Physicochemical property, antioxidant activity, and cytoprotective effect of the germinated soybean proteins

Abstract Appropriate germination can improve the nutritional value and bioactivity of soybeans; however, few studies have assessed the effect of germination on soybean proteins. This study examined the physicochemical property, antioxidation, and cytoprotective effect of the germinated soybean proteins (Gsp). Gsp was extracted from soybeans which germinated for 0–3 days using the method of alkali‐solution and acid‐isolation extraction. The results showed that germination could digest soybean proteins into the smaller molecules; enhance the degree of hydrolysis, emulsifiability, and foaming capacity; increase the removal rate of ABTS, DPPH, O2−˙, and ˙OH radical; and decrease the reducing power and lipid peroxidation of Gsp. Additionally, Gsp was able to protect HL‐7702 human hepatocyte cells against benzo(a)pyrene (BaP)‐induced cytotoxicity through mediating the cell cycle arrest, suppressing apoptosis, and increasing reactive oxygen species (ROS) levels. This work demonstrated that germination could enhance the physicochemical property and antioxidant activity of Gsp, which also displayed the remarkable cytoprotective effect. This study provided a fundamental basis for substantiating dietary of Gsp used for resistance to oxidation and hepatic injury.


| SDS-PAGE electrophoresis of Gsp
The protein concentration of Gsp was determined using BCA kit. Protein samples were diluted in Laemmli buffer with 5% β-mercaptoethanol and then boiled for 5 min prior to loading. Finally, Gsp were separated by SDS-PAGE and exposed using a Molecular Imager ChemiDoc 510 system (UVP Corporation, USA).

| Sulfhydryl and disulfide bond contents measurement
Sulfhydryl (SH) and disulfide bond (S-S) contents of Gsp were measured according to the method of Jia, Huang, and Xiong (2016). SH and S-S were determined by the Equations (1) and (2) as follows: where A 412 is the absorbance at 412 nm, C is the protein concentration (mg/ml), and 73.53 is derived from 10 6 /13,600 (13,600 is Ellman's reagent molar absorptivity). SH T is the free sulfhydryl group. SH F is the total sulfhydryl group.

| Determinations of degree of hydrolysis
The degree of hydrolysis (DH) of Gsp was determined using the modified OPA method described by Nielsen, Petersen, and Dambmann (2010). DH% was calculated using the following equations (Equation (3)-(5)).
where X = g sample, P = protein % in sample, 0.1 is the sample volume in liter (L), h = number of hydrolyzed bonds (meqv/g protein), h tot = total number of peptide bonds per protein equivalent (7.8 specific to soy protein), β = 0.342 (specific for soy protein), and α = 0.970 (specific for soy protein).

| Water and oil holding capacities
Water and oil absorption capacities were determined according to the method of Zhang, Yang, Tang, Chen, and You (2015). 2 g of samples was mixed with 20 ml distilled water or corn oil (Sigma) in 50-ml centrifuge tubes. Each mixture was shocked for 1 min, allowed to stand for 30 min, and then centrifuged at 2,000 × g for 30 min. The results were expressed as ml of liquid retained per g of sample.

| Protein solubility at different pH levels
Protein solubility was determined by dispersing samples in distilled water to obtain a final solution of 0.2% (w/w) in protein.
The pH values of protein solution were adjusted from 8 to 3 and then were centrifuged at 10,800 × g for 30 min. The content of protein in the resulting solution was analyzed using BCA protein kit.

| Analysis of amino acids
Samples were acid hydrolyzed at 110°C for 24 hr in 6 M HCl in vacuum-sealed tubes. Amino acids levels were determined by an automatic amino acid analyzer (L-8900, Hitachi, Ltd., Japan).

| Emulsifiability and emulsifying stability
The emulsifying activity index (EAI) and emulsion stability index (ESI) of Gsp were measured following the method by Pearce and Kinsella (1978) and were calculated using the following equations (Equation 6 and 7): where V 0 refers to the total volume of Gsp solution samples, and V 1 and V 2 are the volume of emulsion layer and the volume of emulsion layer after standing.

| Foaming capacity and foam stability
The method of Zhang, Zhang, Wang, and Guo (2012) was used to evaluate foaming capacity (FC) and foaming stability (FS) of Gsp.
They were calculated using the following equations (Equation 8 and 9): where V 0 refers to the volume before whipping, V 1 is the volume after whipping, and V 2 is the volume after standing.

| DPPH radical scavenging assay
DPPH radical scavenging activity of Gsp was evaluated according to the method reported by Alam, Bristi, and Rafiquzzaman (2013) and calculated using the following equation (Equation 10): where A 0 refers to the absorbance without Gsp, and A 1 is the absorbance in the presence of Gsp. EC 50 value (mg/ml) obtained by interpolation from linear regression analysis represents the effective concentration at which DPPH radicals were scavenged by 50%.

| ABTS radical scavenging assay
ABTS radical scavenging activity was measured using the method developed by Re et al. (1999). It was calculated as follows (Equation 11): where A 0 refers to the absorbance without Gsp, and A 1 is the absorbance in the presence of Gsp. EC 50 value (mg/ml) obtained by interpolation from linear regression analysis represents the effective concentration at which ABTS radicals were scavenged by 50%.

| O
scavenging activity was measured based on the described method (Bajpai, Baek, & Kang, 2017) and calculated using the following equation (Equation 12): where A control was the absorbance without sample, and A sample was the absorbance with sample. EC 50 value (mg/ml) obtained by interpolation from linear regression analysis represents the effective concentration at which O − 2˙ radicals were scavenged by 50%.

| ˙OH scavenging activitẏ
OH radical generated by the Fe 3+ /ascorbic acid system has been studied by the method of Stamenic et al. (2014), and it was calculated as follows (Equation 13): where A 0 is the absorbance of the control sample, and A 1 is the absorbance of sample. The EC 50 value (mg/ml) obtained by interpolation from linear regression analysis represents the effective concentration at which ˙OH radicals were scavenged by 50%.

| Reducing power assay
The reducing power of Gsp was determined using the ferric reducing ability of plasma assay (Almansoub, Asmawi, & Murugaiyah, 2014).
A standard curve was prepared using different concentrations (10-100 mmol/L) of FeSO 4 . Reduction percentage was calculated as fol-

lows (Equation 14):
where As is the absorbance of the sample, and Ac is the absorbance of the standard at maximum concentration. EC 50 value (mg extract per ml) obtained from the linear regression analysis represents the effective concentration at which the absorbance is 0.5 at 593 nm.

| Determination of lipid peroxidation
Malondialdehyde (MDA) is one of the end products of lip peroxidation, and it was determined according to Szőllősi, Varga, Erdei, and Mihalik (2009). Gsp was mixed with 10% (w/v) trichloroacetic acid and 0.5% (w/v) thiobarbituric acid, heated for 15 min at 96°C, then cooled down to room temperature, and centrifuged at 3,600 × g for 5 min. Absorbance of the supernatant was read at 532 nm and 600 nm, respectively. The value of MDA was expressed as μmol/g.

| Antioxidant activity of Gsp after digestion in vitro
Antioxidant activity of Gsp after digestion in vitro was measured according to the method proposed by Shim et al. (2012). Briefly, pepsin and trypsin reacted with proteins at 37°C water bath for 4 hr, respectively, adjusted pH to 7.0. Hydrolyzate was used for the determination of antioxidant capacity (DPPH, ABTS, and ˙OH) through the above method.

| Cell culture
HL-7702 human hepatocyte cells were obtained from Kunming Institute of Zoology, Chinese Academy of Science, and were cultured in RPMI-1640 medium with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator (5% CO 2 , 95% air).
Subsequently, 100 μl of 0.5% (w/v) MTT was added to each well and incubated for 4 hr. Then, removed MTT and added DMSO to dissolve the formazan crystals. Absorbance at 490 nm was measured with a microplate reader (Thermo Fisher, USA).

| Cell cycle analysis
Cell cycle was analyzed using flow cytometry. After treatment, the detached cells in the medium were collected with PBS and combined with the remaining adherent cells. After centrifugation, cell pellets were resuspended and fixed with 70% ethanol at 4°C overnight, and then resuspended in 1.0 mg/ml RNase (Sigma). Subsequently, 50 μg/ml propidium iodide (PI, Sigma) stain solution was added, incubated in the dark for 30 min at room temperature, and finally analyzed by the GUAVA ® easyCyte™ 8HT flow cytometry (Millipore Corporation, USA).  Notes. Values shown are mean ± SD, n = 9. The means in the row not sharing a common letter ( abc ) are significantly different among groups (p ≤ 0.05). † Solubility determined in DW (deionized water at pH 7.0), 0.05 M Tris-HCl buffer (pH 8.0), or 0.05 M Gly-HCl (pH 3.0).

| Assessment of cell apoptosis
Early and late apoptotic cells were detected using an Annexin V-

| Measurement of ROS
Reactive oxygen species (ROS) was measured using DCFH-DA probes. After treatment, cells were incubated with 5 μmol/L of DCFH-DA at 37°C with 5% (v/v) CO 2 for 30 min. Subsequently, cells were washed three times with serum-free medium, and the fluorescence intensity in cells was determined using fluorescence microscope (Olympus Optical Co., Ltd., Japan) and flow cytometry, respectively.

| Statistical analysis
All experiments were performed three times, and data are presented as the means ± standard errors (SE). Significant differences between measurements for the control and treated samples were analyzed using one-way factorial analysis of variance and Duncan's post hoc test with SPSS 16.0.

| Effect of germination on the chemical composition of soybean protein
The secondary structure and chemical composition of Gsp were changed by germination. As shown in Table 1 Notes. Values shown are mean ± SD, n = 9. EAA: total essential amino acid; NEAA: no essential amino acid; TAA: total amino acid; -: not detected. a Essential amino acid. b Conditionally essential amino acid.
TA B L E 2 Composition of amino acid in the germinated soybean proteins (mg/g.pr) S-S content. As the continuation of germination, DH and water absorption capacity of Gsp increased gradually, while oil absorption capacity appeared declined. For the solubility of Gsp in different pH values, the lowest solubility was at pH 7.0, and the highest solubility was at pH 3.0.

| SDS-PAGE of Gsp
As shown in Figure 2, the small molecular weight proteins in control group were slightly lighter than that in the 3-d germinated samples.

| Functionality properties of Gsp
Emulsifying activity index (EAI) and emulsifying stability index (ESI) of Gsp were notably (p ≤ 0.05) increased by germination, while no significant differences were been found between 2-d and 3-d germination ( Figure 3A,B). Besides, foaming capacity (FC) showed a gradual and significant (p ≤ 0.05) increase, and foaming stability (FS) showed a contrary tendency ( Figure 3C,D).
All these results showed that germination was able to enhance the functionality properties of soybean proteins.

| Antioxidant activities of Gsp
As shown in Figure 4, with the increase in germination time, the removal rate of ABTS, DPPH, O − 2˙, and ˙OH radical, decrease in the reducing power, and inhibition rate of lipid peroxidation of Gsp showed a gradual and significant (p ≤ 0.05) increment trend. Table 3 shows that compared

| Effect of Gsp on BaP-induced injury in HL-7702 cells
A significant (p ≤ 0.01) cytoprotective effect of Gsp on HL-7702 cells induced by BaP was observed at 63 mg/L, compared to the untreated cells ( Figure 6). It was found that cells viability notably (p ≤ 0.01) increased with Gsp concentrations, indicating that Gsp could protect liver cells from BaP-induced injury.

| Effects of Gsp on BaP-induced cell cycle arrest in HL-7702 cells
As shown in Figure 7, a strong S-phase arrest in HL-7702 cells was been Note. Values shown are mean ± SD, n = 9. The means in the row not sharing a common letter ( abc ) are significantly different among groups (p ≤ 0.05).

| Effects of Gsp on BaP-induced apoptosis in HL-7702 cells
As shown in Figure 8, BaP resulted in about 4.47% of the cells going into early apoptotic phase, and 45.49% of cells was going into late apoptotic phase. However, both the early and late apoptotic cells were significantly decreased by Gsp treatment. Taken together, these results revealed that the pretreatment with Gsp could protect HL-7702 cell from BaP-induced apoptosis.

| Effects of Gsp on ROS in BaP-treated cells
Accumulating evidence indicates that intracellular ROS can trigger apoptosis, we next determined whether Gsp protected HL-7702 cell from BaP-induced apoptosis through decreasing the generation of ROS. As shown in Figure 9, cells exposed to BaP displayed a significant increase in fluorescence intensity signals, compared with control group. Gsp markedly inhibited the generation of intracellular ROS induced by BaP, indicating that Gsp could inhibit apoptosis induced by BaP through decreasing intracellular ROS levels.

| D ISCUSS I ON
Soybean has various biological functions; however, the effects of germination on the physicochemical properties, antioxidant activity, and cytoprotective properties of soy protein remain unclear. The present study found that germination could not only change secondary structure of soybean proteins, but also promote physicochemical properties and antioxidant activity. In addition, Gsp could effectively inhibit cell damage induced by BaP.
Gsp percentage decreased slightly as the germination time goes on (Table 1); it may be because some protein was metabolized during germination.
It has reported that germination can change the secondary structure of soybean proteins, and protein was digested into smaller molecules and reused for new protein synthesis for plant growth during germination (Chen & Chang, 2015).  (Table 2). Lys content was slightly increased after germination, and no significant effect was found in Asp and Glu.
The functionality properties (EAI and FC) of protein are very important for the practical performance as emulsifying agents.
Emulsifier is used to stabilize the emulsion depends on the oil-water interfacial area, and the emulsifying capacity also depends on the size of the droplets during agitation (Ritzoulis et al., 2014). In food systems, foams are often very complex, including several phases such as a mixture of gases, subdivided solids, subdivided liquids, multicomponent solutions of water, polymers, and surfactants (Richert, 1979). The high FC and low FS of Gsp were observed in the present investigation (Figure 3), and it may have been due to the formation of stable molecular layers in the air-water interface, which impart texture, stability, and elasticity of foams.
Antioxidant capacity is the most extensively investigated bioactivity in germinated edible seeds and sprouts, and it has reported that germination can change the antioxidant capacity in many edible seeds, such as wheat, rice bean, and so on (Chen et al., 2017;Sritongtae, Sangsukiam, Morgan, & Duangmal, 2017  remains unknown. In our study, the result found that Gsp effectively eliminated DPPH, ABTS, O − 2˙, and ˙OH free radicals, and decreasing reducing power and lipid peroxidation (Figure 4), showing that germination is able to strengthen antioxidant activity of soy proteins.
Additionally, to examine whether antioxidant capacity of Gsp will decrease or disappear in body, we simulated the digestive reaction in vitro. The results showed that, after digestion with pepsin and trypsin, Gsp also had a high clearance rate for DPPH, ˙OH, and reducing power (Figure 5), and it was in agreement with a report that Indian bean proteins digested by pepsin and trypsin also showed a high clearance rate for different radicals (Vadde, Pochana, & Pillatla, 2010). All the results suggest that Gsp possessed the notable antioxidant activity.
BaP is universally acknowledged to be a cancerogen. It is widely presented in a large number of high-temperature-processed foods and leads to the severe organism injury. It is reported that plant flavonoids and polyphenols can prevent from BaP damage (Kasala et al., 2016;Omidian, Rafiei, & Bandy, 2017;Sreelatha, Jeyachitra, & Padma, 2011); however, there were few reports about whether Gsp could protect cells from BaP-induced damage. Our study found that Gsp could protect HL-7702 cells from BaP through increasing cell viability, relieving cell cycle arrest, inhibiting apoptosis, and reducing ROS levels ( Figure 6-9), indicating a significant hepatoprotective effect.

| CON CLUS ION
The present work demonstrated that germination could digest soybean proteins into the smaller molecules and enhance functionality properties and antioxidant activity of soy proteins.
Additionally, Gsp could protect cells from damage induced by BaP through increasing cell viability, inhibiting cell cycle arrest and apoptosis, and reducing ROS levels, showing that Gsp could be used for resistance to oxidation and hepatic injury, as a functional foods and dietary supplements. However, we only primarily assessed the cytoprotective properties of Gsp in this paper, the possible underlying mechanisms required to research in further studies. the authors would like to express their appreciation for the assistance provided.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflict of interests.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.