently. The effect of MDA modification on protein-flavor interactions depended on the structure of the flavor compound. Upon low concentration of MDA (≤1 mM), the binding of all six flavors to SPI increased. However, a further increase in the extent of MDA (≥2.5 mM), more soluble and even insoluble aggregates formed, which reduced the binding of hexanal and nonanal to SPI. The other four flavors with double bond revealed little changes in binding (trans-2-octenal, and trans-2-nonenal) or even an increase in binding (trans-2-hexenal, and 1-octen-3-ol). The results suggested that hydrophobic interactions were weakened upon high extent of oxidation, whereas covalent interactions were enhanced.
1. Introduction
affinity between different flavor molecules and a protein; the latter is to calculate the number of binding sites and binding constants for compounds with high water solubility and high UV absorption (Pelletier, Sostmann, & Guichard, 1998). More recently, since the technique called solid-phase microextraction (SPME) has been developed (Arthur & Pawliszyn, 1990), SPME and gas chromatography/mass spectrometry (GC–MS) have been used together to determine the interactions between flavor compounds and proteins (Kühn, Considine, & Singh, 2008; Wang & Arntfield, 2015; Zhou, Zhao, Su, & Sun, 2014). In this work, SPME and GC–MS have been chosen to determine the corresponding binding ability of six selected flavor compounds by soy protein isolates. Learned from previous studies, flavor–soy protein interactions are hydrophobic in nature (Damodaran & Kinsella, 1981a, 1981b), which depend on the nature of proteins and flavor compounds. For flavors, the polarity, chain length, functional group and concentration are included (Damodaran & Kinsella, 1980); for proteins, different structure has different binding ability, for example, the intrinsic binding constant for 2-nonanone by bovine serum albumin is about 1800 M− 1 (Damodaran & Kinsella, 1980), whereas with soy protein it is only 930 M-l (Damodaran & Kinsella, 1981b). In addition, different fraction in
The presence of flavor substances in food products determines their sensory properties, which in turn affects the acceptability of the consumer. Especially for soybeans, the beany flavor is considered to be one of the major factors limiting the widespread use of soy protein in food products (MacLeod, Ames, & Betz, 1988; Schutte & Van den Ouweland, 1979). In fact, proteins themselves are odorless, but they can bind flavor compounds and thus affect the sensory properties. Therefore, it is necessary to study the interaction between flavor compounds and soy proteins as well as their influencing factors. Investigation of interactions between flavor compounds and soy proteins had been conducted mainly in aqueous model systems by the use of equilibrium dialysis techniques (Damodaran & Kinsella, 1981a, 1981b) and static and dynamic headspace (O'keefe, Resurreccion, Wilson, & Murphy, 1991). Besides, inverse gas chromatography (IGC) had been used to study the binding of volatile compounds by dehydrated soy protein isolates (Zhou & Cadwallader, 2006). There was also investigation for β-lactoglobulin using affinity and exclusion size chromatography, the former is a rapid method to calculate the global ⁎
Corresponding author. E-mail address: [email protected] (W. Sun).
https://doi.org/10.1016/j.foodres.2017.11.001 Received 26 August 2017; Received in revised form 5 November 2017; Accepted 5 November 2017 Available online 06 November 2017 0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
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Hua, and Lin (2014) with minor modification. Briefly, 8.4 mL of TMP was mixed with 10.0 mL of 5.0 M HCl and 31.6 mL of deionized water and shaken at 40 °C in the dark for 30 min to obtain MDA through acidic hydrolysis. After that, the pH was adjusted to 7.4 with 6 M NaOH. Then take a small amount of stock solution to dilute to 10− 5, and the concentration of MDA in the stock solution was estimated by absorbance at 267 nm using a molar extinction coefficient value of 31,500 M− 1 cm− 1 (Adams, De Kimpe, & van Boekel, 2008).
proteins has different contribution to the binding ability. In soy protein isolations, β-conglycinin component might be the main protein fraction responsible for the off-flavor binding by soy proteins (Damodaran & Kinsella, 1981a, 1981b). It is well known that flavor-protein binding interactions can be altered by protein modification. However, most investigations focused on flavor compound chemical structure and medium in aqueous model systems, like ionic strength, temperature and pH. A little information is still available concerning the modification of proteins. For instance, the effect of succinylation of soy protein on the binding of 2-nonanone was studied, which suggested that there are about two binding sites for 2nonanone in succinylated soy compared to four binding sites in the native protein (Damodaran & Kinsella, 1981a). Except for the above, the partial deamidation of soy protein isolate (SPI) by protein-glutaminase decreased overall flavor-binding affinity for both vanillin and maltol by approximately 9- and 4-fold (Suppavorasatit & Cadwallader, 2012). Soy protein is an important food ingredient used in a lot of protein-based food. Soy protein oxidation occurred during processing and storage due to oxidative attack of lipid peroxidation-derived free radicals as well as lipid hydroperoxides and reactive aldehydes. In our previous study, the effects of SPI oxidation on their structure had been evaluated. SPI was modified by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) or lipid peroxidation product malondialdehyde (MDA) both had important influence on their structural and functional characteristics (Chen, Zhao, & Sun, 2013; Chen, Zhao, Sun, & Zhao, 2013). However, those studies did not assess the impact of oxidation on the flavor-binding properties of the protein. Therefore, the objective of the present study was to investigate the effect of protein oxidation on the binding of selected carbonyl-containing flavor compounds to SPI in an aqueous system using SPME combined with GC/MS.
2.4. Modification of SPI with MDA Control and MDA-modified SPI were prepared according to the method described by Wu, Zhang, and Hua (2009) SPI suspension (40 mg/mL containing 0.5 mg/mL sodium azide, suspended in 10 mM sodium phosphate buffer, pH 7.4) was mixed with a serial concentration of MDA and then incubated by continuous shaking under air at 25 °C in dark for 24 h to react sufficiently. The final concentrations of MDA were 0, 0.5, 1.0, 2.5, 5.0 and 10.0 mM. The freeze-dried SPI without shaking treated sample was control SPI, and the others were MDA-modified SPI. After that, the MDA-modified SPI suspension was dialyzed against deionized water at 4 °C for 72 h to remove free MDA and salt. Then the dialyzed solution was freeze-dried and stored at 4 °C until used.
2.5. Evaluation of oxidative changes 2.5.1. Carbonyls The content of carbonyls was determined by the reaction with 2,4 dinitrophenylhydrazine (DNPH) using a UV765 spectrophotometer (Shanghai, China), as described by Huang, Hua, and Qiu (2006). twenty milligram samples were suspended in 10 mL of 10 mM sodium phosphate buffer (pH 7.0), stirred for 1 h at room temperature, then centrifuged at 10000 g for 15 min at 4 °C. Took the supernatant and determined the protein concentration by using the Biuret method. Then, in 10 mL capped polyethylene centrifuge tubes, 1 mL SPI solution was mixed with 3 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) dissolved in 2 M HCl and incubated at room temperature for 2 h. And a same amount of SPI solution mixed with 3 mL of 2 M HCl was an absorbance blank. The DNPH-reacted samples after 20% trichloroacetic acid (TCA) precipitation were collected by centrifugation at 10000 g for 15 min at 4 °C and then washed three times with 5 mL of ethanol/ethyl acetate solution (1:1, v/v). The final pellets, free of DNPH, was dissolved in 3 mL of 6 M guanidine hydrochloride in 0.1 M sodium phosphate buffer (pH 7.0). The absorbance at 367 nm was corrected by the absorbance in the HCl blank. The results were calculated using a molar extinction coefficient of 22,000 M− 1 cm− 1 (Boatright & Hettiarachchy, 1995) and expressed as nanomoles of carbonyl group per milligram of protein.
2. Materials and methods 2.1. Samples and materials Defatted soy flakes were purchased from Yuwang Group (Shandong, China). 1,1,3,3-Tetramethoxypropane (TMP, purity 98%) and 8Anilino-1-naphthalenesulfonic acid (ANS) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) and bicinchoninic acid (BCA) protein assay kit were purchased from Dingguo (Beijing, China). The aroma compounds hexanal, (E)-2-Hexenal, (E)-2-Octenal, (E)-2-Nonenal were purchased from Sigma − Aldrich (Steinheim, Germany). 1-Octen-3-ol and nonanal were obtained from Aladdin (Shanghai, China). All other chemicals were of analytical reagent grade and obtained in China. 2.2. SPI preparation Defatted soy flake powder (200 g) was mixed with 15-fold (in weight) deionized water, and the mixture (pH 6.8) was adjusted to pH 8.0 with 2.0 M NaOH. After stirring for 2 h, the resulting suspension was centrifuged at 8000 g for 20 min at 4 °C to remove the insoluble material. Then the pH of the supernatant was adjusted to 4.5 with 2.0 M HCl, and the precipitate was collected by centrifugation at 8000 g for 20 min at 4 °C. The precipitate was then redissolved with 5-fold (in weight) deionized water, and the pH was adjusted to 7.0 with 2.0 M NaOH, maintaining the pH to 7.0 and stirring until protein was completely dissolved. Then the neutral SPI solution was dialyzed against deionized water at 4 °C for 48 h and then freeze-dried and stored at 4 °C until use.
2.5.2. Surface hydrophobicity The hydrophobicity was determined using 1-anilino-8-naphthalenesulfonate (ANS) as fluorescence probe (Kato & Nakai, 1980). SPI (500 mg) was suspended in 100 mL 10 mM sodium phosphate buffer (pH 7.0), stirred thoroughly at room temperature. Then centrifuged at 10000 g for 15 min at 4 °C and collected the supernatant. The protein concentration in the supernatant was evaluated using a BCA protein assay kit, and then diluted the supernatant to serial concentration with the same buffer in the range of 0.005–0.5 mg/mL. After that, 4 mL of protein dilution was mixed with 50 μL ANS solution (8 mM in 0.01 M, pH 7.0 phosphate buffer). Fluorescence intensity (FI) was measured at 365 nm (excitation) and 484 nm (emission) on a F7000 fluorescence spectrophotometer (Hitachi Co., Japan). The curve was fitted with fluorescence intensity and protein concentration, and the initial slope of which was used an index of protein surface hydrophobicity (H0).
2.3. Preparation of MDA stock solution MDA stock solution must be made fresh daily, and a fresh MDA stock solution was prepared by hydrolyzing1,1,3,3-tetramethoxypropane (TMP) according to the method described by Wu, 151
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concentrations were 420 mg/L for hexanal, 450 mg/L for trans-2-hexenal, 425 mg/L for trans-2-octenal, 390 mg/L for 1-octen-3-ol, 415 mg/ L for nonanal, 525 mg/L for trans-2-nonenal. The selection of the six flavor compounds was firstly based on their relatively large proportion of all the flavors in traditional soymilk. Furthermore, hexanal, trans-2hexenal and 1-octen-3-ol are representative of beany flavors, while trans-2-octenal, nonanal and trans-2-nonenal are non-beany flavors (Lv, Song, Li, Wu, & Guo, 2011). And the six flavor compounds differ in chain length and function group.
2.5.3. Fluorescence spectra of tryptophan and Schiff base Control and MDA-modified SPI samples (500 mg) were suspended in 100 mL 10 mM sodium phosphate buffer (pH 7.0) and magnetically stirred thoroughly at room temperature. Then the SPI suspension was centrifuged at 10000 g for 15 min and collected the supernatant. And the protein concentration in the supernatant was determined by using a BCA protein assay kit. Then, protein concentration was separately adjusted to 0.1 mg/mL and 0.2 mg/mL for the measurement of tryptophan and Schiff base. To determine the fluorescence emission spectrum of tryptophan, the excitation wavelength was set at 285 nm, and the emission spectrum was recorded from 300 to 400 nm (Chen, Zhao, Sun, Ren, & Cui, 2013). And for Schiff base, the excitation wavelength was set at 350 nm, and the emission spectrum was recorded from 400 to 600 nm (Utrera, Rodríguez-Carpena, Morcuende, & Estévez, 2012). The slit width was set at 5 nm, and data were collected at 240 nm/min.
2.6.2. Preparation of GC/MS samples Samples were prepared by transferring 6.86 mL of SPI suspensions (0.715%) into 20 mL headspace vials, then 140 μL of the flavor stock solution was added to obtain a final flavor concentration of 8.4 mg/L for hexanal, 9 mg/L for trans-2-hexenal, 8.5 mg/L for trans-2-octenal, 7.8 mg/L for 1-octen-3-ol, 8.3 mg/L m for nonanal, 10.5 mg/L for trans2-nonenal. The blank vial contained exactly the same buffer instead of protein. All the vials were immediately sealed with a PTFE-faced silicone septum (Supelco, Bellefonte, PA) and equilibrated for 24 h at 4 °C. All the samples were prepared in duplicate.
2.5.4. Particle size distributions Particle size distribution was measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcestershire, United Kingdom), aimed to observe the aggregates in oxidized SPI with different degree. Briefly, referred to the method of Wu, Hua, Lin, and Xiao (2011) with minor modification, SPI (100 mg) was suspended in 100 mL 10 mM sodium phosphate buffer (pH 7.0) at a concentration of 1 mg/mL and was magnetically stirred thoroughly. Then the samples were centrifuged at 10000 g for 15 min at 4 °C and the supernatants were collected. Before measurement, the supernatants were filtered through nylon membranes with pore size of 0.45 μm to remove any insoluble particles. Then 1 mL of protein solution was injected into a cuvette that marked by triangle for DLS measurement.
2.6.3. SPME and GC/MS analysis After equilibration, the SPME fiber was punctured into the headspace of the sample vial and was immediately transferred into the gas chromatograph injector port for quantification. The SPME parameters were as follows: 75 μm carboxen/polydimethylsiloxane (CAR/PDMS) fiber (Supelco, Bellefonte, PA), equilibrated at 45 °C for 20 min, extracted at 45 °C for 40 min, desorbed at 250 °C for 3 min. GC–MS conditions: TG-5 ms (30 m × 0.25 mm × 0.25 μm) column was used for separation. The carrier gas was high purity helium (1.0 mL/min). The mass spectrometry conditions were electron bombardment ionization (EI) ion source, electron energy 70 eV, electron multiplication Device voltage 350 V, transmission line temperature 250 °C, ion source temperature 250 °C, scanning speed 3.00 scans/s, mass range 33–350 m/z. Liquid injection program temperature conditions: the initial temperature 40 °C, rose to 120 °C in 2 min at 4 °C/min, keep for 2 min, then rose to 280 °C at 10 °C/min, keep for 10 min; injection volume was 1 μL; injection temperature 230 °C.
2.5.5. SDS–PAGE analysis Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) was performed on a discontinuous buffered system (Laemmeli, 1970), using 12% separating gel and 5% stacking gel. Briefly, 100 μL soy protein isolation suspensions (20 mg/mL in 10 mM phosphate buffer at pH 7.0) were mixed with 400 μL sample buffer with and without 5% β-mercaptoethanol (β ME). The ones with β ME were heated for 5 min in boiling water, while the others were heated for a few seconds in boiling water. Then both were centrifuged at 10000 g for 10 min before electrophoresis. For each sample, 10 μL of the supernatant was loaded to each lane. A prestained protein marker I with ten bands (170 kD, 130 kD, 95 kD, 72 kD, 55 kD, 43 kD, 34 kD, 26 kD, 17 kD, 10 kD) was used as reference. Those bands are purified protein mixtures and are covalently conjugated to the dye. Among them, the band of 72 kD is an orange band and 10 kD is a green band. After the electrophoresis, the gel was stained using the Coomassie brilliant blue R-250 stain solution (0.1% Coomassie brilliant blue R-250, 10% acetic acid, 45% methanol) for 1.0 h and destained by methanol/acetic acid/ water (1:1:8, v/v/v) for 24 h with two or three changes of destain solution.
2.7. Statistical analysis Statistical calculation was analysed using the statistical package SPSS 20 (SPSS Inc., Chicago, IL) for one-way ANOVA. Least-squares difference was used for comparison of mean values among treatments and to identify significant differences (p < 0.05) among treatments. Triplicate preparations of soy protein isolations were carried out to confirm the accordance. Data were expressed as means ± standard deviations (SD) of triplicate determinations unless specifically mentioned. 3. Results and discussion
2.5.6. Microstructure The superficial morphology of oxidized proteins were observed by field emission scanning electron microscopy (FE-SEM, LEO-Merlin, Germany) at an accelerator voltage of 5 kV. Before imaging, the freezedried protein powder was adhered to a small piece of iron with a double-sided adhesive tape and then plated with gold (JEOL JFC-1200 fine coater, Japan).
3.1. Oxidative modifications of soy protein isolations 3.1.1. Carbonyls Carbonyl groups are commonly used to characterize the interaction of proteins with various oxidizing systems. Effects of MDA modification on the carbonyl group content of SPI are given in Fig. 1A. In this work, the carbonyl group content of the control protein was 2.33 nmol/mg protein, which is close to the reports of Wu, Zhang, Kong, and Hua (2009) on soy protein isolation oxidized by peroxyl radicals. Compared with the control, the treatment for the 0 mM that was shaked for 24 h at 25 °C made negligible differences. However, in the presence of MDA, the carbonyls group content increased as the MDA concentration increased. Between 0–1 mM, carbonyl content increased linearly, then from 2.5–10 mM, it increased sharply.
2.6. Flavor binding to control and MDA-modified protein 2.6.1. Preparation of protein and flavor stock solutions 0.715% SPIs suspensions were prepared in 10 mM sodium phosphate buffer (pH 7.0) and magnetically stirred thoroughly. A stock solution containing each flavor compound was prepared in methanol and stored in brown glass bottles to prevent decomposition. Their 152
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Fig. 1. Carbonyl content (A), protein surface hydrophobicity (B) of the soy protein isolations upon oxidation by MDA (0–10 mM). For the letters a–f, means (n = 3) without a common letter differ significantly (p < 0.05).
3.1.3. Fluorescence spectra of tryptophan and Schiff base Fluorescence spectroscopy has been recently supposed to be a simple, fast, and solvent-free method for characterizing protein oxidation in foods by means of tryptophan loss and Schiff base formation (Rui, Estévez, Kylli, Heinonen, & Morcuende, 2010). In this work, the oxidizing agent MDA has two reactive aldehyde groups, which forms a Schiff base complex preferentially with primary amino groups such as εamines of lysine residues. In addition, MDA induces cross-linking proteins and produces fluorescent adducts such as DHP–lysine (Lamore et al., 2010; Zhao, Chen, Zhu, & Xiong, 2012). The intrinsic fluorescence of tryptophan and Schiff base in control and MDA modified SPI are shown in Fig. 2. As shown in Fig. 2, with the increasing concentration of MDA, the loss of tryptophan increased, accompanied by the increasing of Schiff base. At relatively low addition of MDA (0–0.5 mM), both tryptophan and Schiff base were not changed much, but it showed significant differences from 1–10 mM. Interestingly, in Fig. 2A, with the MDA
3.1.2. Surface hydrophobicity Hydrophobicity is known to be significantly related to the structure and functional properties of proteins (Kato & Nakai, 1980). Fig. 1B shows the surface hydrophobicity of the control and oxidized SPI. The surface hydrophobicity of 0 mM was slightly higher than the control. For the oxidized proteins, the surface hydrophobicity increased with the increasing MDA concentration. The above results were consistent with the trend of carbonyl growth. Meanwhile, the solubility of the oxidized SPI in 10 mM sodium phosphate buffer (pH 7.0) decreased with the increasing MDA concentration. This was in accordance to the reports of Chen et al. (2013). This result might be attributed to the unfolding of soy protein isolates when subjected to MDA modification, which led to the exposing of hydrophobic amino acids buried in protein inside, and thus enhanced protein surface hydrophobicity.
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Fig. 2. Fluorescent emission spectra of tryptophan (A) and Schiff base (B) of control and increasing concentration of MDA modified SPI.
concentration up to 2.5 mM, the λmax of fluorescence emission underwent a slight red shift up to 10 mM MDA. The red shift might be attributed to the changes in the microenvironment of the protein in the aqueous solution due to oxidation.
This result was in agreement with the previous studies (Chen et al., 2013). And it indicated that SPI modified by low MDA concentration (≤ 2.5 mM) induced the formation of some soluble aggregates. Further increasing extend of oxidation could promote insoluble component formation and cleavage of peptide bonds by peroxyl radicals (Davies, 2005). Therefore, SPI modified by 5 and 10 mM MDA might also induced some insoluble and soluble aggregates, the former were removed by centrifugation, while the later were some lager aggregates and broke into smaller soluble peptides.
3.1.4. Particle size distributions The average particle size and size volume distribution for the control and MDA-modified SPIs were used to evaluate the aggregation behavior of proteins during oxidation, and the results are shown in Fig. 3. In this work, only soluble component of sample was investigated. It can be clearly seen from the form in the Fig. 3, with the increasing of MDA concentration, the average particle size decreased, accompanied by the decreasing of the particle dispersibility index (PdI). However, for the size volume distribution, SPI gradually shifted to larger particle size within MDA ≤ 2.5 mM, while at the concentration between 5 and 10 mM, it shifted to smaller particle size and was smaller than the control.
3.1.5. SDS–PAGE The aggregation behavior of MDA-modified SPI was further studied by SDS-PAGE analysis (Fig. 4). In the Fig. 4A, in low MDA concentration (≤ 1 mM), the α′ and α subunits of β-conglycinin became less intense, with further oxidation (≥ 2.5 mM), they disappeared. However, the β subunit became more intense, it might be due to the complexes formed by β subunit and basic subunits (Utsumi, Damodaran, & 154
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Fig. 3. Particle size distribution of control and MDA-modified SPI. Average particle size with different letters (a–g) are significant difference (p < 0.05).
linkage (Tezuka, Yagasaki, & Tomotada, 2004), we speculated that the acid subunits interacted with each other or other subunits through disulfide bonds thus resulted in soluble aggregates, which appeared on the top of separating gel. Meanwhile, as for the low MDA modified SPI (≤ 1 mM), high molecular weight polymers formed on the top of the separating gel, which were formed by disulfide bonds. In the Fig. 4B, with the addition of 5% β-ME, the disulfide bonds in proteins were destroyed, characteristic bands for the subunits of βconglycinin and glycinin were presented. It could be clearly seen that, no significant change of electrophoretic pattern was observed among the first four samples (control SPI and those incubated with 0, 0.5, 1.0 mM MDA), whereas there was a gradual disappearance of several subunits in a MDA dose-dependent manner among those incubated with 2.5, 5.0, 10.0 mM MDA. Furthermore, at 10 mM MDA, the α′ subunits of β-conglycinin mostly went, while the subunits of glycinin just reduced. These phenomena demonstrated that β-conglycinin was more sensitive to MDA modification than glycinin. Simultaneously, upon oxidation, high molecular weight polymers formed and accumulated on the top of the separating gel and stacking gel. The formation of these polymers was attributed to the interaction of subunits through nondisulfide covalent bonds as the disulfide bonds were destroyed by β-ME. These results demonstrated the occurrence of cross-linking under oxidation, which was consistent with the changes in particle size distributions as previously mentioned.
3.1.6. Microstructure The changes in the physicochemical properties of oxidized SPI were further investigated by the observation of their morphologic properties. A set of SEM images of oxidized SPI at a magnification factor of 1000-fold are showed in Fig. 5. It could be clearly seen that, there were many small fragments in 0 mM sample. With the increasing MDA concentration, small fragments decreased, followed by the formation of larger fragments. At high MDA (≥ 5 mM), large fragments were stacked with a few small fragments on their surface. These morphological changes might be caused by the unfolding of the SPI molecules, and increased exposure of hydrophobic groups (Fig. 1B) at the surface of the molecules, which could induce protein-protein interactions and form larger aggregates during oxidation.
Fig. 4. Representative SDS− PAGE patterns of control and MDA-modified SPI (A) without and with 5% β-ME. Lanes: M, marker proteins; C, control SPI(without incubation at 25 °C for 24 h); 0, 0.5, 1, 2.5, 5 and 10, SPI treated with 0, 0.5, 1, 2.5, 5 and 10 mM MDA. SPI constituents: α′, α, and β, subunits for β-conglycinin; A and B, acidic subunits and basic subunits for glycinin.
Kinsella, 1984), since the basic subunits largely became less intense. In addition, the acid subunits disappeared completely, while presented in reduction electrophoresis. Since each subunit in glycinin consists of an acidic (A) and basic (B) polypeptide chain connected by a disulfide 155
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Fig. 5. Scanning electron microscope micrographs at × 1000 magnification of the control and MDA-modified soy protein isolations. Scale bars indicate 10 μm.
investigations, who found that the unsaturated aldehydes were retained more strongly in soy protein than the correspongding saturated ones (Gremli, 1974). In addition, trans-2-hexenal has been shown to exhibit covalent binding with milk proteins (Meynier, Rampon, Dalgalarrondo, & Genot, 2004). For the oxidized SPIs, at low MDA concentration (≤1 mM), the percentages of all flavor compounds decreased, demonstrating the increased binding affinity of oxidized protein. Nevertheless, with the further increasing in MDA concentration (> 1 mM MDA), an inverse effect on the percentages of flavor compounds was observed for hexanal, trans-2-hexenal, 1-octen-3-ol, nonanal and trans-2-nonenal. Interestingly, for nonanal and trans-2-nonenal, both of which were bound to a gradual decreasing extent, especially for nonanal. While for hexanal, trans-2-hexenal and 1-octen-3-ol, the binding affinity began to increase, and then decreased. Although the high MDA-modified SPIs showed a decrease in binding affinity, the changes were not marked except for nonanal. The samples treated at high MDA concentration (≥ 2.5 mM) contained sediment of aggregated proteins, whereas the samples treated at low MDA concentration (≤ 1 mM) remained clear. At the MDA concentration between 0 and 1 mM, the increasing binding affinity with the increasing concentration of MDA might be due to the partial exposing of hydrophobic amino acids buried in protein inside. And this was consistent with changes in protein surface hydrophobicity (Fig. 1B). While for the decrease in binding of hexanal and nonanal to SPI upon high concentration of MDA(≥ 2.5 mM), it might be explained by the increase in the extent of soluble and insoluble aggregations of
3.2. Flavor binding to control and MDA-modified protein On the basis of the physicochemical properties changes of oxidized SPI by MDA, the effect of structural changes of SPI on their binding ability with selected flavor compounds was evaluated. Thus, the free percentages of each flavor compound in the headspace of the control and oxidized SPIs after 24 h equilibrium at 4 °C were determined, the result was based on a blank (each flavor compound mixed with 10 mM sodium phosphate buffer, pH 7.4 instead of protein) of 100%. As shown in Fig. 6, the presence of the control SPI produced a remarkable reduction of the percentages of free hexanal, trans-2-hexenal, trans-2-octenal, nonanal, trans-2-nonenal, suggesting that the control SPI was able to bind those aroma compounds. In contrast, the increase in 1-octen-3ol implied its releasing behavior. Furthermore, compared to hexanal, control SPI showed a higher binding affinity for nonanal. And among trans-2-hexenal, trans-2-octenal, trans-2-nonenal, the binding affinity for control SPI increased with an increase in the chain length. This result was in agreement with the previous study (Damodaran & Kinsella, 1981b). trans-2-Hexenal exhibited a higher binding affinity for control SPI than hexanal, and the same consequence is also observed between nonanal and trans-2-nonenal. It indicated that the presence of a double bond further increased the binding affinity of flavor compounds for control SPI. trans-2-Nonenal is less hydrophobic than nonanal (Kühn et al., 2008) but is bound to a higher extent. This suggested that possible interactions between flavors and protein are not only hydrophobic in nature, but also might involve the double bond (“Michael addition”). This observation was in agreement with the previous
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Fig. 6. Effect of MDA modification on the binding of flavor compounds. Results are expressed as percentage of free flavor compounds found in the headspace without protein in solution.
unfolded SPI, making the flavor binding site inaccessible (Kuhn, Zhu, Considine, & Singh, 2007). And for trans-2-hexenal, trans-2-octenal and trans-2-nonenal, no significant changes in binding of MDA (≥2.5 mM) modified SPI might result from the combination of both a decrease in binding attributed to the destruction of the hydrophobic pocket and an increase in binding caused by covalent interactions between aggregated protein molecules with these unsaturated aldehydes (Kuehn, Considine, & Singh, 2008). This was in agreement with the results of SDS-PAGE (Fig.4).
Acknowledgements The authors are grateful to the Special Support Project of Guangdong Province for Science and Technology Innovative Young Talents (2014TQ01N538), Pearl River S&T Nova Program of Guangzhou (201610010105) and the Fundamental Research Funds for the Central Universities (2015ZM155) for their financial supports. References Adams, A., De Kimpe, N., & van Boekel, M. A. J. S. (2008). Modification of casein by the lipid oxidation product malondialdehyde. Journal of Agricultural and Food Chemistry, 56(5), 1713–1719. http://dx.doi.org/10.1021/jf072385b. Arthur, C. L., & Pawliszyn, J. (1990). Solid phase microextraction with thermal desorption using fused silica optical fibers. Analytical Chemistry, 62(19), 2145–2148. Boatright, W. L., & Hettiarachchy, N. S. (1995). Effect of lipids on soy protein isolate solubility. Journal of the American Oil Chemists' Society, 72(12), 1439–1444. Chen, N., Zhao, M., & Sun, W. (2013). Effect of protein oxidation on the in vitro digestibility of soy protein isolate. Food Chemistry, 141(3), 3224–3229. Chen, N., Zhao, M., Sun, W., Ren, J., & Cui, C. (2013). Effect of oxidation on the emulsifying properties of soy protein isolate. Food Research International, 52(1), 26–32. Chen, N., Zhao, Q., Sun, W., & Zhao, M. (2013). Effects of malondialdehyde modification on the in vitro digestibility of soy protein isolate. Journal of Agricultural and Food Chemistry, 61(49), 12139–12145. Damodaran, S., & Kinsella, J. E. (1980). Flavor protein interactions. Binding of carbonyls to bovine serum albumin: Thermodynamic and conformational effects. Journal of Agricultural and Food Chemistry, 28(3), 567–571. Damodaran, S., & Kinsella, J. E. (1981a). Interaction of carbonyls with soy protein: Conformational effects. Journal of Agricultural and Food Chemistry, 29(6), 1253–1257. Damodaran, S., & Kinsella, J. E. (1981b). Interaction of carbonyls with soy protein: Thermodynamic effects. Journal of Agricultural and Food Chemistry, 29(6), 1249–1253. Davies, M. J. (2005). The oxidative environment and protein damage. Biochimica et Biophysica Acta, 1703(2), 93–109. Gremli, H. A. (1974). Interaction of flavor compounds with soy protein. Journal of the American Oil Chemists' Society, 51(1), 95A–97A. Huang, Y. R., Hua, Y. F., & Qiu, A. Y. (2006). Soybean protein aggregation induced by lipoxygenase catalyzed linoleic acid oxidation. Food Research International, 39(2), 240–249. http://dx.doi.org/10.1016/j.foodres.2005.07.012. Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochimica et Biophysica Acta, 624(1), 13–20. Kuehn, J., Considine, T., & Singh, H. (2008). Binding of flavor compounds and whey protein isolate as affected by heat and high pressure treatments. Journal of
4. Conclusions The effects of flavor compound structure and MDA-induced oxidative modifications on the interactions of SPI and flavor compounds were elucidated. The binding between native SPI and the selected flavor compounds was strong and decreased in the order trans-s-undecenal > aldehyde > olefinic alcohol. Furthermore, both the binding affinity of aldehyde and trans-s-undecenal for SPI increased with an increase in the chain length. The flavor compounds with different structure are bound to proteins on different binding sites and/or by different binding mechanisms. Upon low MDA concentration (≤ 1 mM), the structure was unfolded with hydrophobic groups exposed and thus increased the surface hydrophobicity, which is beneficial to the hydrophobic interactions between flavor compounds and SPI. In addition, the aldehyde group would react with specific amino acids residues by covalent interaction in the unfolded proteins. Whereas, high concentration of MDA-induced denaturation (≥2.5 mM) reduces hydrophobic interactions of SPI with flavor compounds, like hexanal and nonanal. Covalent reactions of the double bond with certain amino acid residues might be more readily accessible in the aggregated proteins. For next study, we will separate 7 s and 11 s, using fluorescence quenching as well as isothermal titration calorimetry to further elaborate its binding mechanism with flavor compounds.
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aqueous conditions. Journal of Agricultural and Food Chemistry, 60(32), 7817–7823. Tezuka, M., Yagasaki, K., & Tomotada, O. (2004). Changes in characters of soybean Glycinin groups I, IIa, and IIb caused by heating. Journal of Agricultural & Food Chemistry, 52(6), 1693–1699. Utrera, M., Rodríguez-Carpena, J. G., Morcuende, D., & Estévez, M. (2012). Formation of lysine-derived oxidation products and loss of tryptophan during processing of porcine patties with added avocado byproducts. Journal of Agricultural & Food Chemistry, 60(15), 3917–3926. Utsumi, S., Damodaran, S., & Kinsella, J. E. (1984). Heat-induced interactions between soybean proteins: Preferential association of 11S basic subunits and beta subunits of 7S. Journal of Agricultural & Food Chemistry, 32(6), 1406–1412. Wang, K., & Arntfield, S. D. (2015). Effect of salts and pH on selected ketone flavours binding to salt-extracted pea proteins: The role of non-covalent forces. Food Research International, 77, 1–9. Wu, W., Hua, Y. F., & Lin, Q. L. (2014). Effects of oxidative modification on thermal aggregation and gel properties of soy protein by malondialdehyde. Journal of Food Science and Technology-Mysore, 51(3), 485–493. http://dx.doi.org/10.1007/s13197011-0533-7. Wu, W., Hua, Y., Lin, Q., & Xiao, H. (2011). Effects of oxidative modification on thermal aggregation and gel properties of soy protein by peroxyl radicals. International Journal of Food Science & Technology, 46(9), 1891–1897. Wu, W., Zhang, C. M., & Hua, Y. F. (2009). Structural modification of soy protein by the lipid peroxidation product malondialdehyde. Journal of the Science of Food and Agriculture, 89(8), 1416–1423. http://dx.doi.org/10.1002/jsfa.3606. Wu, W., Zhang, C., Kong, X., & Hua, Y. (2009). Oxidative modification of soy protein by peroxyl radicals. Food Chemistry, 116(1), 295–301. Zhao, J., Chen, J., Zhu, H., & Xiong, Y. L. (2012). Mass spectrometric evidence of malonaldehyde and 4-hydroxynonenal adductions to radical-scavenging soy peptides. Journal of Agricultural & Food Chemistry, 60(38), 9727–9736. Zhou, Q., & Cadwallader, K. R. (2006). Effect of flavor compound chemical structure and environmental relative humidity on the binding of volatile flavor compounds to dehydrated soy protein isolates. Journal of Agricultural and Food Chemistry, 54(5), 1838–1843. Zhou, F., Zhao, M., Su, G., & Sun, W. (2014). Binding of aroma compounds with myofibrillar proteins modified by a hydroxyl-radical-induced oxidative system. Journal of Agricultural and Food Chemistry, 62(39), 9544–9552.
Agricultural and Food Chemistry, 56(21), 10218–10224. http://dx.doi.org/10.1021/ jf801810b. Kühn, J., Considine, T.r.s., & Singh, H. (2008). Binding of flavor compounds and whey protein isolate as affected by heat and high pressure treatments. Journal of Agricultural and Food Chemistry, 56(21), 10218–10224. Kuhn, J., Zhu, X. Q., Considine, T., & Singh, H. (2007). Binding of 2-nonanone and milk proteins in aqueous model systems. Journal of Agricultural and Food Chemistry, 55(9), 3599–3604. http://dx.doi.org/10.1021/jf063517o. Laemmeli, U. K. (1970). Cleavage of structuralb proteins during the assembly of the head to bacteriophage t4. Nature, 5259(227), 680–685. Lamore, S. D., Azimian, S., Horn, D., Anglin, B. L., Uchida, K., Cabello, C. M., & Wondrak, G. T. (2010). The malondialdehyde-derived fluorophore DHP-lysine is a potent sensitizer of UVA-induced photooxidative stress in human skin cells. Journal of Photochemistry & Photobiology B Biology, 101(3), 251–264. Lv, Y. C., Song, H. L., Li, X., Wu, L. A., & Guo, S. T. (2011). Influence of blanching and grinding process with hot water on beany and non-beany flavor in soymilk. Journal of Food Science, 76(1), S20–S25. http://dx.doi.org/10.1111/j.1750-3841.2010.01947.x. MacLeod, G., Ames, J., & Betz, N. L. (1988). Soy flavor and its improvement. Critical Reviews in Food Science & Nutrition, 27(4), 219–400. Meynier, A., Rampon, V., Dalgalarrondo, M., & Genot, C. (2004). Hexanal and t-2-hexenal form covalent bonds with whey proteins and sodium caseinate in aqueous solution. International Dairy Journal, 14(8), 681–690. O'keefe, S. F., Resurreccion, A. P., Wilson, L. A., & Murphy, P. A. (1991). Temperature effect on binding of volatile flavor compounds to soy protein in aqueous model systems. Journal of Food Science, 56(3), 802–806. Pelletier, E., Sostmann, K., & Guichard, E. (1998). Measurement of interactions between β-lactoglobulin and flavor compounds (esters, acids, and pyrazines) by affinity and exclusion size chromatography. Journal of Agricultural and Food Chemistry, 46(4), 1506–1509. Rui, G., Estévez, M., Kylli, P., Heinonen, M., & Morcuende, D. (2010). Characterization of selected wild Mediterranean fruits and comparative efficacy as inhibitors of oxidative reactions in emulsified raw pork burger patties. Journal of Agricultural & Food Chemistry, 58(15), 8854–8861. Schutte, L., & Van den Ouweland, G. (1979). Flavor problems in the application of soy protein materials. Journal of the American Oil Chemists' Society, 56(3), 289–290. Suppavorasatit, I., & Cadwallader, K. R. (2012). Effect of enzymatic Deamidation of soy protein by protein–Glutaminase on the flavor-binding properties of the protein under
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Effect of malondialdehyde modification on the binding of aroma compounds to soy protein isolates Juan Wanga, Mouming Zhaoa, Chaoying Qiub, Weizheng Suna, a b
T
⁎
School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Soy protein isolation MDA modification Flavor binding Aldehydes trans-s-Undecenal SPME
The interactions of soy protein isolate (SPI) and flavor compounds (hexanal, trans-2-hexenal, 1-octen-3-ol, trans2-octenal, nonanal, and trans-2-nonenal) were investigated. The influence of SPI structure modified by malondialdehyde (MDA) and flavor compound structure on the interactions were determined by using headspace solid-phase microextraction (SPME) and gas chromatography (GC) combined with mass spectrometry (MS). The binding of native SPI to the flavor compounds decreased in the order trans-2-nonenal > nonanal > trans-2octenal > trans-2-hexenal > hexanal > 1-octen-3-ol. It might be attributed to that aldehydes are more hydrophobic than alcohols. The former is more conducive to hydrophobic binding with the SPI. Furthermore, the aldehydes, in particular trans-s-undecenal, could also react covalently. The effect of MDA modification on protein-flavor interactions depended on the structure of the flavor compound. Upon low concentration of MDA (≤1 mM), the binding of all six flavors to SPI increased. However, a further increase in the extent of MDA (≥2.5 mM), more soluble and even insoluble aggregates formed, which reduced the binding of hexanal and nonanal to SPI. The other four flavors with double bond revealed little changes in binding (trans-2-octenal, and trans-2-nonenal) or even an increase in binding (trans-2-hexenal, and 1-octen-3-ol). The results suggested that hydrophobic interactions were weakened upon high extent of oxidation, whereas covalent interactions were enhanced.
1. Introduction
affinity between different flavor molecules and a protein; the latter is to calculate the number of binding sites and binding constants for compounds with high water solubility and high UV absorption (Pelletier, Sostmann, & Guichard, 1998). More recently, since the technique called solid-phase microextraction (SPME) has been developed (Arthur & Pawliszyn, 1990), SPME and gas chromatography/mass spectrometry (GC–MS) have been used together to determine the interactions between flavor compounds and proteins (Kühn, Considine, & Singh, 2008; Wang & Arntfield, 2015; Zhou, Zhao, Su, & Sun, 2014). In this work, SPME and GC–MS have been chosen to determine the corresponding binding ability of six selected flavor compounds by soy protein isolates. Learned from previous studies, flavor–soy protein interactions are hydrophobic in nature (Damodaran & Kinsella, 1981a, 1981b), which depend on the nature of proteins and flavor compounds. For flavors, the polarity, chain length, functional group and concentration are included (Damodaran & Kinsella, 1980); for proteins, different structure has different binding ability, for example, the intrinsic binding constant for 2-nonanone by bovine serum albumin is about 1800 M− 1 (Damodaran & Kinsella, 1980), whereas with soy protein it is only 930 M-l (Damodaran & Kinsella, 1981b). In addition, different fraction in
The presence of flavor substances in food products determines their sensory properties, which in turn affects the acceptability of the consumer. Especially for soybeans, the beany flavor is considered to be one of the major factors limiting the widespread use of soy protein in food products (MacLeod, Ames, & Betz, 1988; Schutte & Van den Ouweland, 1979). In fact, proteins themselves are odorless, but they can bind flavor compounds and thus affect the sensory properties. Therefore, it is necessary to study the interaction between flavor compounds and soy proteins as well as their influencing factors. Investigation of interactions between flavor compounds and soy proteins had been conducted mainly in aqueous model systems by the use of equilibrium dialysis techniques (Damodaran & Kinsella, 1981a, 1981b) and static and dynamic headspace (O'keefe, Resurreccion, Wilson, & Murphy, 1991). Besides, inverse gas chromatography (IGC) had been used to study the binding of volatile compounds by dehydrated soy protein isolates (Zhou & Cadwallader, 2006). There was also investigation for β-lactoglobulin using affinity and exclusion size chromatography, the former is a rapid method to calculate the global ⁎
Corresponding author. E-mail address: [email protected] (W. Sun).
https://doi.org/10.1016/j.foodres.2017.11.001 Received 26 August 2017; Received in revised form 5 November 2017; Accepted 5 November 2017 Available online 06 November 2017 0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
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Hua, and Lin (2014) with minor modification. Briefly, 8.4 mL of TMP was mixed with 10.0 mL of 5.0 M HCl and 31.6 mL of deionized water and shaken at 40 °C in the dark for 30 min to obtain MDA through acidic hydrolysis. After that, the pH was adjusted to 7.4 with 6 M NaOH. Then take a small amount of stock solution to dilute to 10− 5, and the concentration of MDA in the stock solution was estimated by absorbance at 267 nm using a molar extinction coefficient value of 31,500 M− 1 cm− 1 (Adams, De Kimpe, & van Boekel, 2008).
proteins has different contribution to the binding ability. In soy protein isolations, β-conglycinin component might be the main protein fraction responsible for the off-flavor binding by soy proteins (Damodaran & Kinsella, 1981a, 1981b). It is well known that flavor-protein binding interactions can be altered by protein modification. However, most investigations focused on flavor compound chemical structure and medium in aqueous model systems, like ionic strength, temperature and pH. A little information is still available concerning the modification of proteins. For instance, the effect of succinylation of soy protein on the binding of 2-nonanone was studied, which suggested that there are about two binding sites for 2nonanone in succinylated soy compared to four binding sites in the native protein (Damodaran & Kinsella, 1981a). Except for the above, the partial deamidation of soy protein isolate (SPI) by protein-glutaminase decreased overall flavor-binding affinity for both vanillin and maltol by approximately 9- and 4-fold (Suppavorasatit & Cadwallader, 2012). Soy protein is an important food ingredient used in a lot of protein-based food. Soy protein oxidation occurred during processing and storage due to oxidative attack of lipid peroxidation-derived free radicals as well as lipid hydroperoxides and reactive aldehydes. In our previous study, the effects of SPI oxidation on their structure had been evaluated. SPI was modified by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) or lipid peroxidation product malondialdehyde (MDA) both had important influence on their structural and functional characteristics (Chen, Zhao, & Sun, 2013; Chen, Zhao, Sun, & Zhao, 2013). However, those studies did not assess the impact of oxidation on the flavor-binding properties of the protein. Therefore, the objective of the present study was to investigate the effect of protein oxidation on the binding of selected carbonyl-containing flavor compounds to SPI in an aqueous system using SPME combined with GC/MS.
2.4. Modification of SPI with MDA Control and MDA-modified SPI were prepared according to the method described by Wu, Zhang, and Hua (2009) SPI suspension (40 mg/mL containing 0.5 mg/mL sodium azide, suspended in 10 mM sodium phosphate buffer, pH 7.4) was mixed with a serial concentration of MDA and then incubated by continuous shaking under air at 25 °C in dark for 24 h to react sufficiently. The final concentrations of MDA were 0, 0.5, 1.0, 2.5, 5.0 and 10.0 mM. The freeze-dried SPI without shaking treated sample was control SPI, and the others were MDA-modified SPI. After that, the MDA-modified SPI suspension was dialyzed against deionized water at 4 °C for 72 h to remove free MDA and salt. Then the dialyzed solution was freeze-dried and stored at 4 °C until used.
2.5. Evaluation of oxidative changes 2.5.1. Carbonyls The content of carbonyls was determined by the reaction with 2,4 dinitrophenylhydrazine (DNPH) using a UV765 spectrophotometer (Shanghai, China), as described by Huang, Hua, and Qiu (2006). twenty milligram samples were suspended in 10 mL of 10 mM sodium phosphate buffer (pH 7.0), stirred for 1 h at room temperature, then centrifuged at 10000 g for 15 min at 4 °C. Took the supernatant and determined the protein concentration by using the Biuret method. Then, in 10 mL capped polyethylene centrifuge tubes, 1 mL SPI solution was mixed with 3 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) dissolved in 2 M HCl and incubated at room temperature for 2 h. And a same amount of SPI solution mixed with 3 mL of 2 M HCl was an absorbance blank. The DNPH-reacted samples after 20% trichloroacetic acid (TCA) precipitation were collected by centrifugation at 10000 g for 15 min at 4 °C and then washed three times with 5 mL of ethanol/ethyl acetate solution (1:1, v/v). The final pellets, free of DNPH, was dissolved in 3 mL of 6 M guanidine hydrochloride in 0.1 M sodium phosphate buffer (pH 7.0). The absorbance at 367 nm was corrected by the absorbance in the HCl blank. The results were calculated using a molar extinction coefficient of 22,000 M− 1 cm− 1 (Boatright & Hettiarachchy, 1995) and expressed as nanomoles of carbonyl group per milligram of protein.
2. Materials and methods 2.1. Samples and materials Defatted soy flakes were purchased from Yuwang Group (Shandong, China). 1,1,3,3-Tetramethoxypropane (TMP, purity 98%) and 8Anilino-1-naphthalenesulfonic acid (ANS) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) and bicinchoninic acid (BCA) protein assay kit were purchased from Dingguo (Beijing, China). The aroma compounds hexanal, (E)-2-Hexenal, (E)-2-Octenal, (E)-2-Nonenal were purchased from Sigma − Aldrich (Steinheim, Germany). 1-Octen-3-ol and nonanal were obtained from Aladdin (Shanghai, China). All other chemicals were of analytical reagent grade and obtained in China. 2.2. SPI preparation Defatted soy flake powder (200 g) was mixed with 15-fold (in weight) deionized water, and the mixture (pH 6.8) was adjusted to pH 8.0 with 2.0 M NaOH. After stirring for 2 h, the resulting suspension was centrifuged at 8000 g for 20 min at 4 °C to remove the insoluble material. Then the pH of the supernatant was adjusted to 4.5 with 2.0 M HCl, and the precipitate was collected by centrifugation at 8000 g for 20 min at 4 °C. The precipitate was then redissolved with 5-fold (in weight) deionized water, and the pH was adjusted to 7.0 with 2.0 M NaOH, maintaining the pH to 7.0 and stirring until protein was completely dissolved. Then the neutral SPI solution was dialyzed against deionized water at 4 °C for 48 h and then freeze-dried and stored at 4 °C until use.
2.5.2. Surface hydrophobicity The hydrophobicity was determined using 1-anilino-8-naphthalenesulfonate (ANS) as fluorescence probe (Kato & Nakai, 1980). SPI (500 mg) was suspended in 100 mL 10 mM sodium phosphate buffer (pH 7.0), stirred thoroughly at room temperature. Then centrifuged at 10000 g for 15 min at 4 °C and collected the supernatant. The protein concentration in the supernatant was evaluated using a BCA protein assay kit, and then diluted the supernatant to serial concentration with the same buffer in the range of 0.005–0.5 mg/mL. After that, 4 mL of protein dilution was mixed with 50 μL ANS solution (8 mM in 0.01 M, pH 7.0 phosphate buffer). Fluorescence intensity (FI) was measured at 365 nm (excitation) and 484 nm (emission) on a F7000 fluorescence spectrophotometer (Hitachi Co., Japan). The curve was fitted with fluorescence intensity and protein concentration, and the initial slope of which was used an index of protein surface hydrophobicity (H0).
2.3. Preparation of MDA stock solution MDA stock solution must be made fresh daily, and a fresh MDA stock solution was prepared by hydrolyzing1,1,3,3-tetramethoxypropane (TMP) according to the method described by Wu, 151
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concentrations were 420 mg/L for hexanal, 450 mg/L for trans-2-hexenal, 425 mg/L for trans-2-octenal, 390 mg/L for 1-octen-3-ol, 415 mg/ L for nonanal, 525 mg/L for trans-2-nonenal. The selection of the six flavor compounds was firstly based on their relatively large proportion of all the flavors in traditional soymilk. Furthermore, hexanal, trans-2hexenal and 1-octen-3-ol are representative of beany flavors, while trans-2-octenal, nonanal and trans-2-nonenal are non-beany flavors (Lv, Song, Li, Wu, & Guo, 2011). And the six flavor compounds differ in chain length and function group.
2.5.3. Fluorescence spectra of tryptophan and Schiff base Control and MDA-modified SPI samples (500 mg) were suspended in 100 mL 10 mM sodium phosphate buffer (pH 7.0) and magnetically stirred thoroughly at room temperature. Then the SPI suspension was centrifuged at 10000 g for 15 min and collected the supernatant. And the protein concentration in the supernatant was determined by using a BCA protein assay kit. Then, protein concentration was separately adjusted to 0.1 mg/mL and 0.2 mg/mL for the measurement of tryptophan and Schiff base. To determine the fluorescence emission spectrum of tryptophan, the excitation wavelength was set at 285 nm, and the emission spectrum was recorded from 300 to 400 nm (Chen, Zhao, Sun, Ren, & Cui, 2013). And for Schiff base, the excitation wavelength was set at 350 nm, and the emission spectrum was recorded from 400 to 600 nm (Utrera, Rodríguez-Carpena, Morcuende, & Estévez, 2012). The slit width was set at 5 nm, and data were collected at 240 nm/min.
2.6.2. Preparation of GC/MS samples Samples were prepared by transferring 6.86 mL of SPI suspensions (0.715%) into 20 mL headspace vials, then 140 μL of the flavor stock solution was added to obtain a final flavor concentration of 8.4 mg/L for hexanal, 9 mg/L for trans-2-hexenal, 8.5 mg/L for trans-2-octenal, 7.8 mg/L for 1-octen-3-ol, 8.3 mg/L m for nonanal, 10.5 mg/L for trans2-nonenal. The blank vial contained exactly the same buffer instead of protein. All the vials were immediately sealed with a PTFE-faced silicone septum (Supelco, Bellefonte, PA) and equilibrated for 24 h at 4 °C. All the samples were prepared in duplicate.
2.5.4. Particle size distributions Particle size distribution was measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcestershire, United Kingdom), aimed to observe the aggregates in oxidized SPI with different degree. Briefly, referred to the method of Wu, Hua, Lin, and Xiao (2011) with minor modification, SPI (100 mg) was suspended in 100 mL 10 mM sodium phosphate buffer (pH 7.0) at a concentration of 1 mg/mL and was magnetically stirred thoroughly. Then the samples were centrifuged at 10000 g for 15 min at 4 °C and the supernatants were collected. Before measurement, the supernatants were filtered through nylon membranes with pore size of 0.45 μm to remove any insoluble particles. Then 1 mL of protein solution was injected into a cuvette that marked by triangle for DLS measurement.
2.6.3. SPME and GC/MS analysis After equilibration, the SPME fiber was punctured into the headspace of the sample vial and was immediately transferred into the gas chromatograph injector port for quantification. The SPME parameters were as follows: 75 μm carboxen/polydimethylsiloxane (CAR/PDMS) fiber (Supelco, Bellefonte, PA), equilibrated at 45 °C for 20 min, extracted at 45 °C for 40 min, desorbed at 250 °C for 3 min. GC–MS conditions: TG-5 ms (30 m × 0.25 mm × 0.25 μm) column was used for separation. The carrier gas was high purity helium (1.0 mL/min). The mass spectrometry conditions were electron bombardment ionization (EI) ion source, electron energy 70 eV, electron multiplication Device voltage 350 V, transmission line temperature 250 °C, ion source temperature 250 °C, scanning speed 3.00 scans/s, mass range 33–350 m/z. Liquid injection program temperature conditions: the initial temperature 40 °C, rose to 120 °C in 2 min at 4 °C/min, keep for 2 min, then rose to 280 °C at 10 °C/min, keep for 10 min; injection volume was 1 μL; injection temperature 230 °C.
2.5.5. SDS–PAGE analysis Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) was performed on a discontinuous buffered system (Laemmeli, 1970), using 12% separating gel and 5% stacking gel. Briefly, 100 μL soy protein isolation suspensions (20 mg/mL in 10 mM phosphate buffer at pH 7.0) were mixed with 400 μL sample buffer with and without 5% β-mercaptoethanol (β ME). The ones with β ME were heated for 5 min in boiling water, while the others were heated for a few seconds in boiling water. Then both were centrifuged at 10000 g for 10 min before electrophoresis. For each sample, 10 μL of the supernatant was loaded to each lane. A prestained protein marker I with ten bands (170 kD, 130 kD, 95 kD, 72 kD, 55 kD, 43 kD, 34 kD, 26 kD, 17 kD, 10 kD) was used as reference. Those bands are purified protein mixtures and are covalently conjugated to the dye. Among them, the band of 72 kD is an orange band and 10 kD is a green band. After the electrophoresis, the gel was stained using the Coomassie brilliant blue R-250 stain solution (0.1% Coomassie brilliant blue R-250, 10% acetic acid, 45% methanol) for 1.0 h and destained by methanol/acetic acid/ water (1:1:8, v/v/v) for 24 h with two or three changes of destain solution.
2.7. Statistical analysis Statistical calculation was analysed using the statistical package SPSS 20 (SPSS Inc., Chicago, IL) for one-way ANOVA. Least-squares difference was used for comparison of mean values among treatments and to identify significant differences (p < 0.05) among treatments. Triplicate preparations of soy protein isolations were carried out to confirm the accordance. Data were expressed as means ± standard deviations (SD) of triplicate determinations unless specifically mentioned. 3. Results and discussion
2.5.6. Microstructure The superficial morphology of oxidized proteins were observed by field emission scanning electron microscopy (FE-SEM, LEO-Merlin, Germany) at an accelerator voltage of 5 kV. Before imaging, the freezedried protein powder was adhered to a small piece of iron with a double-sided adhesive tape and then plated with gold (JEOL JFC-1200 fine coater, Japan).
3.1. Oxidative modifications of soy protein isolations 3.1.1. Carbonyls Carbonyl groups are commonly used to characterize the interaction of proteins with various oxidizing systems. Effects of MDA modification on the carbonyl group content of SPI are given in Fig. 1A. In this work, the carbonyl group content of the control protein was 2.33 nmol/mg protein, which is close to the reports of Wu, Zhang, Kong, and Hua (2009) on soy protein isolation oxidized by peroxyl radicals. Compared with the control, the treatment for the 0 mM that was shaked for 24 h at 25 °C made negligible differences. However, in the presence of MDA, the carbonyls group content increased as the MDA concentration increased. Between 0–1 mM, carbonyl content increased linearly, then from 2.5–10 mM, it increased sharply.
2.6. Flavor binding to control and MDA-modified protein 2.6.1. Preparation of protein and flavor stock solutions 0.715% SPIs suspensions were prepared in 10 mM sodium phosphate buffer (pH 7.0) and magnetically stirred thoroughly. A stock solution containing each flavor compound was prepared in methanol and stored in brown glass bottles to prevent decomposition. Their 152
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Fig. 1. Carbonyl content (A), protein surface hydrophobicity (B) of the soy protein isolations upon oxidation by MDA (0–10 mM). For the letters a–f, means (n = 3) without a common letter differ significantly (p < 0.05).
3.1.3. Fluorescence spectra of tryptophan and Schiff base Fluorescence spectroscopy has been recently supposed to be a simple, fast, and solvent-free method for characterizing protein oxidation in foods by means of tryptophan loss and Schiff base formation (Rui, Estévez, Kylli, Heinonen, & Morcuende, 2010). In this work, the oxidizing agent MDA has two reactive aldehyde groups, which forms a Schiff base complex preferentially with primary amino groups such as εamines of lysine residues. In addition, MDA induces cross-linking proteins and produces fluorescent adducts such as DHP–lysine (Lamore et al., 2010; Zhao, Chen, Zhu, & Xiong, 2012). The intrinsic fluorescence of tryptophan and Schiff base in control and MDA modified SPI are shown in Fig. 2. As shown in Fig. 2, with the increasing concentration of MDA, the loss of tryptophan increased, accompanied by the increasing of Schiff base. At relatively low addition of MDA (0–0.5 mM), both tryptophan and Schiff base were not changed much, but it showed significant differences from 1–10 mM. Interestingly, in Fig. 2A, with the MDA
3.1.2. Surface hydrophobicity Hydrophobicity is known to be significantly related to the structure and functional properties of proteins (Kato & Nakai, 1980). Fig. 1B shows the surface hydrophobicity of the control and oxidized SPI. The surface hydrophobicity of 0 mM was slightly higher than the control. For the oxidized proteins, the surface hydrophobicity increased with the increasing MDA concentration. The above results were consistent with the trend of carbonyl growth. Meanwhile, the solubility of the oxidized SPI in 10 mM sodium phosphate buffer (pH 7.0) decreased with the increasing MDA concentration. This was in accordance to the reports of Chen et al. (2013). This result might be attributed to the unfolding of soy protein isolates when subjected to MDA modification, which led to the exposing of hydrophobic amino acids buried in protein inside, and thus enhanced protein surface hydrophobicity.
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Fig. 2. Fluorescent emission spectra of tryptophan (A) and Schiff base (B) of control and increasing concentration of MDA modified SPI.
concentration up to 2.5 mM, the λmax of fluorescence emission underwent a slight red shift up to 10 mM MDA. The red shift might be attributed to the changes in the microenvironment of the protein in the aqueous solution due to oxidation.
This result was in agreement with the previous studies (Chen et al., 2013). And it indicated that SPI modified by low MDA concentration (≤ 2.5 mM) induced the formation of some soluble aggregates. Further increasing extend of oxidation could promote insoluble component formation and cleavage of peptide bonds by peroxyl radicals (Davies, 2005). Therefore, SPI modified by 5 and 10 mM MDA might also induced some insoluble and soluble aggregates, the former were removed by centrifugation, while the later were some lager aggregates and broke into smaller soluble peptides.
3.1.4. Particle size distributions The average particle size and size volume distribution for the control and MDA-modified SPIs were used to evaluate the aggregation behavior of proteins during oxidation, and the results are shown in Fig. 3. In this work, only soluble component of sample was investigated. It can be clearly seen from the form in the Fig. 3, with the increasing of MDA concentration, the average particle size decreased, accompanied by the decreasing of the particle dispersibility index (PdI). However, for the size volume distribution, SPI gradually shifted to larger particle size within MDA ≤ 2.5 mM, while at the concentration between 5 and 10 mM, it shifted to smaller particle size and was smaller than the control.
3.1.5. SDS–PAGE The aggregation behavior of MDA-modified SPI was further studied by SDS-PAGE analysis (Fig. 4). In the Fig. 4A, in low MDA concentration (≤ 1 mM), the α′ and α subunits of β-conglycinin became less intense, with further oxidation (≥ 2.5 mM), they disappeared. However, the β subunit became more intense, it might be due to the complexes formed by β subunit and basic subunits (Utsumi, Damodaran, & 154
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Fig. 3. Particle size distribution of control and MDA-modified SPI. Average particle size with different letters (a–g) are significant difference (p < 0.05).
linkage (Tezuka, Yagasaki, & Tomotada, 2004), we speculated that the acid subunits interacted with each other or other subunits through disulfide bonds thus resulted in soluble aggregates, which appeared on the top of separating gel. Meanwhile, as for the low MDA modified SPI (≤ 1 mM), high molecular weight polymers formed on the top of the separating gel, which were formed by disulfide bonds. In the Fig. 4B, with the addition of 5% β-ME, the disulfide bonds in proteins were destroyed, characteristic bands for the subunits of βconglycinin and glycinin were presented. It could be clearly seen that, no significant change of electrophoretic pattern was observed among the first four samples (control SPI and those incubated with 0, 0.5, 1.0 mM MDA), whereas there was a gradual disappearance of several subunits in a MDA dose-dependent manner among those incubated with 2.5, 5.0, 10.0 mM MDA. Furthermore, at 10 mM MDA, the α′ subunits of β-conglycinin mostly went, while the subunits of glycinin just reduced. These phenomena demonstrated that β-conglycinin was more sensitive to MDA modification than glycinin. Simultaneously, upon oxidation, high molecular weight polymers formed and accumulated on the top of the separating gel and stacking gel. The formation of these polymers was attributed to the interaction of subunits through nondisulfide covalent bonds as the disulfide bonds were destroyed by β-ME. These results demonstrated the occurrence of cross-linking under oxidation, which was consistent with the changes in particle size distributions as previously mentioned.
3.1.6. Microstructure The changes in the physicochemical properties of oxidized SPI were further investigated by the observation of their morphologic properties. A set of SEM images of oxidized SPI at a magnification factor of 1000-fold are showed in Fig. 5. It could be clearly seen that, there were many small fragments in 0 mM sample. With the increasing MDA concentration, small fragments decreased, followed by the formation of larger fragments. At high MDA (≥ 5 mM), large fragments were stacked with a few small fragments on their surface. These morphological changes might be caused by the unfolding of the SPI molecules, and increased exposure of hydrophobic groups (Fig. 1B) at the surface of the molecules, which could induce protein-protein interactions and form larger aggregates during oxidation.
Fig. 4. Representative SDS− PAGE patterns of control and MDA-modified SPI (A) without and with 5% β-ME. Lanes: M, marker proteins; C, control SPI(without incubation at 25 °C for 24 h); 0, 0.5, 1, 2.5, 5 and 10, SPI treated with 0, 0.5, 1, 2.5, 5 and 10 mM MDA. SPI constituents: α′, α, and β, subunits for β-conglycinin; A and B, acidic subunits and basic subunits for glycinin.
Kinsella, 1984), since the basic subunits largely became less intense. In addition, the acid subunits disappeared completely, while presented in reduction electrophoresis. Since each subunit in glycinin consists of an acidic (A) and basic (B) polypeptide chain connected by a disulfide 155
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Fig. 5. Scanning electron microscope micrographs at × 1000 magnification of the control and MDA-modified soy protein isolations. Scale bars indicate 10 μm.
investigations, who found that the unsaturated aldehydes were retained more strongly in soy protein than the correspongding saturated ones (Gremli, 1974). In addition, trans-2-hexenal has been shown to exhibit covalent binding with milk proteins (Meynier, Rampon, Dalgalarrondo, & Genot, 2004). For the oxidized SPIs, at low MDA concentration (≤1 mM), the percentages of all flavor compounds decreased, demonstrating the increased binding affinity of oxidized protein. Nevertheless, with the further increasing in MDA concentration (> 1 mM MDA), an inverse effect on the percentages of flavor compounds was observed for hexanal, trans-2-hexenal, 1-octen-3-ol, nonanal and trans-2-nonenal. Interestingly, for nonanal and trans-2-nonenal, both of which were bound to a gradual decreasing extent, especially for nonanal. While for hexanal, trans-2-hexenal and 1-octen-3-ol, the binding affinity began to increase, and then decreased. Although the high MDA-modified SPIs showed a decrease in binding affinity, the changes were not marked except for nonanal. The samples treated at high MDA concentration (≥ 2.5 mM) contained sediment of aggregated proteins, whereas the samples treated at low MDA concentration (≤ 1 mM) remained clear. At the MDA concentration between 0 and 1 mM, the increasing binding affinity with the increasing concentration of MDA might be due to the partial exposing of hydrophobic amino acids buried in protein inside. And this was consistent with changes in protein surface hydrophobicity (Fig. 1B). While for the decrease in binding of hexanal and nonanal to SPI upon high concentration of MDA(≥ 2.5 mM), it might be explained by the increase in the extent of soluble and insoluble aggregations of
3.2. Flavor binding to control and MDA-modified protein On the basis of the physicochemical properties changes of oxidized SPI by MDA, the effect of structural changes of SPI on their binding ability with selected flavor compounds was evaluated. Thus, the free percentages of each flavor compound in the headspace of the control and oxidized SPIs after 24 h equilibrium at 4 °C were determined, the result was based on a blank (each flavor compound mixed with 10 mM sodium phosphate buffer, pH 7.4 instead of protein) of 100%. As shown in Fig. 6, the presence of the control SPI produced a remarkable reduction of the percentages of free hexanal, trans-2-hexenal, trans-2-octenal, nonanal, trans-2-nonenal, suggesting that the control SPI was able to bind those aroma compounds. In contrast, the increase in 1-octen-3ol implied its releasing behavior. Furthermore, compared to hexanal, control SPI showed a higher binding affinity for nonanal. And among trans-2-hexenal, trans-2-octenal, trans-2-nonenal, the binding affinity for control SPI increased with an increase in the chain length. This result was in agreement with the previous study (Damodaran & Kinsella, 1981b). trans-2-Hexenal exhibited a higher binding affinity for control SPI than hexanal, and the same consequence is also observed between nonanal and trans-2-nonenal. It indicated that the presence of a double bond further increased the binding affinity of flavor compounds for control SPI. trans-2-Nonenal is less hydrophobic than nonanal (Kühn et al., 2008) but is bound to a higher extent. This suggested that possible interactions between flavors and protein are not only hydrophobic in nature, but also might involve the double bond (“Michael addition”). This observation was in agreement with the previous
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Fig. 6. Effect of MDA modification on the binding of flavor compounds. Results are expressed as percentage of free flavor compounds found in the headspace without protein in solution.
unfolded SPI, making the flavor binding site inaccessible (Kuhn, Zhu, Considine, & Singh, 2007). And for trans-2-hexenal, trans-2-octenal and trans-2-nonenal, no significant changes in binding of MDA (≥2.5 mM) modified SPI might result from the combination of both a decrease in binding attributed to the destruction of the hydrophobic pocket and an increase in binding caused by covalent interactions between aggregated protein molecules with these unsaturated aldehydes (Kuehn, Considine, & Singh, 2008). This was in agreement with the results of SDS-PAGE (Fig.4).
Acknowledgements The authors are grateful to the Special Support Project of Guangdong Province for Science and Technology Innovative Young Talents (2014TQ01N538), Pearl River S&T Nova Program of Guangzhou (201610010105) and the Fundamental Research Funds for the Central Universities (2015ZM155) for their financial supports. References Adams, A., De Kimpe, N., & van Boekel, M. A. J. S. (2008). Modification of casein by the lipid oxidation product malondialdehyde. Journal of Agricultural and Food Chemistry, 56(5), 1713–1719. http://dx.doi.org/10.1021/jf072385b. Arthur, C. L., & Pawliszyn, J. (1990). Solid phase microextraction with thermal desorption using fused silica optical fibers. Analytical Chemistry, 62(19), 2145–2148. Boatright, W. L., & Hettiarachchy, N. S. (1995). Effect of lipids on soy protein isolate solubility. Journal of the American Oil Chemists' Society, 72(12), 1439–1444. Chen, N., Zhao, M., & Sun, W. (2013). Effect of protein oxidation on the in vitro digestibility of soy protein isolate. Food Chemistry, 141(3), 3224–3229. Chen, N., Zhao, M., Sun, W., Ren, J., & Cui, C. (2013). Effect of oxidation on the emulsifying properties of soy protein isolate. Food Research International, 52(1), 26–32. Chen, N., Zhao, Q., Sun, W., & Zhao, M. (2013). Effects of malondialdehyde modification on the in vitro digestibility of soy protein isolate. Journal of Agricultural and Food Chemistry, 61(49), 12139–12145. Damodaran, S., & Kinsella, J. E. (1980). Flavor protein interactions. Binding of carbonyls to bovine serum albumin: Thermodynamic and conformational effects. Journal of Agricultural and Food Chemistry, 28(3), 567–571. Damodaran, S., & Kinsella, J. E. (1981a). Interaction of carbonyls with soy protein: Conformational effects. Journal of Agricultural and Food Chemistry, 29(6), 1253–1257. Damodaran, S., & Kinsella, J. E. (1981b). Interaction of carbonyls with soy protein: Thermodynamic effects. Journal of Agricultural and Food Chemistry, 29(6), 1249–1253. Davies, M. J. (2005). The oxidative environment and protein damage. Biochimica et Biophysica Acta, 1703(2), 93–109. Gremli, H. A. (1974). Interaction of flavor compounds with soy protein. Journal of the American Oil Chemists' Society, 51(1), 95A–97A. Huang, Y. R., Hua, Y. F., & Qiu, A. Y. (2006). Soybean protein aggregation induced by lipoxygenase catalyzed linoleic acid oxidation. Food Research International, 39(2), 240–249. http://dx.doi.org/10.1016/j.foodres.2005.07.012. Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochimica et Biophysica Acta, 624(1), 13–20. Kuehn, J., Considine, T., & Singh, H. (2008). Binding of flavor compounds and whey protein isolate as affected by heat and high pressure treatments. Journal of
4. Conclusions The effects of flavor compound structure and MDA-induced oxidative modifications on the interactions of SPI and flavor compounds were elucidated. The binding between native SPI and the selected flavor compounds was strong and decreased in the order trans-s-undecenal > aldehyde > olefinic alcohol. Furthermore, both the binding affinity of aldehyde and trans-s-undecenal for SPI increased with an increase in the chain length. The flavor compounds with different structure are bound to proteins on different binding sites and/or by different binding mechanisms. Upon low MDA concentration (≤ 1 mM), the structure was unfolded with hydrophobic groups exposed and thus increased the surface hydrophobicity, which is beneficial to the hydrophobic interactions between flavor compounds and SPI. In addition, the aldehyde group would react with specific amino acids residues by covalent interaction in the unfolded proteins. Whereas, high concentration of MDA-induced denaturation (≥2.5 mM) reduces hydrophobic interactions of SPI with flavor compounds, like hexanal and nonanal. Covalent reactions of the double bond with certain amino acid residues might be more readily accessible in the aggregated proteins. For next study, we will separate 7 s and 11 s, using fluorescence quenching as well as isothermal titration calorimetry to further elaborate its binding mechanism with flavor compounds.
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