Investigation on the interaction of heated soy proteins with anthocyanins from cornelian cherry fruits

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 231 (2020) 118114

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Investigation on the interaction of heated soy proteins with anthocyanins from cornelian cherry fruits Loredana Dumitrascu, Nicoleta Stănciuc, Leontina Grigore-Gurgu, Iuliana Aprodu ⁎ Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domnească Street 111, 800201, Galati, Romania

a r t i c l e

i n f o

Article history: Received 4 January 2020 Accepted 24 January 2020 Available online 25 January 2020 Keywords: Cornelian cherries Anthocyanins Soy proteins Fluorescence Binding

a b s t r a c t The interaction between preheated soy proteins and anthocyanins from cornelian cherries was evaluated using a spectroscopic approach and molecular modeling. Structural changes of glycinin, β-conglycinin and soy protein isolate were investigated based on spectra of native and heat treated proteins in the presence of anthocyanins rich extracts from fresh cornelian cherry fruits. The fluorescence maximum emission in the presence of anthocyanins showed significant red shifts when compared with individual proteins, indicating the change of polarity in the surroundings of Trp residues from soy proteins toward more hydrophilic, which were attributed to proteinpolyphenols interactions. Soy proteins interacted with cornelian cherries anthocyanins mainly through a static quenching mechanism. Glycinin presented a better affinity toward anthocyanins as revealed by the binding constant. The in silico approach was further employed to provide single molecule level details on the interaction between the main soy proteins and anthocyanins prevailing in cornelian cherry extracts. The docking results are consistent with the fluorescence spectroscopy data indicating better affinity of glycinin for cyanidin 3glucoside and cyanidin 3-rutinoside, compared to the β-conglycinin. These findings deliver important insights for efficient development of microencapsulated powders based on soy proteins and anthocyanins from cornelian cherries, from the perspectives of obtaining value-added ingredients. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Cornelian cherries are an excellent source of health promoting compounds with antioxidant, anti-inflammatory, anti-bacterial, antiallergic, anti-diabetic and hepato protective activity. Therefore, consumption of cornelian cherry fruits allows maintaining the human's health by improving the immune system in fighting with many diseases, such as cancer or inflammatory diseases [1]. These fruits contain high levels of anthocyanins, with profile depending on overall fruit composition, genotype, sun exposure, temperature, humidity, etc. The major anthocyanins identified so far in berries of cornelian cherries are: cyanidin 3-glucoside, cyanidin 3rutinoside, pelargonidin 3-glucoside, petunidin-3-glucoside, cyanidin3-robinobioside, pelargonidin-3-robinobioside, pelargonidin 3-O-galactoside, cyanidin 3-O-galactoside and delphinidin 3-O-galactoside [2–5]. One of the main drawbacks of these compounds is that they are easily susceptible to degradation, and require protection in order to exert positive physiological effects on target sites. Soy is one of the most important and widely available food protein source. Soy proteins have a good balance in amino acid composition, ⁎ Corresponding author at: Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, Domneasca Street 111, Building F, Room 104, 800201 Galati, Romania. E-mail address: [email protected] (I. Aprodu).

https://doi.org/10.1016/j.saa.2020.118114 1386-1425/© 2020 Elsevier B.V. All rights reserved.

has physiologically beneficial components that reduce the cholesterol levels and the risk of hyperlipidemia and cardiovascular diseases. Also, soy proteins are nontoxic and have very good functional properties such as gelling, emulsifying ability and water and oil holding capacity [6], being considered excellent carriers for different bioactive compounds. Soy protein isolate (SPI) can be used as vehicle for waterinsoluble curcumin [7], or for carrying cranberry polyphenols [8,9]. Wan et al. [10] evaluated the potential of SPI to bind resveratrol and showed that soy protein–polyphenol complex exhibited a good potential to act as an efficient emulsifier to improve the oxidative stability of emulsions. Moreover, another study [11] showed that particles made with soy proteins are promising candidate for encapsulating and delivering hydrophobic bioactive compounds in functional foods. Preheating of soy proteins prior to encapsulation was proved to enhance the stability of the anthocyanins via altering the physicochemical properties of the carrier, such as emulsifying, foaming because of the changes in their secondary structure as well as interfacial properties [12]. Preheating of SPI at temperatures equal or higher than 100 °C improved the thermal and oxidation stabilities of SPI- cyanidin-3-O-glucoside complexes, a proper heat treatment of SPI being helpful in preventing the loss of anthocyanins [12]. Globulins 7S (β-conglycinin) and 11S (glycinin) are the main proteins from soy, accounting for 70–80% of the SPI, and are responsible for the dissociation, denaturation, and aggregation behavior under

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L. Dumitrascu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 231 (2020) 118114

thermal treatment [13].11S contains up to twelve subunits, of which six subunits are acidic and six subunits are basic in nature, whereas βconglycinin contains three acidic subunits in nature [14]. Recently, Ren et al. [15] investigated the binding mechanism of cyanidin-3-glucoside (C3G) to 7S and 11 S fractions using multi spectroscopic and thermodynamics methods. The authors highlighted the essential role of each fraction on the binding of C3G to soybean proteins. On the other hand, the influence of each preheated soy fraction on the binding mechanism with anthocyanins other than C3G was not tested so far. Taking into consideration that a mixture of anthocyanins are usually found in different vegetal materials, the aim of the present study was to investigate the effect of the thermal treatment on the ability of individual soy proteins to bind the anthocyanins from cornelian cherries (CAA) using fluorescence spectroscopy and molecular modeling approach.

2.4. Fluorescence spectroscopy measurements The quenching mechanism was investigated based on the fluorescence quenching spectra of soybean 7S and 11S globulins and of SPI in the presence of different aliquots of CAA. For fluorescence measurement, 0.2 mL of 7S, 11S or SPI at a concentration of 0.4 mg/mL was dispersed in 2.0 mL Tris buffer and then gradually titrated with different volumes of CAA (from 0 to 300 μL). Fluorescence emission spectra were collected using a LS-55 Luminescence Spectrometer (Perkin Elmer, Waltham, MA, USA). The width of the excitation and emission slits was set at 10 nm. The excitation wavelength was set at 295 nm, the emission spectra being collected between 310 and 420 nm. The raw fluorescence intensity was corrected for inner filter effects caused by the absorption of energy by anthocyanins, when exciting the soy proteins at 295 nm and collecting emission at wavelength of 340 nm. Thus, emission fluorescence intensity was corrected as recently reported [18] using the following equation:

2. Materials and methods 2.1. Materials Fresh cornelian cherries were purchased from the local market (Roman, Romania) in august 2016 and stored at −20 °C until needed for analysis. The pulp was removed from the stones and freeze-dried (CHRIST Alpha 1–4 LD plus, Germany) at −42 °C under a pressure of 0.10 mBar for 72 h. The anthocyanins rich extract from cornelian cherries was obtained as previously reported [16]. HPLC analysis revealed that the main anthocyanins from cornelian cherries extract were cyanidine-3-rutinozide (C3R) (180.60 mg/100 g d.w.) and C3G (103.6 mg/100 g d.w) [16]. For fluorescence study, the concentrated extract was diluted in water to obtain a concentration of 5 mg/mL. SPI was kindly provided by Ubimedia S.R.L., Galati, Romania (82.10% protein and 9.52% moisture).

2.2. Separation of soybean protein fractions Preparation of 7S and 11S globulins was performed as previously reported [17]. SPI was dispersed in distilled water (1:15, w/ v), and the pH of the solution was adjusted to 8.0 with 2 M NaOH. After continuous stirring for 1 h the mixture was centrifuged for 30 min at 9000 rpm. The supernatant was treated with sodium bisulphite and calcium chloride to achieve 5 mM SO 2 and 10 mM Ca 2+ respectively. After adjusting the pH to 6.4 the solution was again centrifuged.11S fraction was obtained as precipitated curd, washed two times with distilled water and neutralized with NaOH. The pH of the resulting supernatant was adjusted to 4.5 with 2 M HCl, and the mixture was centrifuged again at 9000 rpm for 30 min at 4 °C to collect the acid-precipitated proteins. The resulted precipitate was washed and neutralized following the protocol mentioned for 7S fraction. Both fractions were diluted to 0.4 mg/mL (determined by Bradford method) and used for later analysis.

2.3. Heat treatment and preparation of complex Protein solutions (7S, 11S and SPI) were prepared in 0.03MTris-HCl buffer of pH 7.2, and heat treated using a water bath (Digibath-2 BAD 4, Raypa Trade, Barcelona, Spain). After heating at temperature of 65 °C, 75 °C and 95 °C for 15 min the protein solutions placed in plastic tubes, all samples were immediately cooled in cold water to avoid any further denaturation. The heat-treated protein solutions of 0.4 mg/mL were mixed with different aliquots of anthocyanins extract at room temperature and the fluorescent properties were immediately analyzed.

F ¼ Fu 10QLðελex þελem Þ

ð1Þ

where, Fu represents the measured emission fluorescence intensity, ελex and ελem are the molar extinction coefficients of CAA at the excitation (295 nm) and emission (350 nm) wavelengths, respectively. Q is the concentration of the quencher (CAA) and L is the path length of the cell. The fluorescence quenching mechanism was described by Stern Volmer Eq. (2), and the quenching constants of experimental data were obtained by regression analysis: F0 ¼ 1 þ K SV  ½Q  ¼ 1 þ kq  τ 0  ½Q  F

ð2Þ

where Fo and F are the fluorescence intensities in the absence and presence of the quencher, KSV is Stern-Volmer quenching constant determined by linear regression of the Fo/F against [Q] plot, [Q] is the concentration of CAA, kq is the fluorescence quenching rate constant, and τ0 is the lifetime of the fluorophore without quencher and is equal to 10−8 s. 2.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) Sodium dodecyl sulphate−polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4.5% acrylamide for the stacking gel and 12% for the resolving gel. The electrophoresis samples were prepared under reducing conditions using β-mercaptoethanol that was added to the 4 x Laemmli sample buffer (Bio-Rad). The samples were heated for 5 min at 95 °C and then centrifuged at 5000 rpm for 5 min. The 7S fraction sample having a protein concentration of 3.4 mg/mL was diluted eight times. The 11S fraction sample (0.468 mg/mL) was loaded undiluted and the SPI was diluted 50 times before the loading. An amount of 20 μL from each samples was loaded in order to have a better representation of the proteins' pattern. The electrophoresis was run at 90 V for the stacking gel and at 130 V for the resolving gel. Coomassie Brilliant Blue G 250 (Bio-Rad) was used to stain the gel. 2.6. Molecular modeling on the interactions between main soy proteins and anthocyanins from cornelian cherries The in silico approach was employed to investigate at single molecule level the interaction between main proteins from soybeans and the anthocyanins prevailing in the cornelian cherries, namely C3G and C3R [16]. The three-dimensional representative models of 7S (α’ homotrimer of β-conglycinin; [19]) and 11S (homotrimer of A1aB1b subunits; [20]) proteins were chosen from RCSB Protein Data Bank (1UIK and 1FXZ, respectively). For convenience, these models were further termed 7S and 11S. In order to comply with the experimental set

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up, the molecular models were first optimized in vacuum and in explicit water environment and afterwards heated and equilibrated at 25 °C, 65 °C, 75 °C and 95 °C. Gromos43a1 force field and Gromacs 5.1.1 package [21] were used to perform all molecular mechanics and molecular dynamics steps under periodic boundary conditions. The protein models equilibrated at different temperatures between 25 and 95 °C were used as receptors for the rigid docking of the C3G and C3R molecules. The ligand-protein docking procedure was performed by means of the shape complementarity based PatchDock algorithm [22]. The top scoring models were further analyzed in detail to gather information on the ligand binding pocket on proteins surface, by means of DoGSiteScorer tool on ProteinPlus server [23]. 2.7. Statistical analysis Minitab19 software was employed to perform the statistical analysis of the resulted data. One-way ANOVA was used to assess the differences between samples after passing the normality and homoscedasticity tests. Post-hoc analysis via Tukey method at 95% confidence was carried out when appropriate (significant if p b 0.05). Values are presented as mean ± standard deviation of triple measurements. 3. Results and discussion 3.1. Fluorescence quenching Fluorescence spectroscopy is widely used to investigate protein structural changes in complex matrices, such as food products. Often, the fluorescence spectrum is determined by the chemical environment of a fluorescent component and changes of the emission spectra of tryptophan (Trp) occur in response to conformational transitions, subunit association, substrate binding, or denaturation of the proteins present in the sample [24]. The change of protein intrinsic fluorescence is frequently used to understand the characteristics of the ligand binding by the receptor. The interaction between 7S, 11S or SPI (collectively named SPF) and CAA was analyzed by testing the influence of increasing concentration of CAA on the fluorescence intensity spectra at 295 nm of SPF solutions preliminary treated at temperatures ranging from 25 to 95 °C. The temperatures used for the thermal treatment were selected considering the denaturation temperature reported for 7S and 11S [24]. Fig. 1 shows the maximum fluorescence intensity (FI) of the heat treated 7S, 11S and SPI samples, in the absence of the quencher. The temperature increase from 25 to65 °C resulted in the decrease of FI of 11S by about 40%, whereas at even higher temperature FI increased, reaching a maximum value at 95 °C. For 7S fraction and SPI, FI increased with increasing temperature by about 20% and 50% respectively. 700

a

600

FI max , (a.u.)

500

a

a

b

400 300 200

a

b

ab

a b

c

a a

100 0 25°C

65°C

75°C

95°C

Temperature (oC) 11S

7S

SPI

Fig. 1. Maximum fluorescence intensity (FImax) at different temperatures of soy proteins without. Values that for each fraction do not share the same letter (a, b, c) are significantly different based on Tukey method (p b 0.01).

3

Regardless of the temperature applied, the highest FI value was registered for 7S fraction, whereas the lowest for 11S fraction. The FI increase was previously reported for 11S and 7S and SPI heat treated at increasing temperature, however relevant structural information can be collected only based on the position of the fluorescence emission maximum [24,25]. Temperature caused red shifts in maximum emission, shifts that were dependent on the analyzed SPF. In the absence of the quencher, λmax was red shifted by 2 nm for 7S and SPI samples when increasing the temperature from 25 °C to 95 °C, and by 3 nm for 11S, indicating either potential alteration of the native quaternary structure through dissociation, or the formation of a more open and solventaccessible tertiary structure [25]. In another study, heating of 0.5 mg/mL SPI (laboratory obtained) at temperatures up to 121°Cfor 15 min caused a small blue shift indicating that the thermal treatment of soy protein solutions places the Trp chromophore to a more hydrophobic environment [12]. For all SPF, λmax before heating was about 350 nm, an indication that Trp residues are exposed to solvent, which is a characteristic of denatured proteins. So far, the studies on the interaction between any biological active compound and soy proteins have been conducted on SPI obtained at laboratory scale, the λmax of these proteins being in the range of 330–340 nm. There are important differences between laboratory and commercial isolates in terms of protein solubility, composition of the protein soluble fractions and gelation behavior, mainly related to significant changes in protein tertiary structure [26]. In many works dealing with SPI as encapsulation wall material, commercially available products are used, which in most cases are found in denaturated state. Fig. 2 shows the fluorescence spectra of 7S (Fig. 2a), 11S (Fig. 2b), SPI (Fig. 2c) in the presence of different CAA concentrations. It can be seen that the fluorescence of SPF was gradually quenched upon increasing CAA concentration, suggesting anthocyanins binding by proteins. When adding the quencher to the native 11S and SPI (25 °C), λmax was red shifted by 13 nm and by 6 nm in case of 7S fraction (Fig. 3), indicating that the most pronounced interaction with CAA occurred for 11S and SPI. The smaller red shift registered in case of 7S quenching with CAA might be attributed to Trp residues that are part of the solvent-N-terminal extended domains of α and α/. On the other hand, the similar shifts registered for SPI and 11S might be related to the ratio of the two main protein fractions prevailing in SPI, to the differences in processing history or might be explained by the presence of aggregates of the various proteins [25]. The fluorescence changes induced by CAA binding to the preheated PF are presented in Fig. 3 as ratio between F/Fo (F is the fluorescence intensity at a specific ligand concentration, Fo is the fluorescence intensity in the absence of the quencher) at maximum emission wavelength. The extinction of the fluorescence of Trp residues on SPI as a consequence of CAA addition occurred more rapidly when compared with 7S and 11S. At a concentration of 1 mM, CAA induced a fluorescence quenching degree of about 42% in case of native 11S and 7S, and about 39% for the native SPI. Increasing temperature slightly decreased the fluorescence quenching degree for all SPF, however, the highest quenching degree by CAA was obtained for SPI and the lowest for 11S fraction (p b 0.05). Preheating of SPF at 95 °C followed by quenching with CAA at maximum concentration used in this study generated smaller red shifts of ʎmax compared with the native proteins, of about 9 nm, 5 nm and 7 nm in 11S, 7S and SPI respectively. A good linearity of the Stern Volmer plot was obtained for all samples, indicating that only a single class of Trp residues within SPF was quenched. The KSV and kq values are presented in Table 1. No significant changes of KSV values (p N 0.05) were observed after heating. Similar results were reported in literature [12]. The calculated kq were higher than the limiting collisional quenching constant of 2.0 ×1010M−1s−1 [27], suggesting that collisional quenching had a minor effect on the binding between SPF and CAA, the static quenching mechanism with the formation of the complex being predominant. Although higher KSV values with increasing temperature are specific to collisional quenching it can be seen from Table 1 that heat treatment

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a)

a f

300 310 320 330 340 350 360 370 380 390 400 410 420 430

1

366

0.9

364

0.8

362

0.7

360

0.6

358

0.5

356

0.4

354

0.3

352

0.2

Wavelength (nm)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

ʎmax, (nm)

500 450 400 350 300 250 200 150 100 50 0

F/Fo

Fluorescence intensiy (a.u.)

a)

350

CAA concentraon, (mM) 11S

SPI

b)

120 a

1

366

0.9

364

0.8

362

0.7

360

0.6

358

0.5

356

300 310 320 330 340 350 360 370 380 390 400 410 420 430

0.4

354

Wavelength (nm)

0.3

352

100 80

f

60 40 20 0

0.2

c)

7S

140

F/Fo

Fluorescence intensity (a.u.)

160

160

0.2

0.4

0.6

0.8

1

1.2

1.4

350

CAA concentraon, (mM)

140

Fluorescence intensity, (a.u.)

0

ʎmax, (nm)

b)

11S

120 100

a

80 60

7S

SPI

Fig. 3. Details on fluorescent properties of 11S, 7S and SPI preheat at 25 °C (a), and 95 °C (b) upon addition of different concentrations of CAA measured as ratio between F/Fo and maximum emission wavelength.

f

40 20 0

300 310 320 330 340 350 360 370 380 390 400 410 420 430

Wavelength (nm) Fig. 2. Fluorescence intensity of Trp residues in 7S (a), 11S (b) and SPI (c) solutions at 25 °C in the presence of different concentrations of CAA (0–1.4 mM) (from a-f) was 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mM.

did not induced a significant increase of KSV values in 7S and 11S (p N 0.05), demonstrating that collisional quenching had a minor role in the binding mechanism between SPF and CAA. In a recent report [28], the molecular interaction of soy fractions with vitamin B12 was investigated, and the authors reported that fluorescence quenching of 7S/ 11S by vitamin B12 should be attributed to binding related changes and not to the collisional quenching. As showed previously the soy proteins fluorescence quenching by CAA took place through a static quenching mechanism. Thus, Scatchard equation was further used to calculate the binding constants (Kb) and binding sites as previously reported [29]. In Table 1 are presented the Kb values for soy proteins-CAA complexes obtained after preliminary treating the proteins at different temperatures. It can be seen that the binding affinity of 11S for CAA was significantly higher (p b 0.01) than that of 7S. The Kb values of SPI are in most cases similar (p b 0.01) to those of 11S, behavior which indicates that 11S fraction present in SPI plays an important role in binding to CAA. Also, based on the same equation, soy proteins showed to have only one class of binding sites for CAA. 7S and 11S have hydrophilic

charged and uncharged groups, as well as hydrophobic groups which enable the development of electrostatic interactions, hydrogen bonds, disulfide bonds, and hydrophobic interactions with small molecules. These interactions explain the ability of soy protein to be used alone or in combination with other biopolymers as vehicles [28]. Our results are in agreement with those reported in literature [15]. These authors mentioned that the higher charge density of 7S, the higher electrostatic repulsion compared to 11S, and the higher proportion of hydrophobic and uncharged amino acids in 11S, strengthen the interaction of 11S with C3G. In another study, 11S was found to have a stronger binding ability of lecithin than 7S, however, both 11S and 7S contributed to the interaction of lecithin with soybean proteins to different degrees, by changing the polarity of the amino residues and the conformation of soybean proteins [13].

3.2. SDS-PAGE analysis The electrophoresis print of SPI (lane 4, Fig. 4) showed conventional bands corresponding to the β-conglycinin (α', α, and β) and glycinin subunits. The bands with a much higher molecular weight than previously expected were seen in the 11S profile (lane 3, Fig. 4) maybe because of the heating treatment that induced different interactions between the 11S subunits. The presence of the bands with ~51 kDa, 68 kDa and 89 kDa within the pattern of 11S fraction (lane 3, Fig. 4) could be explained by the interactions between the basic subunits with molecular masses of ~17 kDa or between those with the acidic subunits, which had the molecular weight of ~34 kDa.

L. Dumitrascu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 231 (2020) 118114

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Table 1 Stern–Volmer quenching constants (KSV), fluorescence quenching rate constants (kq) and binding constants (Kb) corresponding to Trp residues of 7S, 11S and SPI solutions at different temperatures. Temperature (°C)

KSV (x 105 M−1) 7S

kq (x 1013 M−1) 11S

8.98 ± 0.23

aA

SPI

9.63 ± 0.28

aA

9.78 ± 0.04

7S aB

Kb (x 105 M−1) 11S

8.98 ± 0.23

aA

SPI

9.63 ± 0.28

aA

9.78 ± 0.04

7S aB

25 65

9.32 ± 0.09 aB

75

9.35 ± 0.54 aA

95

9.25 ± 0.07 aB

10.04 ± 0.32aA 10.06 ± 0.14aA 9.84 ± 0.29aA

9.54 ± 0.10aAB 9.48 ± 0.17aA

9.32 ± 0.09 aB

9.44 ± 0.08aAB

9.25 ± 0.07 aB

9.35 ± 0.54 aA

10.04 ± 0.32aA 10.06 ± 0.14aA 9.84 ± 0.29aA

9.54 ± 0.10aAB 9.48 ± 0.17aA 9.44 ± 0.08aAB

100.21 ± 0.01aB 100.2 ± 0.00aC 100.23 ± 0.00aB 100.2 ± 0.00aC

11S 100.7 ± 0.16 101.61 ± 0.01aA 100.92 ± 0.23aA 100.80 ± 0.16aA

SPI bA

100.63 ± 0.05aA 100.5 ± 0.0aB 100.69 ± 0.23aAB 100.46 ± 0.00aB

Values that on the same row do not share an uppercase letter are significantly different based on Tukey method (p b .01). Values that on the same column do not share the same lowercase letter are significantly different based on Tukey method (p b .01).

3.3. Single molecule level investigation on anthocyanins binding to the heat treated soy proteins The heat induced changes on the structure of main soy proteins were further simulated through in silico approach. The 7S and 11S models were equilibrated through a sequence of molecular dynamics steps at temperatures ranging from 25 °C to 95 °C, in agreement with the experimental protocol. The temperature increase resulted in significant energy increase because of the accelerated thermal motion (Table 2). The structure of both 7S and 11S models appeared rather stable at thermal treatment; only slight changes in proteins structure and surface were noticed when heated up to 95 °C. In case of 7S the total number of amino acids involved in defining strands decreased with the temperature, whereas the α-helical content increased. Anyway, regardless of the temperature, the secondary structure of 7S is dominated by the strands based motifs. As a consequence of molecular rearrangements, the protein volume and surface varied with the temperature. An increase of the total protein surface from 421.01 ± 3.94 to 433.25 ± 3.14 was notices when increasing the temperature from 25 to 75 °C, followed by a decrease to 419.92 ± 3.94 at 95 °C. These results suggest limited protein unfolding at 75 °C, followed by refolding on a different

1

2 (7S)

3 (11S) 4 (SPI)

250 150 100 75 -

α` α

50 -

β

37-

A

2520-

B

15Fig. 4. SDS-PAGE profile of 7S, 11S and SPI. Lane 1 - Molecular weight standards - Precision Plus Protein™ Dual Color Standards (Bio-Rad). Lane 2 – 7S fraction profile. Lane 3 – 11S fraction profile. Lane 4 - Soy protein isolate profile (SPI).

pattern in respect to the native proteins at even higher temperature. These molecular events were accompanied by changes in the hydrogen bonding network (Table 2). About 6.3% of the hydrogen bonds (Hb) stabilizing the native protein structure were disrupted at 75 °C, yielding new acceptor for the interaction with surrounding water molecules. A large molecular heterogeneity has been reported for β-conglycinin trimer. There are three kinds of subunits, which assemble together to form the trimer with almost random combinations. These subunits are α/, α and β, the latter having the highest thermal stability [30]. All βconglycinin subunits have core regions which share high sequence similarity of 90.4% between α and α’, of 76.2% between α and β, and of 75.5% between α’ and β subunits [30]. It is therefore safe to assume that all types of β-conglycinin trimers might exhibit high thermal stability. Analyzing the structural particularities of the11S model equilibrated at different temperatures though molecular dynamics approach it was observed that the protein is tightly packed within the two β-barrel and two extended helix domains [20], and no important variation of the volume or surface hydrophobicity are noticed with increasing temperature (Table 2). The intra-chain disulfide bond Cys12 - Cys45 and the inter-chain Cys88 - Cys298 connecting the acidic and basic chains of 11S play an important role in providing structural stability to the protein [20]. Anyway, the 8.5% decrease of the Hbs within protein structure when raising the temperature from 25 °C to 95 °C, as well as the gradual decrease of the Hb between protein and water, provide clear indications that the protein structure undergone heat dependent molecular rearrangements. This observation at single molecule level is in good agreement with the fluorescent spectroscopy results. In order to find out atomic level details which could help explaining the experimental results on fluorescence quenching, further docking tests were performed, where the main soy proteins equilibrated at varying temperatures were used as receptor for C3G and C3R, the main anthocyanins from cornelian cherries. For each of the sixteen types of complexes considered in the in silico study, the top scoring models decided based on binding energy values were carefully characterized in terms of interaction particularities (Table 3). The native 7S and 11S proteins appeared to have better affinity for C3G, the binding energy being lower compared to the corresponding complex which involved the C3R as ligand. Anyway, because of the heat induced changes in the conformation and surface cavities of the proteins, both models equilibrated at temperatures higher than 65 °C exhibited better affinity toward the C3R. The interaction energy varied with the complex and the applied temperature (Table 3). In case of 7S the lowest binding energy of −45.45 kcal/mol was observed for complex formed between the protein heated at 95 °C and C3R, indicating good affinity between the two components of the molecular assembly. This binding site has the largest surface of 713.51Å2 among all potential binding site of 7S indicated by the docking study results, accommodating the ligands considered in the study. The binding is stabilized through 21 hydrophobic interactions and 12 Hbs. Regardless of the thermal treatment, 11S exhibited better

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Table 2 Energy, surface and secondary structure descriptors of the 7S and 11S models equilibrated at temperatures ranging from 25 to 95 °C. Temperature 65 °C

75 °C

95 °C

Energy, secondary structure and surface descriptors of 7S (−1853.19 ± 1.23) ·103 Total energy, kJ mol−1 Potential energy, kJ mol−1 (−2521.57 ± 1.48) ·103 Main secondary structure elements, % Strand – 39.7; α helix – 12.1; 3–10 helix – 2.2 2 Total surface, nm 421.01 ± 3.94 HSAS, nm2 205.97 ± 1.91 Volume, nm3 180.27 ± 2.54 Hb within protein 902 ± 14 Hb protein-water 1768 ± 30

25 °C

(−1602.15 ± 0.98) ·103 (−2360.48 ± 1.28) ·103 Strand – 39.9; α helix – 13.2; 3–10 helix – 1.9 417.67 ± 2.55 199.75 ± 2.52 180.09 ± 3.23 881 ± 14 1684 ± 21

(−1542.06 ± 0.97) ·103 (−2322.84 ± 1.43) ·103 Strand – 38.3; α helix – 14.4; 3–10 helix – 1.5 433.25 ± 3.14 206.93 ± 3.80 182.00 ± 2.85 845 ± 16 1744 ± 22

(−1424.23 ± 1.06) ·103 (−2249.78 ± 1.33) ·103 Strand – 38.1; α helix – 13.2; 3–10 helix – 1.7 419.72 ± 1.68 202.23 ± 2.01 181.03 ± 2.96 857 ± 20 1631 ± 18

Energy, secondary structure and surface descriptors of 11S Total energy, kJ mol−1 (−1871.62 ± 1.59) ·103 Potential energy, kJ mol−1 (−2551.28 ± 1.70) ·103 Main secondary structure elements, % Strand – 35.4; α helix – 11.9; 3–10 helix – 0.8 2 Total surface, nm 401.24 ± 2.02 HSAS, nm2 201.35 ± 1.64 Volume, nm3 179.11 ± 2.82 Hb within protein 920 ± 13 Hb protein-water 1486 ± 14

(−1617.37 ± 1.08) ·103 (−2388.33 ± 1.15) ·103 Strand – 33.8; α helix – 12.3; 3–10 helix – 0.0 408.64 ± 2.78 204.10 ± 2.19 179.41 ± 3.69 880 ± 13 1483 ± 19

(−1556.72 ± 1.44) ·103 (−2350.36 ± 1.66) ·103 Strand – 35.8; α helix – 11.5; 3–10 helix – 1.0 398.09 ± 1.92 196.05 ± 1.65 178.48 ± 3.90 870 ± 11 1470 ± 22

(−1437.61 ± 1.98) ·103 (−2276.82 ± 1.88) ·103 Strand – 34.4; α helix – 11.9; 3–10 helix – 0.5 402.51 ± 2.72 198.68 ± 2.18 178.08 ± 3.38 842 ± 17 1433 ± 18

HSAS – Hydrophobic surface available to the solvent; Hb – hydrogen bonds.

Table 3 Single molecule level details on the interaction between main soy proteins (7S and 11S) equilibrated at different temperatures and cyanidin 3-glucoside (C3G), cyanidin 3-rutinoside (C3R) from cornelian cherries. Type of complex⁎

Binding energy⁎⁎, kcal/mol

Particularities of the binding site Volume, Å3

Surface, Å2

Depth, Å

Residues from A, B and C chains in direct contact with the anthocyanin

7S(25) – C3G

−27.52

359.18

642.60

17.57

7S(25) – C3R 7S(65) – C3G

−13.47 −34.93

211.63 431.32

480.52 667.42

7.06 16.62

7S(65) – C3R

−37.68

431.32

667.42

16.62

7S(75) – C3G

−32.50

341.80

659.63

11.96

7S(75) – C3R

−35.73

239.81

441.57

11.96

7S(95) – C3G

−25.12

596.17

700.83

12.53

7S(95) – C3R

−45.45

509.86

713.51

11.80

11S(25) – C3G

−40.52

554.11

767.98

13.85

11S(25) – C3R

−36.71

554.11

767.98

13.85

11S(65) – C3G

−25.16

526.37

724.33

15.41

11S(65) – C3R

−48.33

809.32

740.56

25.94

11S(75) – C3G

−51.30

234.85

473.56

9.49

11S(75) – C3R

−57.18

724.06

803.97

16.43

11S(95) – C3G

−30.94

903.83

930.49

21.77

11S(95) – C3R

−48.17

903.83

930.49

21.77

B: Arg185, Tyr187, Asp208, Arg240, Pro242, Ile265, Asp389, Leu390 C: Asn183, Asp391, Lys412, Ile483, Asn484 Ser154, Lys155, Lys162, Asn163, Tyr165, Gly166, His167, Arg169, Glu191, Asn193, Ser194, Lys195, Asn257, Glu456, Gln457 B: Arg185, Asp186, Asp208, Arg240, Ile265, Pro266, Val267 C: Asn183, Leu184, Arg185, Tyr187, Asp389, Leu390, Asp391, Lys412, Asn484 B: Arg185, Asp186, Asp208, Arg240, Ile265, Pro266, Val267, Asp389 C: Asn183, Leu184, Arg185, Asp389, Leu390, Asp391, Ly412, Asn484 B: Arg185, Tyr187, Asp208, Arg240, Al243, Ile265, Val267, Asp389, Leu390 C: Ly412, Glu486, Glu487 B: Tyr187, Asp208, Arg240, Ala243, Ile265, Val267, Asp389, Leu390 C: Lys412, Asn484, Ala485, Glu486 A: Asn183, Leu184, Arg185, Tyr187, Asp391, Lys412, Ile483, Asn484, Glu486 C: Asp208, Arg240, Pro266, Val267, Asp389, Leu390 A: Val267, Asn268, B: Asn183, Ile184, Arg185, Tyr187, Ile265, Asp391, Lys412, Ile483, Asn484, Glu486, Asn48 A: Ser151, Leu152, Glu153, Asn154, Gln155, B: Gly47, Asp148, Asn150, Ser151, Leu152, Arg337, Asn358, Ala359, Gln397, Thr417, Asn418, Asp419 C: Asn150, Leu152, Glu153, Asn154, Gln155, Trp335, Arg337, Asn358, Asn418, Asp419 A: Ser151, Leu152, Glu153, Gln155 B: Ala46, Asp148, Asn150, Ser151, Leu152, Glu153, Asn154, Gln155, Arg337, Asn358, Thr417, Asn418 C: Asn150, Ser151, Leu152, Glu153, Gln155, Arg337 A: Asp148, Asn150, Ser151, Leu152, Gln155, Leu336, Arg337, Asn358, Thr417 B: Ser151, Leu152 C: Asn150, Ser151, Leu152, Glu153, Asn154, Gln155, Leu156, Trp335, Arg337 A: Leu152, Asn154, Gln155, Leu156 B: Cys12, Gln13, Pro69, Pro126, Thr127, Asp148, Asn150, Ser151, Leu152, Glu153, Asn154, Gln155, Leu156, Trp335, Leu336, Arg337, Thr417 C: Leu152, Arg337 A: Asn150, Leu152, Asn154, Arg337, Asn358, Thr417, Asn418 B: Leu152, Glu153 C: Leu152, Glu153, Asn154, Gln155, Trp335 A: Asn150, Ser151, Leu152, Glu153, Asn154, Arg337 B: Asp148, Asn150, Ser151, Leu152, Glu153, Trp335, Leu336, Arg337 C: Leu152, Trp335, Arg337 A: Leu152 B: Asn150, Ser151, Leu152, Glu153, Asn154, Gln155, Trp335, Leu336, Arg337 C: Asn150, Leu152, Trp335, Arg337 A: Leu152, Gln155 B: Asp148, Asn150, Ser151, Leu152, Glu153, Gln155, Trp335, Leu336, Arg337, Thr417 C: Asn150, Ser151, Leu152, Trp335, Arg337, Thr417, Asn418

⁎ Protein (temperature) - anthocyanin complex. ⁎⁎ The binding energy was calculated by subtracting the energy of the individual molecules out of the total energy of the complex.

L. Dumitrascu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 231 (2020) 118114

affinity toward both ligands compared to 7S. The lowest binding energy of −57.18 kcal/mol was registered for the 11S-C3R, upon heating the protein at 75 °C. The in silico results indicated that the 7S is able to bind the C3G or C3R molecules within the inner space of the trimer, the ligand being usually in contact with residues from two different chains of the protein. The only exception concerns the complexes involving the native 7S protein. In case of the native 7S-C3R complex, the most probable binding site, as indicated by the molecular docking test, consists of a rather small opened cavity with depth of 7.06 Å, and volume of 211.63 Å3. Because each protomer has this kind of binding site located on top, outside the trimer hollow (Fig. 5a), one can easily assume that each native 7S trimer is able to bind up to three C3R molecules. Moreover, given the particular location of the C3G binding site within the free space between the three protomers (Fig. 5b), the same 7S trimer could additionally bind one C3G molecule. Regardless of the simulated temperature, the 11S binds both C3G and C3R in the hollow formed through the interaction between the three protomers in the proglycinin trimer. Although some variations were

7

registered in the amino acids directly involved in the interaction with the ligand, it was observed that the 11S can bind only one C3G or C3R molecule. Because of the comparable size, the anthocyanin molecules are well accommodated by the 11S trimer hollow (Fig. 5c). In particular, in case of the complex involving the 11S protein equilibrated at 75 °C, the C3R molecule appeared anchored between the three equivalent polar positively charged Arg337 residues of each chain (inset in Fig. 5c). In case of all investigated 11S-anthocyanin complexes, Trp335 residue is in direct contact with the ligand (Table 3). This observation allows explaining the fluorescence quenching upon addition of increasing volumes of anthocyanins extract from cornelian cherries. Moreover, when simulating the thermal treatment at the highest temperatures, because of the rearrangement at the protein surface, two equivalent Trp residues from different chains are in contact with the C3G or C3R molecules. 4. Conclusions In this work a spectroscopic approach was combined with in silico methods to study the binding interactions between β-conglycinin,

Fig. 5. (a) Details on the interaction between native 7S and C3G. The residues defining the binding pockets on the A (dark red), B (orange) and C (yellow) chains of the 7S trimer are represented in Licorice style. (b) Details on the interaction between native 7S and C3R. (c) Three dimensional model of the complex formed between 11S equilibrated at 75 °C and C3R molecule. In inset presents atomic level details on the contacts established between C3R and Arg337 residues of A, B and C chains of the 11S trimer. The proteins are represented in Cartoon and the ligands in Spacefill style, respectively.

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L. Dumitrascu et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 231 (2020) 118114

glycinin and SPI preheated in the temperature range 25–95 °C and anthocyanins from cornelian cherries. The fluorescence intensities of native and preheated proteins decreased when quenching with cornelian anthocyanins, indicating that the interaction between the two types of compound occurred. A significant red shift was noticed in the presence of the quencher, indicating the interaction between anthocyanins and soy protein fractions. The glycinin fraction and SPI were found to have higher affinity for anthocyanins binding than βconglycinin fraction. Similar findings were further provided through the in silico approach. Regardless of the thermal treatment simulated through molecular dynamics, the docking tests indicated that glycinin has higher affinity toward cyanidin 3-glucoside and cyanidin 3rutinoside, in respect to the β-conglycinin. Except for the native βconglycinin molecule, which can bind one cyanidin 3-glucoside and three cyanidin 3-rutinoside molecules, all other protein models equilibrated at different temperatures ranging from 25 °C to 95 °C can accommodate only one anthocyanin molecule in the inner hollow formed in between the protomers, when interacting to form the trimer. These findings will enable us to further develop microencapsulated powder using the commercially available soy protein derivatives as the main encapsulating wall material for protection of anthocyanins from cornelian cherries, from the perspectives of obtaining valueadded food ingredients. Declaration of competing interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by grants of Ministry of Research and Innovation Romania, CNCS-UEFISCDI, project number PN-III- P1-1.1-PD2016-0950 within PNCDI III and project number PN-II-RU-TE-2014-40618. The Integrated Center for Research, Expertise and Technological Transfer in Food Industry and Grant POSCCE ID 1815, cod SMIS 48745 are acknowledged from providing technical support. References [1] P. Mikaili, M. Koohirostamkolaei, S. Sajjad Babaeimarzangou, S. Aghajanshakeri, M. Moloudizargari, N. Shamsi Gamchi, Therapeutic uses and pharmacological effects of Cornus mas: a review, J. Pharm Biomed. Sci. 35 (2013) 1732–1738. [2] S. Tural, I. Koca, Physico-chemical and antioxidant properties of cornelian cherry fruits (Cornus mas L.) grown in Turkey, Sci. Hort. 116 (2008) 362–366, https://doi. org/10.1016/j.scienta.2008.02.003. [3] A.Z. Kucharska, A. Szumny, A. Sokól-Letowska, N. Piórecki, S.V. Klymenko, Iridoids and anthocyanins in cornelian cherry (Cornus mas L.) cultivars, J. Food Compos. Anal. 40 (2015) 95–102, https://doi.org/10.1016/j.jfca.2014.12.016. [4] H. Antolak, A. Czyzowska, M. Sakač, A. Mišan, O. Đuragić, D. Kregiel, Phenolic compounds contained in little-known wild fruits as antiadhesive agents against the beverage-spoiling bacteria asaia spp, Molecules 22 (2017) 1256, https://doi.org/ 10.3390/molecules22081256. [5] N.P. Seeram, R. Schutzki, A. Chandra, M.G. Nair, Characterization, quantification, and bioactivities of anthocyanins in Cornus species, J. Agric. Food Chem. 50 (2002) 2519–2523, https://doi.org/10.1021/jf0115903. [6] K. Nishinari, Y. Fang, S. Guo, G.O. Phillips, Soy proteins: a review on composition, aggregation and emulsification, Food Hydrocoll. 39 (2014) 301–318, https://doi.org/ 10.1016/j.foodhyd.2014.01.013. [7] A. Tapal, P.K. Tiku, Complexation of curcumin with soy protein isolate and its implications on solubility and stability of curcumin, Food Chem. 130 (2012) 960–965, https://doi.org/10.1016/j.foodchem.2011.08.025. [8] D.E. Roopchand, P. Kuhn, C.G. Krueger, K. Moskal, M.A. Lila, I. Raskin, Concord grape pomace polyphenols complexed to soy protein isolate are stable and hypoglycemic in diabetic mice, J. Agric. Food Chem. 61 (2013) 11428–11433, https://doi.org/10. 1021/jf403238e.

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