https://orcid.org/0000-0003-4460-4634
Abstract
The hierarchical structure modification and antioxidant traits of broad bean protein (BBP) with low denaturation induced by alternating current electric field (ACEF) were examined. The ultraviolet, Fourier transform infrared, and Raman spectrometry showed that ACEF affected the tertiary and secondary structures of BBP, as evidenced by the significant increase in surface hydrophobicity, more β-turns and random coils, fewer β-sheets, and the variation of microenvironments of certain amino acid residues. X-ray diffractometry revealed that ACEF reduced the relative crystallinity and granular size of BBP, increased lattice spacing, caused the weakening of intermolecular forces, and significantly decreased the compactness of spatial conformations in BBP. Furthermore, the antioxidant capacity of BBP increased following ACEF treatment and was influenced by voltage. This may be due to the BBP structure unfolding and the release of aromatic amino acids.
Introduction
The surge in global population and a substantial rise in the consumption of animal protein-based foods have prompted scientists to concentrate on issues pertaining to the environment, resources, and scarcity of protein (Alavi et al., 2021). Legume proteins are more environmentally sustainable in comparison to animal proteins. Broad bean is renowned for its high quantities of protein (26–29% of dry weight), the protein-to-carbohydrate ratio (27%) higher than other legumes such as peas (25%), soybeans (24%), chickpeas (19%), etc., minerals, vitamins, fibre, as well as anti-inflammatory properties and antioxidants, and so on (Rebeca et al., 2023). Furthermore, broad bean exhibits superior economic performance over other legumes due to its low cost, effective soil nitrogen fixation capacity, and tolerance to environmental extremes. Its total global production has reached 6 million tons by 2021 (Kamani et al., 2024). Broad bean protein (BBP), being a plant protein of superior quality, possesses an amino acid composition that closely approximates the optimal ratio that is required by humans and animals. Among these, the mass fraction of lysine is much higher than that of grains, and the first limiting amino acids to exist specifically are the sulphur-containing amino acids (methionine and cysteine) (Sharan et al., 2021). Nevertheless, natural BBP is principally comprised of globulins (85%), including legumin (11S) and vicilin (7S). 11S (55%) is a hexamer with a large number of hydrophobic amino acids (e.g., leucine, glycine, alanine, tryptophan, tyrosine, phenylalanine, methionine, etc.) hidden in the core linked mainly by disulphide bonds and tightly packed via hydrophobic and electrostatic interactions (Liu et al., 2022), and 7S (30%) is a trimer with three subunits (Sharan et al., 2021), resulting in the aggregation of BBP into large insoluble particles, limiting the solubility, functional and bioactive properties (Eckert et al., 2019).
The low functionality and bioactivity of numerous natural proteins impose restrictions on their application in the food business. Hence, it is imperative to modify the conformation and molecular interactions of proteins, induce intermolecular unfolding or aggregation, and enhance protein functionality (Wang et al., 2023). The investigation of protein unfolding behaviour has garnered significant attention due to its profound associations with certain diseases (allergenic responses, etc.) (Kang et al., 2023), functional properties (emulsification, gel formation, etc.) (Hall & Moraru, 2021), and biological activities (antimicrobial, antioxidant, etc.) (Alizadeh & Aliakbarlu, 2020). Uluko et al. (2014) indicated that the main cause of low activity in bioactive peptides was the presence of dense spatial structure in natural protein structure. The application of hydrostatic pressure, ultrasound, and pulsed electric fields for the pretreatment of proteins had the potential to augment both antioxidant and biological activity.
Traditional protein modification methods (enzymatic hydrolysis, thermal treatment, etc.) have some adverse consequences regarding the denaturation degree, properties, and nutritious value of proteins (Li et al., 2022). New technologies for modifying protein structures have been rapidly developed. As a non-thermal technology, electric fields (EFs), including pulsed, alternating current (AC), and direct current (DC) EFs, can reduce processing costs and time, improve energy efficiency, and have the advantage of low denaturation for proteins. Among them, ACEF is notable for its straightforward construction, user-friendly operation, economical price, and wide-ranging practicality (Guo et al., 2024). Brandner et al. (2022) demonstrated that subjecting wheat dough to ACEF can enhance the extensibility and softness of the dough, significantly accelerating the relaxation of the gluten network structure. Guo et al. (2024) treated pea protein with ACEF, which disrupted intermolecular interactions and conformation, and improved the flavour and characteristics of proteins consequently.
The utilisation of ACEF in proteins assessing the biological properties of proteins is an area that is still at stages of development. This research aimed to examine the implications of ACEF regarding the structures, intermolecular interactions, and spatial conformation of BBP, seeking to speculate the mechanism of structural modification and antioxidant activity of BBP in ACEF and provide a theoretical basis for novel technologies that enhance protein properties (based on low denaturation).
Materials and methods
Ingredients and reagents
Shelled broad beans were purchased from Sichuan (China); sodium dodecyl sulphate (SDS) and 8-benzene amino-1-naphthalene sulphonic acid (ANS) were purchased from Sinopharm Chemical Reagent, China. 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Shanghai Yien Chemical, China. The chemicals utilised were of analytical grade.
Preparation of BBP and ACEF treatment
Shelled broad beans were ground to pass a 200 μm sieve and mixed with purified water. The mixture (pH 8.0) was agitated for 60 min. The supernatant was collected, and the pH was adjusted to 3.8 followed by centrifugating at 4000 g for 20 min to obtain the protein sediment. The protein sediment was neutralised (pH 7.0) and freeze-dried to acquire BBP. The protein content of BBP was quantified as 911.8 g kg−1 (N × 5.71, dry basis) employing the Kjeldahl method.
The ACEF treatment was based on the report by Guo et al. (2024). The BBP powder was put onto the lower electrode plate; electrodes were spaced 2 cm apart. Alternating current was produced by the transformer (TDGC2-0.5, Zhejiang Zhengtai Electric, China) for ACEF in the range of 0–250 V. The voltage was adjusted to 40, 80, 120, 160, and 200 V, respectively, for 15 min. The temperature of BBP did not change significantly, as BBP did not contact the upper plate.
Ultraviolet (UV) spectroscopy and surface hydrophobicity (H0)
The UV absorption spectra of BBP (1 mg mL−1) from 200 to 400 nm were collected by a spectrophotometer (UV-759S, Shanghai Lengguang Technology, China) according to Wang et al. (2019).
H0 was assessed by the method of Zhang et al. (2021). 4 mL of BBP solutions (0.05–0.25 mg mL−1) were blended with 20 μL of ANS (8 mmol L−1). The reactants were left in the dark for 5 min. The results were quantified at 470 nm (emission) and 390 nm (excitation) utilising a fluorescence spectrophotometer (F97 Pro, Shanghai Lengguang Technology, China).
Attenuated total-reflection Fourier-transform infrared (ATR-FTIR) spectroscopy
The spectra (4000–400 cm−1) of BBP powder were captured utilising a spectrometer (Cary 610/670 FT-IR, Varian, USA) on a horizontal germanium crystal ATR plate equipped with a deuterated triglycine sulphate detector, following the approach mentioned by Byler et al. (1986).
X-ray diffractometry (XRD)
The X-ray diffractograms of BBP powder were acquired employing a diffractometer (D8 Advance, Bruker, Germany), following the approach described by Blundell (2021). Data was captured within 5–45°.
Raman spectroscopy
The Raman spectra (400–2000 cm−1) were acquired utilising a Raman spectrometer (DXRxi, Thermo Fisher Scientific, USA) at 785 nm wavelength, following the approach described by Zhuang et al. (2019).
Intermolecular forces determination
The solubility of BBP was measured employing the steps stated by Zhang et al. (2022). The solvents with pH 8 of A (deionised water), B (tris-glycine buffer), C (buffer B with 0.5% SDS and 8 M urea), and D (buffer C with 10 mM DTT) were prepared in advance. BBP solutions (15 mg mL−1) were centrifuged at 8000 g for 20 min. As a standard, bovine serum albumin was utilised, and the Bradford method was employed for ascertaining protein content. The intermolecular forces depended on the solubility of proteins in A, B, C, and D solvents.
Antioxidant activity determination
DPPH radical scavenging activity
The antioxidant activity of DPPH was conducted employing the approach mentioned by Chen et al. (2014). 4 mL of DPPH solution (0.2 mM) was combined with 4 mL of BBP solution (5 mg mL−1). The reactant was left for 30 min at 25 °C and examined at 517 nm. The results were computed with eqn (1):
where A0 represents the control absorbance, A represents the sample absorbance.
ABTS radical scavenging activity
The antioxidant activity of ABTS was quantified following an approach reported by Wang et al. (2020). 3.2 mL of ABTS solution (7 mM ABTS, 2.45 mM K2S2O8) was combined with 0.8 mL of BBP solution (5 mg mL−1). The results of the reactant were recorded at 734 nm and computed with eqn (2):
where A0 represents the control absorbance, A represents the sample absorbance.
Ferric reducing antioxidant power
Ferric reducing antioxidant power (FRAP) was conducted employing the approach mentioned by Zhao et al. (2021). 0.3 mL of BBP solution (5 mg mL−1) was combined with 5.7 mL of FRAP solution (10 mM TPTZ, 40 mM HCl, 20 mM FeCl3, pH 3.6). The results of the reactant were examined at 593 nm.
Statistical analysis
There were three repetitions per measurement, and outcomes were shown by mean values ± standard deviations. The collected results were evaluated utilising SPSS 27 (SPSS, Chicago, IL, USA) for one-way analysis of variance (ANOVA) and Duncan's multiple comparison with a degree of significance set at P < 0.05. The plots were generated employing Origin 2021 (OriginLab, Northampton, MA, USA).
Results and discussion
Tertiary structure of ACEF-treated BBP
Figure 1 presented the UV spectra and H0 of the BBP by the ACEF treatments. The majority of proteins exhibited an absorption peak in the ranges of 260–280 nm of UV spectra, which was attributed to the absorption of light by Trp, Tyr, and Phe (Guo et al., 2024). The intensity increased by 28.26%, and the λmax shifted from 269 nm to 271 nm with increasing voltage from 0 to 160 V, which could be attributed to the increase in free Trp, Tyr, and Phe. H0 was closely related to protein expansion and aggregation (Zheng et al., 2021). H0 increased by 43.92% from 349.63 for the control to 623.47 at 120 V. The ACEF might facilitate the unfolding of peptide chains and the exposure of hydrophobic groups within BBP to enhance the fluorescence emission, leading to an increase in the H0 of BBP. The decrease in H0 above 160 V may have arisen from the entropic effect of the solution, prompting part of the exposed hydrophobic groups to rebury (Wang et al., 2019).
Effect of ACEF treatment on tertiary (a, b) of BBP. (a) UV spectra; (b) surface hydrophobicity; Table in the figure: the proportions of the secondary structure.
Secondary structure of ACEF-treated BBP
Figure 1 shows the changes in the infrared spectra of BBP after ACEF treatments. The alterations in the secondary structure of the protein can be precisely represented in the amide I region, among which the β-sheets, the random coils, the α-helices, and the β-turns (the loosely ordered structure) were distributed between 1622–1637, 1637–1645, 1645–1662, and 1662–1681 cm−1, respectively. The α + β was upheld by intramolecular hydrogen bonds (Hu & Li, 2022). The β1 (1608–1622 cm−1) was the β intermolecular antiparallel folded sheet, the β2 (1682–1700 cm−1) was the β intermolecular parallel folded sheet, which were maintained by non-planar weak hydrogen bonds between molecules. The β1 + β2 was sustained through intermolecular hydrogen bonds (Guo et al., 2020). After ACEF treatment, the β1 + β2 decreased by 35.68%, the random coils increased by 6.68%, the β-turns increased by 10.52%, and the α + β increased by 7.32%, respectively. The decrease of β1 + β2 indicated that the ACEF disrupted the weak hydrogen bond between BBP molecules, weakened the intermolecular interaction forces of proteins, induced the unfolding of peptide chain, and then increased the β-turns and the random coils.
Crystalline structure of ACEF-treated BBP
Table 1 displayed the half-width height of diffraction peak (FWHM), granular size (GS), lattice distance (d), and relative crystallinity (CL) of BBP treated with ACEF. At 200 V, in comparison to the control group, the CL reduced by 29.17%. For the peak near 9°, ACEF at 200 V reduced the maximum diffraction intensity (DImax) and GS by 20.68% and 23.33%, while the FWHM and d increased by 25.54% and 8.41%, respectively. For the peak near 19.5°, ACEF at 200 V decreased the DImax and GS by 3.03% and 32.24%, while the FWHM and d increased by 16.75% and 1.55%, respectively. The results indicated that the ACEF significantly decreased the BBP crystallinity and granular size, and increased the lattice spacing, which could be caused by the increase in the BBP intermolecular gap and the decrease in structural compactness.
The XRD parameters of BBP in different voltages
| Treatment | 2θ (°) | FWHM | GS (Å) | d (Å) | CL (%) |
|---|---|---|---|---|---|
| Control | 9.41 ± 0.02a | 2.78 ± 0.02e | 30.00 ± 0.67a | 9.16 ± 0.04f | 39.43 ± 0.12a |
| 19.69 ± 0.03a | 8.24 ± 0.03e | 10.33 ± 0.67a | 4.51 ± 0.02d | 39.43 ± 0.12a | |
| 40 V | 9.41 ± 0.01a | 2.84 ± 0.03d | 28.00 ± 0.58abc | 9.40 ± 0.02d | 37.03 ± 0.24b |
| 19.67 ± 0.01ab | 8.46 ± 0.02bc | 10.00 ± 0.00a | 4.51 ± 0.01cd | 37.03 ± 0.24b | |
| 80 V | 9.24 ± 0.02c | 2.96 ± 0.04c | 27.00 ± 1.15bcd | 9.58 ± 0.02c | 34.80 ± 0.40c |
| 19.57 ± 0.01c | 8.76 ± 0.02b | 8.67 ± 0.33c | 4.54 ± 0.01b | 34.80 ± 0.40c | |
| 120 V | 9.09 ± 0.01d | 3.05 ± 0.02b | 26.00 ± 1.15cd | 9.73 ± 0.01b | 32.30 ± 0.23d |
| 19.57 ± 0.02c | 8.74 ± 0.03b | 8.33 ± 0.67cd | 4.54 ± 0.01b | 32.30 ± 0.23d | |
| 160 V | 8.92 ± 0.03e | 3.07 ± 0.04b | 25.00 ± 0.58de | 9.91 ± 0.02a | 31.47 ± 0.44e |
| 19.40 ± 0.03d | 8.67 ± 0.02b | 7.67 ± 0.33de | 4.58 ± 0.02a | 31.47 ± 0.44e | |
| 200 V | 8.90 ± 0.01e | 3.49 ± 0.04a | 23.00 ± 1.15e | 9.93 ± 0.05a | 27.93 ± 0.15f |
| 19.40 ± 0.05d | 9.62 ± 0.07a | 7.00 ± 0.00e | 4.58 ± 0.01a | 27.93 ± 0.15f |
| Treatment | 2θ (°) | FWHM | GS (Å) | d (Å) | CL (%) |
|---|---|---|---|---|---|
| Control | 9.41 ± 0.02a | 2.78 ± 0.02e | 30.00 ± 0.67a | 9.16 ± 0.04f | 39.43 ± 0.12a |
| 19.69 ± 0.03a | 8.24 ± 0.03e | 10.33 ± 0.67a | 4.51 ± 0.02d | 39.43 ± 0.12a | |
| 40 V | 9.41 ± 0.01a | 2.84 ± 0.03d | 28.00 ± 0.58abc | 9.40 ± 0.02d | 37.03 ± 0.24b |
| 19.67 ± 0.01ab | 8.46 ± 0.02bc | 10.00 ± 0.00a | 4.51 ± 0.01cd | 37.03 ± 0.24b | |
| 80 V | 9.24 ± 0.02c | 2.96 ± 0.04c | 27.00 ± 1.15bcd | 9.58 ± 0.02c | 34.80 ± 0.40c |
| 19.57 ± 0.01c | 8.76 ± 0.02b | 8.67 ± 0.33c | 4.54 ± 0.01b | 34.80 ± 0.40c | |
| 120 V | 9.09 ± 0.01d | 3.05 ± 0.02b | 26.00 ± 1.15cd | 9.73 ± 0.01b | 32.30 ± 0.23d |
| 19.57 ± 0.02c | 8.74 ± 0.03b | 8.33 ± 0.67cd | 4.54 ± 0.01b | 32.30 ± 0.23d | |
| 160 V | 8.92 ± 0.03e | 3.07 ± 0.04b | 25.00 ± 0.58de | 9.91 ± 0.02a | 31.47 ± 0.44e |
| 19.40 ± 0.03d | 8.67 ± 0.02b | 7.67 ± 0.33de | 4.58 ± 0.02a | 31.47 ± 0.44e | |
| 200 V | 8.90 ± 0.01e | 3.49 ± 0.04a | 23.00 ± 1.15e | 9.93 ± 0.05a | 27.93 ± 0.15f |
| 19.40 ± 0.05d | 9.62 ± 0.07a | 7.00 ± 0.00e | 4.58 ± 0.01a | 27.93 ± 0.15f |
2θ, diffraction angle; CL, relative crystallinity; FWHM, half-width height of diffraction peak; GS, granular size; d: lattice distance. The variable letters (a–e) appended to numerals reflected significant differences (P < 0.05).
The XRD parameters of BBP in different voltages
| Treatment | 2θ (°) | FWHM | GS (Å) | d (Å) | CL (%) |
|---|---|---|---|---|---|
| Control | 9.41 ± 0.02a | 2.78 ± 0.02e | 30.00 ± 0.67a | 9.16 ± 0.04f | 39.43 ± 0.12a |
| 19.69 ± 0.03a | 8.24 ± 0.03e | 10.33 ± 0.67a | 4.51 ± 0.02d | 39.43 ± 0.12a | |
| 40 V | 9.41 ± 0.01a | 2.84 ± 0.03d | 28.00 ± 0.58abc | 9.40 ± 0.02d | 37.03 ± 0.24b |
| 19.67 ± 0.01ab | 8.46 ± 0.02bc | 10.00 ± 0.00a | 4.51 ± 0.01cd | 37.03 ± 0.24b | |
| 80 V | 9.24 ± 0.02c | 2.96 ± 0.04c | 27.00 ± 1.15bcd | 9.58 ± 0.02c | 34.80 ± 0.40c |
| 19.57 ± 0.01c | 8.76 ± 0.02b | 8.67 ± 0.33c | 4.54 ± 0.01b | 34.80 ± 0.40c | |
| 120 V | 9.09 ± 0.01d | 3.05 ± 0.02b | 26.00 ± 1.15cd | 9.73 ± 0.01b | 32.30 ± 0.23d |
| 19.57 ± 0.02c | 8.74 ± 0.03b | 8.33 ± 0.67cd | 4.54 ± 0.01b | 32.30 ± 0.23d | |
| 160 V | 8.92 ± 0.03e | 3.07 ± 0.04b | 25.00 ± 0.58de | 9.91 ± 0.02a | 31.47 ± 0.44e |
| 19.40 ± 0.03d | 8.67 ± 0.02b | 7.67 ± 0.33de | 4.58 ± 0.02a | 31.47 ± 0.44e | |
| 200 V | 8.90 ± 0.01e | 3.49 ± 0.04a | 23.00 ± 1.15e | 9.93 ± 0.05a | 27.93 ± 0.15f |
| 19.40 ± 0.05d | 9.62 ± 0.07a | 7.00 ± 0.00e | 4.58 ± 0.01a | 27.93 ± 0.15f |
| Treatment | 2θ (°) | FWHM | GS (Å) | d (Å) | CL (%) |
|---|---|---|---|---|---|
| Control | 9.41 ± 0.02a | 2.78 ± 0.02e | 30.00 ± 0.67a | 9.16 ± 0.04f | 39.43 ± 0.12a |
| 19.69 ± 0.03a | 8.24 ± 0.03e | 10.33 ± 0.67a | 4.51 ± 0.02d | 39.43 ± 0.12a | |
| 40 V | 9.41 ± 0.01a | 2.84 ± 0.03d | 28.00 ± 0.58abc | 9.40 ± 0.02d | 37.03 ± 0.24b |
| 19.67 ± 0.01ab | 8.46 ± 0.02bc | 10.00 ± 0.00a | 4.51 ± 0.01cd | 37.03 ± 0.24b | |
| 80 V | 9.24 ± 0.02c | 2.96 ± 0.04c | 27.00 ± 1.15bcd | 9.58 ± 0.02c | 34.80 ± 0.40c |
| 19.57 ± 0.01c | 8.76 ± 0.02b | 8.67 ± 0.33c | 4.54 ± 0.01b | 34.80 ± 0.40c | |
| 120 V | 9.09 ± 0.01d | 3.05 ± 0.02b | 26.00 ± 1.15cd | 9.73 ± 0.01b | 32.30 ± 0.23d |
| 19.57 ± 0.02c | 8.74 ± 0.03b | 8.33 ± 0.67cd | 4.54 ± 0.01b | 32.30 ± 0.23d | |
| 160 V | 8.92 ± 0.03e | 3.07 ± 0.04b | 25.00 ± 0.58de | 9.91 ± 0.02a | 31.47 ± 0.44e |
| 19.40 ± 0.03d | 8.67 ± 0.02b | 7.67 ± 0.33de | 4.58 ± 0.02a | 31.47 ± 0.44e | |
| 200 V | 8.90 ± 0.01e | 3.49 ± 0.04a | 23.00 ± 1.15e | 9.93 ± 0.05a | 27.93 ± 0.15f |
| 19.40 ± 0.05d | 9.62 ± 0.07a | 7.00 ± 0.00e | 4.58 ± 0.01a | 27.93 ± 0.15f |
2θ, diffraction angle; CL, relative crystallinity; FWHM, half-width height of diffraction peak; GS, granular size; d: lattice distance. The variable letters (a–e) appended to numerals reflected significant differences (P < 0.05).
Regional amino acid environments of ACEF-treated BBP
Figure 2 showed the Raman spectra and microscopic imaging of the BBP treated by ACEF (500–1800 cm−1), and the pertinent characteristic peak data were displayed in Table S1. I850/830 indicated the extent of tyrosine residues exposure in the microenvironment. The residues of tyrosine were encased in proteins when I850/830 < 1. The hydroxy-oxygen atoms on the benzene ring functioned as hydrogen bonding donors, and their strength decreased with the increase of I850/830. Conversely, the tyrosine residues tended to be exposed at I850/830 > 1, and the protein molecules exhibited unfolding behaviour. The hydroxyl oxygen atoms on the benzene ring acted as the hydrogen bond acceptor, and its strength increased with I850/830 (Yang et al., 2020). ACEF increased I850/830 from 0.95 to 1.35 (42.11%), indicating that the ACEF made tyrosine residues gradually shift from the original ‘embedded state’ to the ‘exposed state’. This was due to the fact that ACEF induced the hydroxy-oxygen atoms on the benzene ring to gradually transit from hydrogen bonding donors to receptors, resulting in more tyrosine residues exposed in polar environments.
Effect of ACEF treatment on regional amino acid environments and disulphide bond of BBP.
Zhuang et al. (2019) revealed the boost in intensity of peaks corresponding to aliphatic amino acid residues (1450 cm−1) and tryptophan residues (760 cm−1) was positively correlated with the exposure of hydrophobic groups. After ACEF treatment, the I760 increased by 91.67%, and the I1450 increased by 64.72%. The transition from blue to red in the Raman microscopic imaging (Fig. 2) represented the substance content distributed from low to high. As the voltage increased, the imaging of BBP near 760 cm−1 and 1450 cm−1 matched the progressive growth of green and red areas, respectively. The aforementioned results indicated that the ACEF weakened the internal cross-linking of BBP, and the aliphatic amino acid residues and tryptophan residues lay bare in the polar environment.
Intermolecular forces of ACEF-treated BBP
The solubility of proteins in different solvents reflects the interaction forces between protein molecules. Tris-glycine buffer destroys electrostatic interactions in proteins. Urea is a known disruptor of hydrophobic interactions and hydrogen bonding. SDS is associated with hydrophobic amino acids in proteins. DTT generally cleaves disulphide bonds (Zhang et al., 2022).
The difference between the contents dissolved in B and A, C and B, and D and C reflect proteins solubilised by breaking ionic bonds, hydrogen bonding and hydrophobic interactions, and disulphide bonds, respectively. ACEF significantly enhanced the difference between the contents dissolved in B and A, C and B, and D and C, which corresponded to a pronounced disruption of ionic bonding, hydrophobic interactions and hydrogen bonding, and disulphide bonding (Table 2). Compared to the control, ACEF disrupted mainly the hydrophobic interactions and hydrogen bonding (60.49%) of BBP, followed by ionic bonding (43.79%) at maximum voltage, which may be attributed to the fact that ACEF disrupted the tertiary structure of BBP and weakened the hydrophobic interactions, exposing hydrophobic amino acids within BBP. The binding of exposed hydrophobic amino acid residues strengthened the electrostatic repulsion between protein molecules (Ma et al., 2022). This phenomenon may negatively affect the disruption of BBP ionic bonding by ACEF. The disruption of BBP disulphide bonds (27.49%) by ACEF was relatively minor compared to ionic bonds, hydrophobic interactions, and hydrogen bonds, which may be attributed to the effect of ACEF on the low denaturation of BBP. Consequently, ACEF disrupted the ionic bonding, hydrophobic interactions, hydrogen bonding, and disulphide bonding of BBP and weakened the physical entanglement between protein chains, leading to the unfolding of BBP molecular structure.
The intermolecular forces in BBP at different voltages
| Treatment | Solubility (%) | |||
|---|---|---|---|---|
| A | B | C | D | |
| 0 V | 11.08 ± 0.02d | 13.98 ± 0.02e | 19.65 ± 0.05e | 28.60 ± 0.05e |
| 40 V | 12.23 ± 0.03c | 16.07 ± 0.03d | 20.92 ± 0.02d | 28.85 ± 0.08e |
| 80 V | 12.52 ± 0.02c | 16.63 ± 0.05d | 23.83 ± 0.04c | 30.47 ± 0.05d |
| 120 V | 13.31 ± 0.03b | 18.51 ± 0.04c | 25.12 ± 0.03b | 34.26 ± 0.02c |
| 160 V | 13.73 ± 0.02b | 20.21 ± 0.05b | 30.28 ± 0.02a | 40.43 ± 0.05b |
| 200 V | 18.65 ± 0.04a | 22.80 ± 0.05a | 31.90 ± 0.05a | 43.31 ± 0.06a |
| Treatment | Solubility (%) | |||
|---|---|---|---|---|
| A | B | C | D | |
| 0 V | 11.08 ± 0.02d | 13.98 ± 0.02e | 19.65 ± 0.05e | 28.60 ± 0.05e |
| 40 V | 12.23 ± 0.03c | 16.07 ± 0.03d | 20.92 ± 0.02d | 28.85 ± 0.08e |
| 80 V | 12.52 ± 0.02c | 16.63 ± 0.05d | 23.83 ± 0.04c | 30.47 ± 0.05d |
| 120 V | 13.31 ± 0.03b | 18.51 ± 0.04c | 25.12 ± 0.03b | 34.26 ± 0.02c |
| 160 V | 13.73 ± 0.02b | 20.21 ± 0.05b | 30.28 ± 0.02a | 40.43 ± 0.05b |
| 200 V | 18.65 ± 0.04a | 22.80 ± 0.05a | 31.90 ± 0.05a | 43.31 ± 0.06a |
A, deionised water; B, tris-glycine buffer; C, buffer B with 0.5% SDS and 8 M urea; D, buffer C with 10 mm DTT. Various letters (a–e) appended to values within the same column reflected significant differences (P < 0.05).
The intermolecular forces in BBP at different voltages
| Treatment | Solubility (%) | |||
|---|---|---|---|---|
| A | B | C | D | |
| 0 V | 11.08 ± 0.02d | 13.98 ± 0.02e | 19.65 ± 0.05e | 28.60 ± 0.05e |
| 40 V | 12.23 ± 0.03c | 16.07 ± 0.03d | 20.92 ± 0.02d | 28.85 ± 0.08e |
| 80 V | 12.52 ± 0.02c | 16.63 ± 0.05d | 23.83 ± 0.04c | 30.47 ± 0.05d |
| 120 V | 13.31 ± 0.03b | 18.51 ± 0.04c | 25.12 ± 0.03b | 34.26 ± 0.02c |
| 160 V | 13.73 ± 0.02b | 20.21 ± 0.05b | 30.28 ± 0.02a | 40.43 ± 0.05b |
| 200 V | 18.65 ± 0.04a | 22.80 ± 0.05a | 31.90 ± 0.05a | 43.31 ± 0.06a |
| Treatment | Solubility (%) | |||
|---|---|---|---|---|
| A | B | C | D | |
| 0 V | 11.08 ± 0.02d | 13.98 ± 0.02e | 19.65 ± 0.05e | 28.60 ± 0.05e |
| 40 V | 12.23 ± 0.03c | 16.07 ± 0.03d | 20.92 ± 0.02d | 28.85 ± 0.08e |
| 80 V | 12.52 ± 0.02c | 16.63 ± 0.05d | 23.83 ± 0.04c | 30.47 ± 0.05d |
| 120 V | 13.31 ± 0.03b | 18.51 ± 0.04c | 25.12 ± 0.03b | 34.26 ± 0.02c |
| 160 V | 13.73 ± 0.02b | 20.21 ± 0.05b | 30.28 ± 0.02a | 40.43 ± 0.05b |
| 200 V | 18.65 ± 0.04a | 22.80 ± 0.05a | 31.90 ± 0.05a | 43.31 ± 0.06a |
A, deionised water; B, tris-glycine buffer; C, buffer B with 0.5% SDS and 8 M urea; D, buffer C with 10 mm DTT. Various letters (a–e) appended to values within the same column reflected significant differences (P < 0.05).
Antioxidant activities of ACEF-treated BBP
The antioxidant activity of BBP after ACEF treatment was assessed by means of DPPH, ABTS, and FRAP experiments (Fig. 3). The ability of BBP to scavenge DPPH radicals was positively correlated to the voltage, with scavenging activity increasing from 11.36% of control to 64.20% at 200 V. Tong et al. (2022) confirmed that the boost in activity was due to the stabilisation of radical electron donors, promoting the termination of radical chain reactions. The capability of BBP to scavenge ABTS radicals increased with the increase in voltage, whereby the capacity of capturing free radicals peaked at 200 V, achieving a maximum (41.13%). In addition, antioxidant activity is proportional to the reducing power of the substance (Zhao et al., 2021). The FRAP experiment was conducted to examine the impact of ACEF on the reduction capability of BBP. As the voltage increased, the absorbance of BBP solution at 593 nm rose, corresponding to the absorbance from 0.34 to 0.52, which might be attributed to the fact that ACEF significantly increased the electron-donating and iron-reducing ability of BBP. Therefore, ACEF could enhance the overall antioxidant activity of BBP from the perspective of the ability to reduce and scavenge radicals.
Effect of ACEF treatment on antioxidant activities of BBP.
Previous studies documented that Phe, Tyr, and Trp possessed the capacity to effectively eliminate free radicals as a consequence of the robust electron-donating capability in their benzene rings. Hence, the augmented concentrations of Phe, Tyr, and Trp potentially enhanced the antioxidant capabilities of proteins (Borawska et al., 2016). The enhancement in antioxidant activity of BBP might be a consequence of ACEF causing the substantial molecular structure to unfold and expose antioxidant amino acids (Trp, Tyr, and Phe, etc.), which acted as an additional provider for protons and electrons, as shown by Raman results.
Possible mechanism of enhancement of BBP antioxidant activity caused by ACEF
Based on the above findings, a plausible mechanism of ACEF influencing the protein unfolding and antioxidant activity of BBP was proposed (Fig. 4). Wu et al. (2023) demonstrated that the ACEF effect was associated with the alteration of polarity induced by alternating positive and negative electric fields, which impacted the orientation of molecular dipoles and induced molecular movement. During the processing, ACEF significantly disrupted the intermolecular interactions of BBP. Consequently, the BBP peptide chain underwent unfolding and reduced compactness, resulting in amino acid exposure and a rise in disordered formations, ultimately causing damage to both tertiary and secondary structures.
The schematic illustration of the mechanism of BBP unfolding (a) and antioxidant activity (b) caused by ACEF.
The influence of ACEF on hydrophobic interactions and hydrogen bonds of BBP caused the transition of tyrosine phenyl hydroxyl oxygen atoms from donors to acceptors, and hydrophobic amino acids were released into a polar environment. Additionally, Yang et al. (2020) revealed that Trp, Tyr, and Phe possessed a robust electron-donating capability in their benzene rings. Overall, these amino acids can serve as additional sources of electrons and protons to enhance the ability to reduce Fe and scavenge radicals.
Conclusions
In this work, the hierarchical structures, antioxidant activity, and mechanism of action of BBP after ACEF treatment were explored. The ACEF significantly decreased the compactness of BBP spatial conformations. ACEF significantly disrupted the intermolecular interactions of BBP, and its influence on hydrophobic interactions and hydrogen bonds caused the tyrosine phenyl hydroxyl oxygen atom to transition from the donor to the acceptor. Additionally, ACEF improved the iron-reducing and DPPH and ABTS radical scavenging capacities of BBP. Consequently, we put forth the novel assumption that the improvement in antioxidant potency of proteins resulting from ACEF corresponds to the unfolding of BBP molecules and the exposure of aromatic amino acids. ACEF is a moderate and non-thermal processing technique that exhibits promising applications for enhancing the nutritional value and bioactivity of proteins while lowering denaturation.
Acknowledgments
This work was funded by National Natural Science Foundation of the People's Republic of China (No. 32072133 and No. 31571765) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX22_3499).
Author contributions
Yiping Ren: Investigation; methodology; formal analysis; writing – original draft. Xiunan Zhang: Investigation. Qian Li: Software. Chen Zhang: Formal analysis. Jian-Ya Qian: Conceptualization; resources; supervision; writing – review and editing; funding acquisition.
Conflict of interest
There is no conflict of interest.
Ethical statement
Ethics approval is not required for this research.
Peer review
The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1111/ijfs.17433.
Data availability statement
This manuscript has no associated data.
References
This work proposed a method for calculating the secondary structure of proteins, providing a reference for our data calculations.
The paper investigated the alterations in the structure, function, and flavor of pea protein in different electric fields, which served as the foundation for our experimental ACEF treatment and subsequent protein structure studies.
This work established a diagrammatic model of the auxiliary action of alternating electric fields on muscle protein, which laid the foundation for our further exploration of the mechanism of action of AC electric field on protein.
This study elucidated the structural alterations of protein hydrophobic amino acids in response to environmental alterations, aiding in the exploration of alterations and mechanisms involved in protein antioxidant activity in an AC electric field.
