Functional alteration of soybean 11S globulin through glycation

Abstract

Glucose-conjugated soybean 11S globulin was prepared by mixing soybean 11S globulin and glucose at a ratio of 1:11.7 (w/w) and incubating at 52 °C and 38 000 relative humidity for up to 72 h. The glycated soybean globulin in 15 mM phosphate buffer (pH 6.4) without NaCl was almost insoluble at 0 h, but solubility increased to approximately 68% at 36 h and 40% at 72 h. During this reaction, the available lysine content rapidly decreased, whereas the quantity of fructosamine increased, indicating glucose binding to soybean 11S globulin. Glucose-conjugated soybean 11S globulin after the 72 h reaction showed a hydroxyl radical antioxidant capacity of approximately 8.2 µmol gallic acid equivalent/g protein, and the average particle size of its emulsions tended to be smaller than that of the soybean 11S globulin, resulting in superior stability of its emulsions compared to native globulin emulsions.

Introduction

Soybean protein finds widespread application in the food industry owing to its excellent nutritional, processing, and biofunctional properties (Yamauchi, 1994). To further enhance some of these properties, functional alteration of soybean proteins through glycation using the Maillard reaction has been studied by several researchers. Achouri et al. (2005) reported enhanced solubility, emulsifying activity, and foaming of glucose-conjugated soybean 11S globulin during the early and middle stages of the Maillard reaction. Tian et al. (2011) found that glycation enhanced the solubility of soybean protein isolate (SPI) over a wide pH range. In addition, the emulsifying ability and emulsion stability of glucose-conjugated SPI were considerably higher than those of commercial emulsifiers, especially at high salt concentrations and neutral pH. Li et al. (2019) demonstrated that unfolding of SPI secondary structure occurs during the Maillard reaction, which consequently increases its molecular flexibility, resulting in improved emulsifying properties of SPI due to increased molecular flexibility and hydrophilicity due to glycation. Furthermore, through several studies (Nishimura et al. 2011; Isono et al., 2012; Nishimura and Saeki, 2016; Nishimura and Saeki, 2018a; Nishimura and Saeki, 2018b; Nishimura et al., 2019; Nishimura et al., 2020; Nishimura and Saeki, 2021), we have previously revealed that protein glycation can confer antioxidant capacity to chicken myofibrillar proteins.

Thus, if soybean 11S globulin can acquire antioxidant capacity through glycation, it could be applied as an emulsifier with antioxidant properties. To this end, in this study, we investigated the possibility of conferring similar functional alterations, such as increased solubility and emulsifying properties, as well as antioxidant activity, to soybean 11S globulin through glycation.

Materials and Methods

Materials and chemicals    Soybeans were purchased from the Fujiya Katsuobushi Store (Kyoto, Japan). Glycated human serum was purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid (GA) was obtained from ChromaDex Inc. (Irvine, CA, USA). All other chemicals were of reagent grade and obtained from Nacalai Tesque, Inc. (Kyoto, Japan) or Wako Pure Chemicals Industries, Ltd. (Osaka, Japan).

Preparation of defatted soybeans    The dried soybeans were ground in a mill. Then, a chloroform:methanol mixture (2:1) was added at 20 mL per g soybean powder, and the mixture was homogenized using a hand homogenizer. The mixture was centrifuged at 1 170 ×g for 10 min at 20 °C and the supernatant was removed. The chloroform: methanol mixture (2:1) was again added at 10 mL per g pellet, and the mixture was stirred vigorously for 1 min using a vortex mixer. After centrifugation at 1 170 ×g for 10 min at 20 °C, the supernatant was removed. The precipitate was dried on filter paper and used as defatted soybean powder.

Preparation of soybean 11S globulin    Soybean 11S globulin was prepared according to the method described by Vu and Shibasaki (1976). Tris-HCl buffer (30 mM, pH 8.0) containing 10 mM 2-mercaptoethanol was added to the defatted soybean powder (20 mL/g) and mixed at room temperature for 1 h to extract the major globulins. Following centrifugation at 24 870 ×g and 20 °C for 30 min, the supernatant was adjusted to pH 6.4 with HCl and stored at 4 °C overnight. The 11S globulin was precipitated via centrifugation at 24 870 ×g at 4 °C for 30 min. Phosphate buffer (15 mM, pH 7.0) was added to dissolve the 11S globulin to give a protein concentration of approximately 20 mg/mL. The solution was then transferred to a dialysis tube with 14 kDa molecular weight cutoff and dialyzed against 15 mM phosphate buffer (pH 7.0) for 24 h at 3 °C to remove the 2-mercaptoethanol. This final solution was used as the 11S globulin preparation.

Glycation of soybean 11S globulin    Soybean 11S globulin was glycated following the method described by Saeki (1997). The prepared soybean 11S globulin solution was mixed with glucose at a ratio of 11.7 mg glucose/mg protein. The final protein concentration was adjusted to 6.0 mg/mL, and 5 mL aliquots of the soybean 11S globulin-glucose mixture were transferred to test tubes (16 mm diameter), frozen at −80 °C, and immediately lyophilized using a freeze-dryer (FDU-1110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). Lyophilization was terminated when the temperature of the samples reached 15-18 °C. Each lyophilized protein powder was immediately stored at −40 °C and used within 30 days of preparation. The Maillard reaction between soybean 11S globulin and glucose was performed by incubating the lyophilized powder at a temperature of 52 °C and a relative humidity (RH) of 38% for up to 72 h, based on the optimal conditions determined previously for glucose conjugation of chicken myofibrillar proteins (Nishimura et al., 2019). An incubator/humidity cabinet (KCL-2000A, Tokyo Rikakikai Co., Ltd.) was used to control temperature and RH.

Solubility of glycated soybean 11S globulin    After glycation, the protein powder was mixed immediately with plain 15 mM sodium phosphate buffer (pH 6.4) or buffer containing 0.5 M NaCl and incubated at 8-12 °C overnight. After reaching a final protein concentration of 1.5 mg/mL, the mixture was dispersed using a high-speed blender (T-10 basic Ultra-Turrax, IKA-Labortechnik, Staufen, Germany) at 13 500 rpm for 0.5 min, and this step was repeated. Homogenization was followed by centrifugation at 32 000 ×g for 30 min at 4 °C, and the protein content before centrifugation and in the supernatant was determined. Total soluble soybean 11S globulin was expressed as the percentage of protein content in the supernatant relative to the total protein content prior to centrifugation.

Determination of available lysine and fructosamine    The amounts of available lysine and fructosamine were determined to monitor the progress of the Maillard reaction between soybean 11S globulin and glucose. Glycated soybean 11S globulin was dissolved in 15 mM sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl using a high-speed blender (T-10 basic Ultra-Turrax, IKA-Labortechnik) and precipitated with 7.5% trichloroacetic acid in ice water for 30 min. After centrifugation at 1 000 ×g for 30 min at 4 °C to remove the buffer, unreacted sugars, and trichloroacetic acid, the precipitated protein was reconstituted in 50 mM sodium phosphate buffer (pH 9.5) containing 2% sodium dodecyl sulfate (Saeki, 1997). The available lysine content was determined by spectrophotometric analysis using o-phthalaldehyde and N-acetyl-L-cysteine (Hernandez and Alvarez-Coque, 1992).

Similarly, glycated soybean 11S globulin dissolved in 15 mM sodium phosphate buffer (pH 7.5) containing NaCl (0.5 M) was dialyzed against the same buffer at 8–12 °C overnight to remove any unreacted glucose. Fructosamine, a ketoamine product of protein glycation, was assayed using the method described by Johnson et al. (1983). Glycosylated human serum was used as standard to determine the fructosamine content. Assays for determining the available lysine and fructosamine concentrations were performed immediately after sample preparation.

Measurement of hydroxyl radical antioxidant capacity (HORAC)    The protein powder after glycation was mixed immediately with 15 mM sodium phosphate buffer (pH 7.0) and dialyzed using a membrane with a 40–50 Å pore size in the same buffer solution at 8–12 °C overnight to remove any unreacted glucose. The dialysate was centrifuged at 32 000 ×g for 30 min at 4 °C and the supernatant containing 10–12 mg/mL protein was used as the working solution.

The HORAC of each protein sample was measured on a chemiluminescence reader (AccuFLEX Lumi400, Aloka Co. Ltd., Tokyo, Japan) using an assay based on the Fenton reaction, wherein the hydroxyl radical (·OH) reacts with luminol, resulting in light emission (Yildiz and Demiryürek, 1998; Parejo et al., 2000). Aliquots of the samples were harvested to measure their antioxidant capacity. Initially, 50 µL cobalt solution and 50 µL luminol solution were mixed with 20 µL sample solution and incubated for 8 min at 37 °C. The generation of ·OH was initiated by adding 50 µL H₂O₂ solution. Light emission at 430 nm was measured for 120 s immediately after initiation, and the light emissions from 80 to 120 s were integrated. Sodium phosphate buffer (15 mM, pH 7.0) was used as control. The residual ratio of ·OH was calculated for each sample, and antioxidant activity was measured as RLU_sample/RLU_control × 100, where RLU is relative light units.

Emulsifying properties    The emulsifying properties of soybean 11S globulin and glucose-conjugated soybean 11S globulin were measured using the method of Matsumura et al. (2003), with a few modifications. An oil-in-water emulsion was prepared using 5 wt% oil phase and 95 wt% aqueous phase.

Glycated soybean 11S globulin dissolved in 15 mM sodium phosphate buffer (pH 7.0) was dialyzed against the same buffer at 8-12 °C overnight to remove any unreacted glucose. The supernatant containing 3 mg/mL protein obtained after centrifugation at 32 000 ×g for 30 min at 4 °C was used as the aqueous phase. The oil phase consisted of corn oil. The oil and aqueous phases were mixed and homogenized for 3 min in a high-speed blender (Microtec Co., Ltd., Chiba, Japan) at 15 000 rpm. The average droplet diameter was further reduced using an ultrasonic homogenizer (WakenBtech Co., Ltd., Kyoto, Japan) operated at maximum power for 5 min.

The emulsifying properties of the protein samples were analyzed by measuring the particle size distribution and mean particle diameter using a laser diffraction particle size analyzer (SALD-2200, Shimadzu Co., Ltd., Kyoto, Japan). The refractive index was set at 1.40-0.05i. The stability of the emulsions was analyzed by sealing the vials containing the emulsions and maintaining them at 4 °C without agitation, and by visually observing them immediately after emulsification, at one week, and at four weeks.

Protein content determination    The dissolved soybean 11S globulin protein content was determined using the Kjeldahl method (AOAC 1990). The protein concentrations of soybean 11S globulin and glycated soybean 11S globulin preparations used to measure available lysine and emulsifying properties were determined as described by Lowry et al. (1951). The biuret method (Gornall et al., 1949) was used for other assays, with bovine serum albumin used as a protein standard.

Statistical analyses    Statistical analyses were performed on the results obtained from three independent glycated soybean 11S globulin samples. The results are presented as mean ± standard deviation of three replicates. Differences were tested using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons to compare multiple groups. Linear regression analysis was used to describe the concentration-dependent antioxidative effects of proteins. Statistical analyses were performed using Microsoft Excel 365 with Ekuseru–Toukei software (Social Survey Research Information Co. Ltd., Tokyo, Japan). Statistical significance was set at p < 0.05.

Results and Discussion

Changes in solubility and amounts of available lysine and fructosamine in soybean 11S globulin during glycation    Lyophilized soybean 11S globulin-glucose mixtures at a weight ratio of 1:11.7 (w/w) were incubated at 52 °C and 38% RH for up to 72 h for glycation. The solubility of glucose-conjugated 11S globulin was measured in 15 mM sodium phosphate buffer (pH 6.4) containing no or 0.5 M NaCl (Fig. 1A); pH 6.4 was selected as the protein precipitates at this pH under low ionic strength. Subsequent changes in the amount of available lysine and fructosamine were also examined (Fig. 1B and C). The solubility of glycated 11S globulin in 0.5 M NaCl (pH 6.4) was 87.0 ± 5.6% (n = 3) at 0 h, and the high solubility was maintained for up to 24 h. Subsequently, the value gradually decreased to reach 43.3 ± 18.7% (n = 3) after 72 h. In contrast, the solubility in the absence of NaCl (pH 6.4) started at 14.9 ± 2.1% (n = 3) and rose with increasing reaction time to reach 67.8 ± 1.1% (n = 3) after 36 h, almost matching the solubility in 0.5 M NaCl (pH 6.4). On longer incubation, the value gradually decreased, similar to the change in 0.5 M NaCl solution, reaching 40.3 ± 2.5% (n = 3) after 72 h (Fig. 1A).

Fig. 1.

Changes in solubility of soybean 11S globulin and amounts of available lysine and fructosamine during glycation.

Lyophilized soybean 11S globulin-glucose mixtures at a weight ratio of 1:11.7 (w/w) were incubated at 51.6 °C and 38.1% RH for up to 72 h. Solubility (A) was measured in 15 mM sodium phosphate buffer (pH 6.4) containing no NaCl (●) or 0.5 M NaCl (○). Available lysine (B) and fructosamine (C) were measured simultaneously. Results are expressed as the mean ± standard deviation (n ≥ 3).

The available lysine decreased rapidly during the first 6 h to reach 34.1 ± 2.4% (n = 4), and then decreased gradually to 18.9 ± 4.4% (n = 3) at 48 h. Finally, the value increased to 32.0 ± 3.8% (n = 3) at the end of the reaction (Fig. 1B).

In contrast, the quantity of available fructosamine increased with reaction time to reach 220.9 ± 35.1 µmol/g of protein (n = 3) after 24 h, and subsequently decreased to reach 134.2 ± 37.7% (n = 3) at 72 h (Fig. 1C).

The increase in solubility of glycated soybean 11S globulin (Fig. 1) in the solution without NaCl (pH 6.4) was associated with an increase in fructosamine content, indicating that the protein gains high solubility on glycation independently of salt concentration. The water solubilization of glucose-conjugated soybean 11S globulin at pH 6.4 could be attributed to an increase in protein hydrophilicity. Meanwhile, soybean 11S globulin initially showed high solubility in 0.5 M NaCl, which decreased after 24 h. At 36 h, the solubility was similar to that in the absence of NaCl and continued to decrease. This phenomenon was likely the result of protein denaturation and aggregation caused by the long incubation period.

During the initial 6 h of the reaction, the available lysine content decreased (Fig. 1B), whereas the fructosamine content increased (Fig. 1C). This indicated that the lysine residue on soybean 11S globulin was conjugated with glucose to rapidly produce fructosamine via the Maillard reaction. Hodge (1953) confirmed that the Amadori rearrangement product decomposes into amino compounds, opson, and reductones when the Maillard reaction progresses to the middle stage. This progression could have caused the increase in the amount of available lysine after 48 h observed in this study, which leads to an increase in unmodified amino groups. In addition, the decrease in the amount of fructosamine after 36 h could be associated with the progression of the Maillard reaction to the middle stage.

The correlation between changes in the amount of available lysine and fructosamine suggested the formation of glucose-conjugated soybean 11S globulin, which was confirmed by the shortening of 11S soybean globulin mobility with time using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown).

HORAC measurement in the glucose-conjugated soybean 11S globulin    The antioxidant capacity of the glucose-conjugated soybean 11S globulin was measured at 72 h. Native soybean 11S globulin barely demonstrated any HORAC in the concentration range 0.66–1.36 mg/mL (Fig. 2).

Fig. 2.

Effect of native or glycated soybean 11S globulin concentration on HORAC.

The antioxidant activity of soybean 11S globulin against ·OH was independently measured three times using a fluorescence method at a reaction time of 72 h in 15 mM sodium phosphate buffer (pH 7.0). The symbols (○) and (●) represent the residual concentration of ·OH (%) for native soybean 11S globulin dissolved in 15 mM sodium phosphate buffer (pH 7.0) and glycated soybean 11S globulin at 72 h reaction time, respectively, reported relative to soybean 11S globulin concentration (mg/mL). Results are presented as mean ± standard deviation (n = 3).

The inhibition curve of HORAC values associated with glucose-conjugated soybean 11S globulin at 72 h reaction time is shown in Fig. 2. The production of ·OH decreased with increasing protein concentration, with a 40.3 ± 6.6% (n = 3) reduction achieved at a protein concentration of 1.13 mg/mL. The half-maximal inhibitory concentration was 0.80 ± 0.17 mg/mL, as calculated from Fig. 2. The ·OH production of glycated soybean 11S globulin at a reaction time of 72 h was estimated via linear regression analysis to be lower than that of native soybean 11S globulin at the tested protein concentrations. This could be simply validated by the following calculation: Yglycated soybean 11S globulin at 72 h reaction time − Ynative soybean 11S globulin = (−33.855X + 77.935) − (−22.614X + 101.01) = −11.241X − 23.075 < 0 (X > 0).

The HORAC of the glycated soybean 11S globulin from 72 h reaction was converted into GA equivalent activity (µmol/mL) based on the half-maximal inhibitory concentration of GA (6.4 ± 0.1 nmol/mL (Nishimura and Saeki, 2018a)). Consequently, a value of 8.2 ± 1.6 µmol GA equivalent/g protein was obtained. However, this glycated protein lost its solubility in water (pH 6.4) at 72 h, suggesting that one of the functional alterations did not occur. It is therefore necessary to search for conditions that result in antioxidant activity without the loss of solubility in water (pH 6.4).

Emulsifying properties    Oil-in-water emulsions were prepared by combining soybean 11S globulin solution with corn oil, as described in the Materials and Methods, and their particle sizes were measured to evaluate emulsifying ability of the protein (Fig. 3).

Fig. 3.

Particle size distribution of the emulsion of native or glycated soybean 11S globulin.

The emulsifying ability of the native and glycated proteins was analyzed by measuring the particle size distribution and calculating the mean droplet diameter of the emulsion samples using a light-scattering instrument. A, B, and C show the particle size distribution of the emulsions prepared using native soybean 11S globulin, and using glycated soybean 11S globulin at 36 h and 72 h reaction time, respectively. Results are expressed as the mean ± SD of three independent experiments.

The average particle size of emulsions prepared using native soybean 11S globulin was 2.36 ± 0.42 µm (n = 3) (Fig. 3A). The emulsions prepared with glycated soybean 11S globulin after 36 h and 72 h reactions showed particle sizes of 1.84 ± 0.03 µm (n = 3) and 1.86 ± 0.09 µm (n = 3), respectively (Fig. 3B and C). Although there was no significant difference between the non-glycated and glycated samples, oil droplets larger than 10 µm only appeared in the non-glycated sample, resulting in a smaller average particle size of glycated sample emulsions. This can be attributed to changes in the structure of soybean 11S globulin with progress in glycation, especially unfolding of the acidic and basic subunits (or at the level of quaternary structure). These changes may lead to the rapid spread of the subunits at the oil-water interface, resulting in high emulsification efficiency and smaller particles (Fig. 3). Moreover, the increase in the number of OH groups owing to glycation likely resulted in an increase in hydrophilicity, which contributed to the formation of a thick and strong emulsifier membrane at the oil droplet surface. Such a membrane may resist the coalescence of oil droplets during continuous homogenization, thereby generating emulsions with a smaller average particle size. Emulsion droplets of smaller size are generally believed to be associated with better emulsions (Prak et al., 2015). Therefore, the stabilities of the emulsions generated from soybean globulin were investigated.

Emulsions prepared with native (Fig. 4A) and glycated soybean 11S globulin at 36 h (Fig. 4B) and 72 h reaction time (Fig. 4C) were added to vials (35.0 mm diameter × 81.5 mm long) and sealed, and their stability was observed visually at 4 °C for up to four weeks. Immediately after emulsification, all of the emulsified oil droplets were well dispersed. However, the emulsion of native soybean 11S globulin separated completely into water and cream phases after one week. In contrast, glycated soybean 11S globulin emulsions with reaction times of 36 h and 72 h did not separate completely, even after four weeks. These results indicate that glycated soybean 11S globulin efficiently forms stable emulsions, suggesting that emulsion stability is improved by glycation. Peng et al. (2018) reported improved emulsifying properties on conjugating soy-soluble polysaccharides to soybean 11S globulin using the Maillard reaction. Glucose introduced into protein molecules is known to protrude into the aqueous phase to form a thick hydration layer, which precludes close contact of oil droplets, thereby preventing their aggregation and coalescence (Dickinson, 2003; O'Mahony et al., 2017). Furthermore, it is generally believed that the smaller the average particle size, the more stable is the emulsion (Wang et al., 2019). Thus, the improvement in emulsion stability of glycated 11S globulin (Fig. 4) could be attributed to the above reasons.

Fig. 4.

Emulsion stability of native or glycated soybean 11S globulin.

The stability of the emulsions was analyzed by sealing the emulsions in vials and maintaining them at 4 °C without agitation, with visual observation immediately after emulsification, one week, and four weeks. The emulsion stability of soybean 11S globulin and all glycated soybean 11S globulins was determined in three independent experiments. Average emulsion stability of each soybean 11S globulin sample is shown. A, B, and C show the states of native soybean 11S globulin and glycated globulin after 36 h and 72 h reaction, respectively.

These results suggest the potential of using glycation to develop an emulsifier with antioxidant properties from soybean protein. This will be further investigated in our future studies.

Acknowledgements    This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 18K02195) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest    There are no conflicts of interest to declare.

Abbreviations

GA

gallic acid

HORAC

hydroxyl radical antioxidant capacity

·OH

hydroxyl radical

RH

relative humidity

SPI

soybean protein isolate

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