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Grain & Oil Science and Technology 3 (2020) 100–109

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Establishment of two-dimensional gel electrophoresis for soybean protein isolate and its application

Xinkai Lu, Yaoming Cui, Junjun Guan ⁎ , Xue Liu, Hao Zhu, Xuyang Ji, Jianzhang Zheng,

Yunlong Cheng, Xiaofei Fu

College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China

⁎ Corresponding author. E-mail address: [email protected]. (J. Guan).

http://dx.doi.org/10.1016/j.gaost.2020.02.002 2590-2598/© 2020 Henan University of Technology. the CC BY-NC-ND license (http://creativecommons.o

A B S T R A C T

A R T I C L E I N F O

Article history: Received 13 October 2019 Received in revised form 5 February 2020 Accepted 19 February 2020

To optimize the conditions for the establishment of two-dimensional gel electrophoresis (2-DE) of soy- bean protein isolate (SPI), we investigated Ampholine mixture, anodic and cathodic electrolytes, load- ing amount of sample, acrylamide concentration, pH gradient and gel staining method in two- dimensional gel electrophoresis to optimize the protein imaging conditions in two-dimensional gel. The results of mixed-level design experiments showed that Ampholine, loading amount and gel staining method had significant effect (P < 0.05) on 2-DE of SPI. The optimal conditions were Ampholine mix- ture (pH 3–10 + pH 5–7 or pH 4–6 + pH 5–7), loading amount of 2 mg sample and silver staining. Al- though the acrylamide concentration of the gel, the pH gradient, the anodic and cathodic electrolyte solutions had significant statistical effects on the protein separation degree, the complexity of the pro- tein composition and the visibility of the gel images were more inclined to the 12% gel, the 3–10 pH gradient and the H3PO4/NaOH electrolyte. According to the established conditions, the hydrolyzed products of SPI emulsion were determined by 2-DE, and the dynamic changes of protein in the process of enzymatic hydrolysis were described.

Keywords: Two-dimensional gel electrophoresis Soybean protein isolate Enzymatic hydrolysate Soybean protein isolate emulsion

1. Introduction

As an important plant protein source, soybean proteins with balanced amino acid composition, physiologically beneficial components and excellent processing ability, such as gelling [1], are a complex polymorphic mixture of polypeptides, containing four groups of proteins: enzymes, structural proteins, membrane proteins and storage pro- teins. Usually, relevant foods of soybean proteins have var- ious forms such as tofu, fermented soybean paste, etc. [2].

Soybean protein isolate (SPI) is a highly purified form of soybean proteins, often made from defatted soybean flour, and commonly used in the food industry. The main ingredi- ents of SPI are classified into four protein categories: 2S, 7S, 11S and 15S, among which 7S (β-conglycinin) and 11S (glycinin) account for more than 80% [3]. 7S globulin

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consists of three subunits of ɑ, ɑ′ and β. 11S globulin is a hexamer, and made up of five different subunits, each of which consists of an acidic subunit A with a molecular mass about 35 kDa, and a basic subunit B linked by a disul- fide bond with molecular mass about 20 kDa [4].

Due to the hydrophilic and lipophilic properties of pro- tein molecules, the interfacial properties of soybean pro- tein, such as the foaming, emulsifying, and gel properties, have attracted the interests of researchers. These functional properties are often applied to the food, so how they affect the digestion and absorption of the food gradually becomes a new research focus in recent years, and moreover the en- zymatic digestion of soybean protein emulsion is an impor- tant aspect [5,6].

However, with the development of soy proteins, much basic information about these proteins, such as the most

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X. Lu et al. Grain & Oil Science and Technology 3 (2020) 100–109

important primary and/or secondary physiological func- tions, is still being studied.

Therefore, protein including molecular structure changes is the key to the above research. Currently, proteo- mics is thought of a relatively new set of tools to be used for protein separation and protein identification [7]. In proteo- mics, three methods are preferred for separation of protein or peptide samples: denaturing polyacrylamide gel electro- phoresis (PAGE), two-dimensional gel electrophoresis (2- DE), and liquid chromatography (LC) including ion ex- change, affinity, and reverse-phase chromatography [8].

2-DE is the combination of isoelectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and can give more higher resolution than either procedure alone [9]. One-dimensional gel is used according to the iso- electric point of protein (pI) to separate dissolved proteins or peptides. By isoelectric focusing and then applied to the top of the two-dimensional gel, the proteins in the one- dimensional gel are migrated to the two-dimensional gel by electrophoresis and separated according to the molecu- lar weight of the protein.

In IEF, proteins are isolated due to their differences in isoelectric point in a gel. There are two important variants of IEF, immobilized pH gradient (IPG)-based IEF [10] and non-IPG-IEF [11]. IPG-based IEF has been used in conven- tional 2-DE methods for over 30 years, and is often used in the study of proteins introduced into immobilized pH gradient gels. A pH gradient has been established in the gel, and proteins are first separated by their pIs. Non-IPG- LEF is an adjustable locally scaled pH gradient in IEF. By selecting appropriate cathode and anode pairs, the ideal ca- thodic and anodic migration pH gradient can be effectively achieved, the protein precipitation and quantitative uncer- tainty existing in conventional LEF and 2-DE can be elimi- nated, and the solution and sensitivity of IEF can be improved, especially for the high resolution separation of many protein isomers and low abundance proteins.

However, there are few reports about soybean protein in 2-DE, which mainly focus on the analysis of soybean seeds [12–14], and the operating conditions of 2-DE in reports were based on the 2-DE of non-food protein such as medical protein, whose structural characteristics were different from the food proteins such as soybean protein. Therefore, the systematic study of the operating conditions of 2-DE on soybean protein is still needed. Hence, in this paper, the principal purposes are to explore optimization condi- tions for establishing 2-DE of soybean protein by using the IPG-IEF as the first-dimension, and these optimized condi- tions which simulate the digestion in vivo can be better applied to the analysis of the enzymatic hydrolyzate of soy- bean protein emulsion (one of the hotspots in the field of food) so that a feasible way could be provided to reveal the relationship between the proteomics characteristics of

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soybean protein and solve the problem of how to use prote- omics to improve food protein effective application in food.

2. Materials and methods

2.1. Materials

Soybean was purchased from Dennis supermarket, Zhengzhou, Henan province, China, and stored at 5 °C until using. Pepsin was purchased from Sangon Biotech (Shanghai) Co., Ltd. Chemicals for electrophoresis, in- cluding acrylamide, bis-acrylamide, SDS, N,N,N′,N′- tetramethylethylendiamine (TEMED), ammonium per- sulfate, were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. Urea and ampholytes (pH 3–10, pH 4–6, pH 5–7) were purchased from Solarbio. Tris- HCl (pH 8.8), 2-mercaptoethanol, glycerol, agarose (low melting), and Triton X-100 were purchased from Sangon Biotech (Shanghai) Co., Ltd. All other chemicals were standard reagent grade laboratory chemicals. Deionized water was used for making all solutions.

2.2. Methods

2.2.1. Preparation of soybean protein isolate (SPI) As previously described [1], soybean was crushed,

passed through 80 mesh sieve, and defatted with ethyl ether. 10 g defatted soybean flour obtained was added into 100 mL distilled water, stirring to disperse it completely and adjusting pH to 8.0 by 2 mol/L NaOH. As the temperature of the water increases, the solubility in- creases. So after water bath heating with stirring at 50 °C for 1 h, the dispersion was centrifuged at 5,000 × g at 4 °C for 30 min. The supernatant was obtained and its pH was adjusted to 4.8 by 2 mol/L HCl, after storing at 4 °C overnight, it was centrifuged at 5,000 × g at 4 °C for 30 min. The precipitation obtained was washed with dis- tilled water for three times and dispersed in distilled water again, adjusting the pH to 7.0 with 2 mol/L NaOH. After dialysis and freeze drying of dispersion, soybean pro- tein isolate was obtained. The protein content of SPI deter- mined by the method of Kjeldahl [15] was 94% in this study.

2.2.2. Preparation of simulated gastric fluid 2 g NaCl, 7 mL HCl, and 0.32 g pepsin (10,000 U/mg)

were mixed and added with distilled water to 1,000 mL, adjusting pH to 1.2 with 1 mol/L HCl [16] to simulate the condition of the gastric fluid.

2.2.3. Preparation of soybean protein emulsion 1.6 g SPI was added to the 80 mL distilled water,

stirred at room temperature for 2 h, mixed with 20 mL soybean oil, and homogenized for 120 s at 20,000 r/min

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by a homogenizer (Model FA25, produced by Fluko, Germany) to protect the protein so that wrapped protein is not digested when passing through the stomach, and can be absorbed within the intestinal tract.

2.2.4. Enzymatic reaction and enzyme hydrolysate preparation 100 mL soybean protein emulsion was mixed with

100 mL simulated gastric fluid at 37 °C, stirring at 90 r/min for 2 h. During this process, the pH of mixture was remained at 1.2 by 1 mol/L HCl, and samples were taken regularly, heated at 80 °C for enzyme inactivation, and placed in a refrigerator overnight at −20 °C, then thawed at room temperature, thus contributing to break the emulsion, and finally centrifuged at 10,000 ×g for 30 min at 4 °C in order to separate oil from solution. The upper oil was removed and the residue was freeze-dried. The obtained protein powder was mixed with ethyl ether to remove the residual oil as much as possible. After being dried, the enzyme hydrolysates were obtained for the fol- lowing experiments. We studied the effect of enzymatic hy- drolysates of soybean protein emulsion on two-dimensional electrophoresis of protein detection.

2.2.5. 2-DE and image analysis We conducted two-dimensional electrophoresis experi-

ments by referring to the O'Farrell's method [17] with some modification. On the basis of a large number of exper- iments in the previous years, it was found that the protein bands were clear when the acrylamide concentration in two dimensional gel was 12% and 15%. Therefore, this ex- periment only analyzed the concentration of acrylamide in two dimensional gel by 12% and 15%, isoelectric focusing gels were made in glass tubes (120 × 2.5 mm inside diam- eter). The gel mixture composed of 2.75 g urea, 1 mL Triton X- 100 (10% V/V), 0.665 mL of acrylamide stock (28.38% W/ V), 0.985 mL ddH2O and 0.25 mL Ampholines (40% W/V) (to make 2% (V/V) Ampholines). 5 mL gel mixture were added to the flask, swirled to let the urea dissolve completely, then 10 μL 10% (W/V) ammonium persulfate was added, and the solution was degassed for about 1 min. Immediately after addition of 3 μL of TEMED, the solution was loaded into the gel tube with the space from the top, and was not filled full, and overlaid with gel-overlay solu- tion (8 mol/L urea) for 1 to 2 h, then this overlay solution was removed and replaced with 20 μL of lysis buffer, composed by 9.5 mol/L urea, 2% (V/V) TritonX-100, 2% (V/V) Ampholines and 5% (V/V) β-mercaptoethanol, over- laid with a small amount of water. After being allowed to set for 1 to 2 h, the gels were placed in a standard tube gel electrophoresis chamber with the solutions of anodic and ca- thodic electrolytes, composed with 0.01 mol/L H3PO4 or glycine in the lower reservoir and 0.02 mol/L NaOH or argi- nine in the upper reservoir. Gels were then pre-run

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according to the following schedule: (1) 200 V for 30 min; (2) 300 V for 30 min; (3) 400 V for 1 h. Then the power was turned off, and the samples were loaded and overlaid with 10 μL of sample overlay solution, composed with 9 mol/L urea and 1% (V/V) Ampholines. The gels were run at 400 V for 3 h, then at 800 V for 13 h and at 1,500 V for 2 h.

After the first dimension, the IEF strips were equilibrated for about 1–2 h in equilibration buffer, composed by 50 mmol/L Tris-HCl (pH 6.8), 6 mol/L urea, 30% (V/V) glycerol, 2% (W/V) SDS, 1% (V/V) β-mercaptoethanol, and immediately loaded on polyacrylamide gels (130 × 135 × 1.5 mm). The second-dimension electrophoresis was per- formed by the reference [18]. Electrophoresis was carried out without exceeding 35 mA per gel and with a voltage in- creasing gradually to 350 V.

After migration, gels were stained by Coomassie blue G250 and silver stain protocol [19].

The 2-DE gels were scanned using a JS-680D image scan- ner (Hangzhou Deju Equipment Co., Ltd. Hangzhou, China), and image analysis was performed with PDQuest 2-D analy- sis software (ver. 8.0, Bio-Rad Laboratories, Inc., USA).

2.3. Experimental designs

According to a large number of experiments in the early stage, we designed the different levels of concentrations of acrylamide, pH range of Ampholines, kinds of anodic and cathodic electrolytes, loading amount of sample, pH gradi- ent in IEF, staining methods, which all have great influence on two-dimensional electrophoresis.

A mixed-level design (4 × 2 × 2) experiment was used for the factors of Ampholines at 4 levels (Ampholines pH 4–6 + pH 5–7, 1:1 (V/V); Ampholine pH 3–10; Ampholines pH 3–10 + pH 4–6, 1:1 (V/V); Ampholines pH 3–10 + pH 5–7, 1:1 (V/V)), anodic and cathodic elec- trolytes at 2 levels (H3PO4/NaOH and glycine/arginine) and gel staining method at 2 levels (Coomassie blue and sil- ver staining).

In addition, the same design (4 × 2 × 2) was also selected for the factors of loading amount of sample at 4 levels (0.5 mg, 1 mg, 2 mg, 3 mg), pH gradient at 2 levels (pH 3–10: Ampholines pH 3–10 + pH 5–7, 1:1 (V/V); pH 4–7: Ampholines pH 4–6 + pH 5–7, 1:1 (V/V)) and gel staining method at 2 levels (Coomassie blue and silver staining).

2.4. Statistical analysis

2-DE was performed at least in duplicate to ensure cred- ibility of data. Data were analyzed using ANOVA and means were considered to be significantly different at P < 0.05 as determined by least significant differences (LSD).

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3. Results

3.1. Effect of Acrylamide concentration on the two-dimensional gel electrophoresis

For the gel electrophoresis of soybean protein, previous studies tended to select 12% (W/V) [13,20] or 15% (W/V) [12,14] acrylamide in the second-dimensional gel. The re- sults of 2-DE showed that there seemed to be lower protein resolution in 15% (W/V) gel (Fig. 1a, b), compared with the 12% (W/V) gel in the second-dimensional electrophoresis (Fig. 2a, e), in which ɑ′, β and basic subunit spots were not clear. But from the spots detected (Fig. 1c), no signifi- cant difference was found between 12% and 15% gels (P > 0.05). After all, 12% of the gels were more favorable for the clear appearance of protein spots.

In SDS-PAGE or the second-dimensional gel, lower per- centage gels are often better for resolving very high molec- ular weight proteins, while much higher percentages are needed to resolve smaller proteins. As acrylamide concen- tration increased, larger particle size impeded migration in- creasingly in order to reduce gel mobility gradually. Trends of increased mobility among larger soybean-protein sub- units maybe therefore arose from using low-percentage

Notes: a: Coomassie blue staining; b: silver staining; c: amount of protein spots Ampholines pH 4–6 + pH 5–7, 1:1 (V/V), 2 mg sample, 15% (W/V) gel in th

Fig. 1. Effect of acrylamide concentra

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acrylamide gel. However, high-percentage acrylamide gel could reduce mobility among smaller soybean-protein sub- units (i.e. basic subunit), but the resolution of these sub- units had not been improved, maybe due to SDS-PAGE migration in a complex manner that would depend on both acrylamide concentration and molecular weight [21]. In this experiment, although 12% gel had good clarity, but the number of protein spots detected from the 12% gel had no significant difference of 15% gel and no difference with that from the 15% gel. Therefore, this explains why these two kinds of gel concentration are often chosen in re- ports [12–14,20] on the gel electrophoresis of SPI.

3.2. Effect of pH range of Ampholine mixtures

When a single Ampholine pH 3–10 was used, the overall resolution of the protein was not high and even the less pro- tein spots were got in 2-DE (Fig. 2b, f, j, n). Therefore, Ampholines affected significantly 2-DE of SPI (P < 0.05). On the other hand, the mixture of the two Ampholines used had higher resolution (P < 0.05) than the single Ampholine (Fig. 2), however, there was no significant differ- ence (P > 0.05) in the number of spots detected between these mixtures (Fig. 2q). Furthermore, with the higher ratio

detected for a and b. Vertical bars represent standard errors of replicates. e second-dimensional gel and pair of electrolytes H3PO4/NaOH.

tion (pH 4–7) on the 2-DE of SPI.

Notes: a, e, i, m: Ampholines pH 4–6 + pH 5–7, 1:1(V/V); b, f, j, n: Ampholine pH 3–10; c, g, k, o: Ampholines pH 3–10 + pH 4–6, 1:1(V/V); d, h, l, p: Ampholines pH 3–10 + pH 5–7, 1:1(V/V); a–h: pair of electrolytes H3PO4/NaOH; i–p: pair of electrolytes glycine/arginine; a–d, i–l: Coomassie blue staining; e–h, m–p: silver staining; q: protein spots detected for a to p.

Fig. 2. Effect of anodic and cathodic electrolytes on the 2-DE of SPI.

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of Ampholine pH 3–10 to Ampholine pH 5–7 in mixture, spots of 11S acidic subunit was closer to the right of gel (acidic), and some spots of basic subunits were not clear, even some not appearing (Fig. 3a, b), but there was no signif- icant difference (P > 0.05) be found in the number of spots detected (Fig. 3c), compared with the lower ratio mixture (Fig. 2d, h, q). For the subunits, Ampholine mixtures also af- fected significantly (P < 0.05) the resolution of α and acidic subunits, and pH 4–6 + pH 5–7 and pH 3–10 + pH 5–7

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had more spots of α subunit detected (P < 0.05) than other Ampholine mixtures, while for the acidic subunit, pH 4–6 + pH 5–7 had the most spots (P < 0.05) (Fig. 2q).

3.3. Anodic and cathodic electrolytes

Compared with pair of electrolytes H3PO4/NaOH (Fig. 2a–h), when pair of electrolytes glycine/arginine was used in IEF (Fig. 2i–p), the protein spots were shifted to

Notes: Ampholines pH 3–10 + pH 5–7, 2:1 (V/V), 12% (W/V) gel in the second-dimensional gel, 2 mg sample and pair of electrolytes H3PO4/NaOH. a: Coomassie blue staining; b: silver staining; c: protein spots detected for a and b.

Fig. 3. 2-DE of SPI with pH gradient 3–10.

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the right end of the gel (acidic), and in this scheme, α, α′ and acidic subunits were easily identified in gels, while spots of β and alkaline subunits were not clear, and appeared to be lower resolution. However, there was no significant differ- ence (P > 0.05) in the number of spots detected between both pairs of electrolytes.

The solutions of anodic and cathodic electrolytes in IEF had some influences on the separation of proteins. Protein spots would be inclined to be distributed towards the loca- tion of pH 3 in the electrophoresis images when using a pair of electrolytes glycine/arginine, no matters under pH gradient 3–10 or 4–7. Interestingly, the number of protein spots were not significantly affected by the solutions of an- odic and cathodic electrolytes. Relatively, anodic and ca- thodic electrolytes of H3PO4/NaOH used in IEF could improve resolving the protein, due to its weak cathodic mi- gration rather than moderate anodic migration for proteins [11], while the latter was the characteristic of electrolytes glycine/arginine used in IEF.

3.4. Loading amount of protein

In two-dimensional electrophoresis, loading amount sig- nificantly affected protein resolution (P < 0.05). Results

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(Fig. 4) showed that, with the amount of protein increased (from 1 mg to 3 mg), protein resolution increased, and when loading 2–3 mg samples, protein spots were clearer. For the pH 3–10 (Fig. 4a–h), at the left side of the gel ob- tained by loading 3 mg of sample, there was a vertical streak so that the protein spots were not easy to be distinguished, but at the same position of the gel obtained by loading 2 mg of sample, there was no vertical streak, and some pro- tein spots could be found. However, for the pH 4–7 (Fig. 4i–p), when the loading amount was 2 mg, 11S acidic subunit had higher resolution with more obvious protein spots than that of loading 3 mg of sample, but there were streaks found in both gels, such as a vertical streak with iso- electric point near pH 7 and a horizontal streak at the posi- tion of the acidic subunit near pH 4. As a result, for both pH gradients, the loading amount of 2 mg had higher protein resolution with more spots detected (P < 0.05, Fig. 4q) in SPI, α, acidic and basic subunits.

Loading amount of samples onto the 2-DE played an important role on resolution and visualization of elec- trophoretogram, which was also connected with the selec- tion of gel staining method. Increased loading amount led to better resolution and visualization of soybean protein. But more loading amount could lead to occurrence of

Notes: Loading capacity at 4 levels, pH gradient at 2 levels and gel staining at 2 levels, and 2-DE of SPI with 12% (W/V) gel in the second-dimensional gel and pair of electrolytes H3PO4/NaOH. a, e, i, m: 0.5 mg sample; b, f, j, n: 1 mg sample; c, g, k, o: 2 mg sample; d, h, l, p: 3 mg sample; a–h: Ampholines pH 3–10 + pH 5–7, 1:1 (V/V); i–p: Ampholines pH 4–6 + pH 5–7, 1:1 (V/V); a–d, i–l: Coomassie blue staining; e–h, m–p: silver staining; q: protein spots detected for a to p.

Fig. 4. Comparison of Coomassie brilliant blue and silver dye.

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horizontal and vertical streaks. For the soybean protein, 2 mg sample used could be fit for the analysis of 2-DE, be- cause most protein spots had better visualization through Coomassie blue staining, and resolutions of other compo- nents could be improved by silver staining. So, the applica- tion sequence of these two gel staining methods, such as first Coomassie blue staining and then silver staining, can display various components of soybean protein.

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3.5. pH gradient in IEF

Soybean proteins are predominantly composed of 7S and 11S globulin. Usually, isoelectric point (pI) of 7S globulin is 4.8, and pI of 11S globulin is 6.4, so pI of soybean protein changes in the range of 4–7 [22]. Results of different pH gradients in 2-DE were showed in Figs. 2 and 4. Just from the gel images, some protein spots in pH 3–10 gradient

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were not clearer than those in pH 4–7 gradient, and the sim- ilar results were also found in pH 4–7 images. Furthermore, some spots appeared on the images of gel from the pH 3–10 gradient, but not on the gel from pH 4–7 gradient, perhaps due to the different pH ranges and isoelectric points of some proteins in SPI more than pH 7. However, statistical analy- sis showed that the two pH gradients (pH 3–10 and pH 4–7) had no significant effect (P > 0.05) on the detected protein spots, either from the experiments in Fig. 2 or Fig. 4.

In IEF, molecules that will be focused are distributed over a medium with a pH gradient. Although isoelectric point of soybean protein is usually pH 4 to 7, results of IEF using pH 4–7 gradient showed that components of soybean protein did not completely appear, because tests of the pH 3–10 gradient showed that some soybean protein spots could still be found in the pH > 7, indicat- ing some protein components with isoelectric point >7, which was also consistent with some reports [23,24]. Therefore, the selection of pH 3–10 gradient in IEF could display the widest range of soybean protein com- ponents on a single 2-DE gel, while the narrower pH ranges 4–7 would be used for higher resolution

Notes: Ampholines pH 3–10 + pH 5–7, 1:1 (V/V), 12% (W/V) gel in the seco silver staining. Enzymolysis time: 0 min (a), 5 min (b), 15 min (c), 30 min (d

Fig. 5. 2-DE for the temporal development of enzymatic hydrol

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separations in a particular pH range, such as some units of 7S and 11S. Since Ampholines created the pH gradient, their composition and ratio were able to affect the formation of the pH gradient, then the separation of protein components through IEF, and ultimately the res- olution of protein, which had been showed by results obtained. Moreover, it seemed that IEF with the pH gra- dient created by the only Ampholine pH 3–10 or the ad- dition of the Ampholine pH 4–6 could not effectively separate the components of the soybean protein; on the contrary, the addition of the Ampholine pH 5–7 was in favor of resolving soybean proteins, which should be re- lated to the isoelectric point of the major components 7S (pI 4.8) and 11S (pI 6.4) of soybean protein.

3.6. Staining methods

For the same gel, first Coomassie blue staining, and then silver staining, the protein resolution was obviously differ- ent (P < 0.05). The experimental results showed that by Coomassie blue staining (Figs. 1a, 2a–d, i–l, 3a, 4a–d, i–l), the main protein spots were displayed, but there were still

nd-dimensional gel, pair of electrolytes H3PO4/NaOH, 2 mg samples and ), 60 min (e), and 90 min (f). g: Protein spots detected for a to f.

ysates of soybean protein emulsion with pH gradient 3–10.

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some unclear protein spots, while silver staining was able to show these spots (Figs. 1b, 2e–h, m–p, 3b, 4e-h, m-p). Therefore, the spots detected by silver staining were significantly more than those obtained by Coomassie blue staining (P < 0.05). On the other hand, in the gel by silver staining, there were some horizontal and vertical streaks, which influenced the identification of proteins, especially for the high content components. So, silver staining was more suitable for the protein fractions with very low con- tent, for example, protein hydrolysates.

3.7. 2-DE of enzymatic hydrolysates of soybean protein emulsion

With the extension of the time of enzymatic hydrolysis, the number of protein spots in the two-dimensional gel electrophoresis appeared a trend of decrease (P < 0.05) (Fig. 5). The α, β and α′ subunits gradually decreased (P < 0.05), but after 60 min of the enzymatic hydrolysis there were some protein spots below their isoelectric point (Fig. 5e, f). After enzymatic hydrolysis for 5 min (Fig. 5b), most of the β subunits were degraded (P < 0.05, Fig. 5g), and after 15 min (Fig. 5c), protein spots were found in the position of pH more than their isoelectric point, and as the reaction proceeded, the proteins had higher resolution, which should be produced by enzyme reaction. After 5 min of enzymatic hydrolysis, most of the acidic subunits were degraded (P < 0.05, Fig. 5g). And for the basic sub- unit, the polypeptide chains with isoelectric point near pH 10 were easy to be hydrolyzed by pepsin, while those with the isoelectric point of lower pH had the slower rate of enzymatic hydrolysis, such as time from 5 to 30 min (P > 0.05, Fig. 5g).

Through the 2-DE map, the dynamic changes of protein during the enzymatic hydrolysis of soybean protein emul- sion could be described in more detail. Relatively, 7S protein and some subunits of 11S protein were not easy to be hydro- lyzed by pepsin [25–27]. It was found that most of these sub- units had acidic isoelectric points from the maps of 2-DE, and the enzymatic reaction was carried out under pH 1.2, and, according to the relation of isoelectric point to protein solubility that decreases as the pH of solution is near isoelec- tric point, these subunits are insoluble and therefore not eas- ily hydrolyzed by pepsin. On the other hand, some proteins with basic isoelectric points also not easily hydrolyzed might be some enzymatic hydrolysates or some protein that could not be used as a substrate for pepsin.

4. Conclusions

In conclusion, the three factors of Ampholines, load- ing amount and gel staining method had significant ef- fects on 2-DE of SPI. Better imaging effects could be obtained under the following optimal conditions:

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Ampholine mixture of pH 3–10 + pH 5–7 or pH 4–6 + pH 5–7, loading amount of 2 mg sample and silver staining. Although gel concentration, pH gradient and an- odic and cathodic electrolytes had no significant effect on the protein separation in statistics, based on the complexity of protein composition and the visibility of gel image, 12% gel in the second dimensional electrophoresis, pH gradient 3–10 and anodic and cathodic electrolytes of H3PO4/ NaOH were preferred. Basing on these optimized condi- tions, 2-DE of enzymatic hydrolysates of SPI emulsion prod- ucts was performed, which could describe the dynamic changes of protein during the enzymatic hydrolysis of soy- bean protein emulsion protein and will provide molecular support for analyzing the change of aggregation state of in- terfacial proteins.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We acknowledge the National Natural Science Founda- tion of China (No. 31371782), the Key Project of Science and Technology Research of Henan Education Department (No. 14A550007), and the Basic Research Project of Henan University of Technology (No. 171157).

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