
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00292-X
10.1016/j.ultsonch.2024.107044
107044
Original Research Article
Effect of ultrasound on the characterization and peptidomics of foxtail millet bran protein hydrolysates
Peng Zeyu
Wang Fei
Yu Luming
Jiang Bo
Cao Jia
Sun Zhigang sunzhigang1985311@163.com
⁎
Cheng Jianjun jjcheng@neau.edu.cn
⁎
Northeast Agricultural University, Harbin, Heilongjiang, 150030, China
⁎ Corresponding authors. sunzhigang1985311@163.comjjcheng@neau.edu.cn
24 8 2024
11 2024
24 8 2024
110 10704423 7 2024
19 8 2024
23 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

Protein hydrolysates have attracted much attention for their high biological activity and are a crucial product form for the utilization of foxtail millet bran by-products. In this study, changes in the structure, functionality, activity and peptide profile of foxtail millet bran protein hydrolysates (FMBPHs) at different ultrasound powers (0 – 600 W) were investigated. The results showed that ultrasound promoted the transformation of α-helix and β-sheet to random coils and β-turn, and the exposure of hydrophobic groups and sulfhydryl groups in FMBPHs. The average particle size of the samples decreased, and the absolute value of the ζ-potential increased significantly. Simultaneously, smaller porous particles and loose fragments appeared on the surface of FMBPHs when the ultrasonic power was increased to 450 W. Additionally, 450 W ultrasound treatment improved solubility, foaming properties, emulsifying properties, thermal stability of FMBPHs. The DPPH, ABTS and hydroxyl radical scavenging ability (IC50, 2.65, 1.06 and 3.02 mg/mL), Fe2+ chelating activity (IC50, 2.62 mg/mL), and reducing power of the samples were also enhanced. The peptidomics results demonstrated that ultrasonication increased the number of active peptides in the hydrolysate, and the relative abundance of 17 active peptides was obviously elevated at 450 W. Peptide map analysis showed that ultrasound-induced structural modifications affected the peptide profiles of Ubiquitin-like domain-containing protein, Cupin type-1 domain-containing protein, 40S ribosomal protein S19, and Oleosin 1, showing changes in the abundance of certain peptides, which may be related to changes in the characterization of FMBPHs.

Keywords

Foxtail millet bran protein hydrolysates
Ultrasound
Structural changes
Functionality
Antioxidant capacity
Peptidomics
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pmc1 Introduction

Foxtail (Setaria italica (L.) Beauv.) millet are one of the oldest crops in the world and have become an important sustainable food source due to their high nutritional value, high yields, low prices and drought tolerance [1]. However, a large amount of by-product bran is produced during the milling process, amounting to 480,000 tons per year in China, which has a negative impact on the environment as well as on production costs [2]. Therefore, several researchers have explored the composition and nutritional value of foxtail millet bran (FMB) to promote its full utilization.

Studies have demonstrated that FMB is a rich source of protein (8.1 to 19.6 g per 100 g) with a balanced amino acid composition profile, easy digestibility, and hypoallergenic qualities [3]. Moreover, research has identified that foxtail millet bran protein (FMBP) possessed physiological benefits, including antioxidant [2] and anticancer properties [4]. Nevertheless, the therapeutic potential of FMBP is limited. Therefore, researchers have prepared foxtail rice bran protein hydrolysates (FMBPHs) by enzymatic hydrolysis to augment their physiological activity and encourage their broad application in functional foods. Subsequently, several researchers have identified various bioactive fragments within foxtail millet bran protein hydrolysates (FMBPHs). These fragments have been verified to exhibit ACE inhibitory activity [5], anti-inflammatory effects [6], and strong metal ion chelating ability [2]. Additionally, the enzymatic process can enhance the functional properties of natural proteins, such as emulsifying properties. Protein hydrolysates, unlike native proteins, exhibit exposed free amino groups, which can enhance peptide diffusion into the emulsion interface, thereby reducing interfacial tension and forming a consistent emulsion [7]. Therefore, some researchers have revealed that some gluten protein hydrolysates can be used as antioxidants and emulsifiers to produce O/W emulsions [8], [9]. However, Felix et al. [10] found that as the degree of protein hydrolysis increased, the emulsification of the hydrolysate decreased, limiting its broad application in food systems. Furthermore, traditional single enzyme hydrolysis results in low protein hydrolysis efficiency and a reduced yield of the peptide due to the cleavage site specificity of the enzyme [11]. Hence, hydrolysates prepared using traditional enzymatic methods exhibit certain limitations regarding their antioxidant capacity. Some researchers have also found that in alcalase hydrolysis of bran protein, the release of hydrophobic peptides coupled with heat treatment for enzyme inactivation leads to the formation of undesirable insoluble bran protein hydrolysate aggregates, constituting 40 % of the total hydrolysates [12], [13]. Consequently, this not only reduces the utilization rate of bran protein hydrolysates but also affects their industrial value.

To overcome these challenges, several techniques have been developed to enhance the antioxidant and functional properties of protein hydrolysates and peptides including ultrasound [14], pulsed electric field [15] and polyphenol modification [8]. Among them, ultrasonic modification technology is widely used due to its high efficiency, environmentally friendly nature, and low cost. The cavitation and thermal effect caused by ultrasound (20 – 100 kHz) can generate high shear stress and mechanical force, leading to conformational changes in protein hydrolysates, which in turn improves their antioxidant and functional properties [16]. For instance, Liu et al. [17] found that ultrasound treatment induced alterations in the physicochemical and structural properties of mung bean protein hydrolysate (MPH), and with increasing ultrasonic power, the antioxidant properties of MPH significantly improved. Furthermore, Tian et al. [14] reported that ultrasonic treatment disrupted insoluble soy protein peptide aggregates, resulting in an 18.33 % increase in protein solubility of soy protein hydrolysates (SPHs) and an increase in the percentage of proteins with MW<1 kDa in the aqueous layer of SPHs. However, there is still insufficient characterization of specific peptide changes in protein hydrolysates following ultrasonic treatment.

Peptidomics is an essential tool for large-scale analysis of proteins and their hydrolysis product differences [18]. Peptidomics is an untargeted has been used to rapidly detect and quantify a wide range of peptides, which in combination with bioinformatics technology contributes to the discovery and production of active peptides in foods [19]. Recently, peptidomics and proteomics has been utilized to examine the impact of protein modification techniques, including polyphenol modification and glycosylation, on specific peptides within proteins [20], [21]. Therefore, peptidomics enables comprehensive analysis of alterations in protein profiles and specific peptides of FMBPHs resulting from various ultrasound conditions. The purposes of this study were to (1) study the effect of ultrasound powers (0 W, 60 W, 150 W, 250 W, 350 W, 450 W, 600 W) on the structure (secondary structure, tertiary structure and microstructure), physicochemical properties (particle size, ζ-potential), functionality (emulsifying properties, foaming, and thermal properties), and antioxidant properties (DPPH scavenging rate, ABTS scavenging rate, Total reducing power, OH– scavenging rate and metal ions (Fe2+) chelation rate) of FMBPHs; (2) The potential mechanism of action of ultrasound treatment to improve the antioxidant as well as functional properties of FMBPHs was clarified using peptidomics analysis. In this work, the selection of suitable ultrasound parameters to enhance the functionality and antioxidant properties of FMP will greatly expand its potential applications in pharmaceutical, cosmetic and food industries. Additionally, peptidomics technology was utilized to obtain more detailed information about FMBPHs, laying the foundation for their future studies.

2 Materials and methods

2.1 Materials

Foxtail millet bran was obtained from Institute of Millet Crops, Hebei Academy of Agriculture and Forestry Sciences (Hebei, China). Alcalase 2.4L (1 × 105 U/g) was obtained from Novozyme Biotechnology Co. Ltd. (Beijing, China). The 8-Anilino-1-naphthalenesulfonic acid (ANS) and 5,5-dithio-bis2-nitrobenzoic acid (DTNB) were obtained from Sigma-Aldrich (Shanghai, China). Acetonitrile (UHPLC-MS-grade) and methanol (UHPLC-MSHPLC-grade) was purchased from Fisher Scientiffc Company (Waltham, MA). Other chemical reagents were of analytical grade and were purchased from Beijing Solaibao Technology Co., Ltd. (Beijing, China).

2.2 Preparation of foxtail millet bran proteins (FMBPs)

FMBPs were extracted following the procedure of Bera et al. [22] with minor modifications. Initially, FMB samples were crushed and then defatted with 5 volumes of n-hexane at 25 °C for 4 h. All defatted FMB samples were air dried overnight followed by dispersing in distilled water (pH 10) at a ratio of 1:10 (w/v) for 2 h. The mixtures were then centrifuged at 6000 × g for 20 min using. The pH of the supernatant was adjusted to 5.0 using 1 M HCl, followed by standing for 30 min. After centrifugation at 6000 × g for 20 min, the precipitate was collected, washed with distilled water, and neutralized using 1 M NaOH. The resulting product was freeze-dried to obtain FMBPs using Alpha 2–4 LSCplus freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), with a protein content of 80.10 ± 4.53 % determined by the Kjeldahl method.

2.3 Preparation of foxtail millet bran protein concentrate hydrolysates (FMBPHs)

FMBPs samples were adjusted to a concentration of 5 % (w/v) using ultrapure water. After stirring for 2 h, the FMBPs dispersions were hydrolyzed at 3000 U/g (enzyme units/substrate weight) alcalase activity at pH 8.5, 50 °C, and for 3 h. After hydrolysis, the hydrolysate was heated in boiling water for 10 min to ensure complete enzyme inactivation. After sonication, the hydrolysate samples were centrifuged at 8,000 × g for 15 min followed by collection of the supernatant and freeze-drying [14].

2.4 Ultrasound treatment of FMBPHs

FMBPHs samples were sonicated using the method described by Liu et al. [17] with slight modifications. The hydrolyzed samples were stirred. Subsequently, an aliquot of the FMBPHs solution (30 mL) was added to a 50 mL glass container, followed by processing with an ultrasonic homogenizer (JY92-IIDN, Ningbo Scientz Biotechnology Co., China) equipped with a 0.6 cm diameter titanium probe. The ultrasonication process was conducted at a frequency of 20 kHz using various power levels (60 W, 150 W, 250 W, 350 W, 450 W, 600 W) for 30 min each. Throughout the process, the FMBPHs samples were placed in an ice bath and pulsed for 5 s with a 1 s pause. The FMBPHs samples were freeze-dried after sonication at different power levels and named FMBPHs – 60 W, FMBPHs – 150 W, FMBPHs – 250 W, FMBPHs – 350 W, FMBPHs – 450 W, FMBPHs – 600 W.

2.5 Structural analysis

2.5.1 Fourier transform infrared (FTIR) spectroscopy

FTIR spectra (4000 – 525 cm−1) of FMBPHs and ultrasonicated-FMBPHs were obtained by the ATR method using a Fourier-transform spectrophotometer (Nicolet iS10, Thermo Fisher, USA). The operating conditions were 16 scans and a resolution of 4 cm−1 [23].

2.5.2 Ultraviolet (UV) absorption spectroscopy

The UV absorption spectra of FMBPHs and ultrasonicated-FMBPHs (0.2 mg/mL) were collected in the wavelength range of 200 – 420 nm at a scanning rate 1.0 nm/s using a T9 UV–visible spectrophotometer (Puxi Analytical Instrument Co. Ltd, Beijing, China). Deionized water was used as the blank and entire measurement was performed in triplicate [12].

2.5.3 Intrinsic fluorescence spectra

The intrinsic fluorescence spectra of FMBPHs and ultrasonicated-FMBPHs was conducted using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA), as previously described by Cao et al. [24]. Samples were prepared at 0.2 mg/mL in 10 mmol/L phosphate buffer (pH 7.0) and analyzed with excitation wavelength at 280 nm and emission wavelength from 300 to 500 nm, using a 10 nm slit width for both.

2.5.4 Surface hydrophobicity (Ho)

An aliquot (10 μL) of the ANS solution (8 mM, pH 7.4) was respectively added to 0.2 mg/mL FMBPHs and ultrasonicated-FMBPHs solutions (400 μL), followed by a reaction in darkness for 1 h. The fluorescence intensity of the mixture was determined using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA) over the emission wavelength range of 420 – 600 nm at an excitation wavelength of 390 nm [8].

2.5.5 Free sulfhydryl (SH) groups

The free sulfhydryl groups of FMBPHs and ultrasonicated-FMBPHs were determined using DTNB assay, following the procedure of Zhang et al. [12]. Briefly, the FMBPHs and ultrasonicated-FMBPHs solutions was adjusted to 0.2 mg/mL and then an aliquot (200 μL) of the sample was mixed with 10 μL of Ellman’s reagent (containing 4 mg/ mL DTNB, 90 mM glycine, 86 mM Tris, and 5 mM EDTA, pH 8.0). The mixtures were incubated in the dark 37 °C for 20 min and the absorbance of all samples was recorded at 412 nm. The free SH content was calculated as μmol per g protein using a molar extinction coefficient of 136,000 / (M*cm).

2.5.6 Scanning electron microscopy (SEM)

The SEM images were captured using an S-3400 N scanning electron microscope (Hitachi, Tokyo, Japan) as previously reported [16]. The FMBPHs and ultrasonicated-FMBPHs powders were attached to a metal stage with conductive adhesive tape and coated with gold. Then, an accelerating voltage of 15 kV and a magnification of 1000 × were used to monitor the morphology of all samples.

2.6 ζ-potential and particle size analysis

The ζ-potential value, particle mean diameter and particle size distribution of FMBPHs and ultrasonicated-FMBPHs were measured using a Zetasizer Nano ZS90 (Malvern Instruments Co. Ltd., Worcestershire, UK). Each freeze-dried sample was dissolved in phosphate buffer (10 mM, pH 7.0) to 0.1 mg/mL prior to analysis at 25 °C [25]. All measurements were carried out in triplicates.

2.7 Functional properties analysis

2.7.1 Solubility

The solubility of FMBPHs and ultrasonicated-FMBPHs were assessed according to the method of Yang et al. [16]. The centrifugation of all samples (5 mg/mL) were carried out at 8,000 × g for 20 min. The protein concentration in the supernatant after centrifugation was determined with BCA protein assay kit (Beyotime Biotechnology, Shanghai, Chian) using a SpectraMax reg iD3 microplate reader (Molecular Devices, LLC., San Jose, CA) at 562 nm. The solubility was determined using the following formulas:(1) Solubility (%) =A1A2×100%

where A1 and A2 were the protein concentrations of the supernatant after centrifugation and the sample before centrifugation, respectively.

2.7.2 Emulsifying activity index (EAI) and emulsifying stability index (ESI)

The EAI and ESI of FMBPHs and ultrasonicated-FMBPHs samples were measured based on the procedure of Pi et al. [20] with slight modifications. In summary, each sample solution (7.5 mL, 2.5 mg/mL) was mixed in a ratio of 3:1 (v/v) with soy oil before homogenization (10,000 rpm, 2 min) using Ultra Turrax T25 high-performance disperser (IKA Works GmbH & Co. KG, Staufen, Germany). Subsequently, the bottom emulsion was harvested at 0 and 10 min using a pipette and mixed in a ratio of 4:1 (v/v) with SDS solution (0.1 %, w/v). Finally, the absorbance of the mixture was recorded at 500 nm. The EAI and ESI were determined using the following formulas:(2) EAI (m2/g) =2×2.303×A0×DN×C×10000

(3) ESI (min)=A0×TA0-A10×100%

where A0 and A10 were the absorbance of diluent emulsions after 0 and 10 min respectively, D was the dilution factor, N represented the volumetric oil fraction, C was the protein content (g/mL): and, T was the time (10 min).

2.7.3 Foaming capacity (FC) and foam stability (FS)

The FC and FS of FMBPHs and ultrasonicated-FMBPHs samples were determined based on the procedure of Pi et al. [20], with minor modifications. In brief, all samples were fixed at 2.5 mg/mL with phosphate buffer (10 mM, pH 7.0), and 10 mL of sample solutions (V) was homogenized at 8,000 rpm for 2 min using Ultra Turrax T25 high-performance disperser (IKA Works GmbH & Co. KG, Staufen, Germany). After homogenization, the volumes of samples at 0 min (V0) and 10 min (V10) were recorded separately. FC and FS were determined using the following formulas:(4) FC (%) =V0 - VV×100

(5) FS (%) =V10 - VV0 - V×100

2.7.4 Thermal behaviour

Differential scanning calorimetry (DSC) was applied to determine the thermal stability of FMBPHs and ultrasonicated-FMBPHs samples. Exactly 5 mg of sample was loaded into a standard aluminum pot, hermetically sealed and then heated in a DSC 3 differential scanning calorimeter (METTLER TOLEDO, Schwerzenbach, Switzerland) at a rate of 10 °C /min from 25 °C to 200 °C with a nitrogen purge rate of 50 mL/min [24].

2.8 Antioxidant activity analysis

2.8.1 DPPH radical scavenging activity

An aliquot of 0.1 mL sample solution (0.1 – 5.0 mg/mL) was mixed with an equal volume of DPPH reagent (0.2 mM, in 95 % ethanol) and then reacted for 30 min at 25 °C in the dark. The absorbance of the reactant was recorded at 517 nm, with deionized water as a blank [26]. The DPPH radical scavenging activity was calculated by the equation (6):(6) DPPH radical scavenging activity (%) =(Ab - AS)Ab×100

Here Ab and As represent absorbance of the blank and sample, respectively.

2.8.2 ABTS radical scavenging activity

An aliquot of 0.1 mL sample solution (0.1 – 5.0 mg/mL) was mixed with tan equal volume of ABTS solution (containing 3.5 mM ABTS and 1.225 mM potassium persulfate) and then incubated at room temperature for 10 min in the absence of light. The absorbance of the reactant was recorded at 734 nm, with deionized water as a blank [26]. The ABTS scavenging capacity was calculated by the equation (7):(7) ABTS radical scavenging activity (%) =(Ab - AS)Ab×100

Here Ab and As represent absorbance of the blank and sample, respectively.

2.8.3 Hydroxyl radical scavenging activity

An aliquot of 1 mL sample solution (0.5 – 5.0 mg/mL) was mixed with equal volumes of FeSO4 (6.0 mM) and H2O2 (6.0 mM). After incubation at 25 °C for 10 min, an equal amount of salicylic acid solution (6.0 mM) was added to the mixture and the reaction was carried out at 25 °C for 30 min. Then, the absorbance was recorded at 510 nm [18]. The formula was shown as follows:(8) Hydroxyl radical scavenging activity (%) =(Ab - AS)Ab×100

Here Ab and As represent absorbance of the blank and sample, respectively.

2.8.4 Metal ion-chelating activity

An aliquot of 1.96 mL of protein hydrolysate solution (0.5–5 mg/mL) was mixed with 280 µL of FeCl2 (0.2 mM) in a water bath at 37 °C for 3 h. Then 500 µL of ferrozine (0.5 mM) was added to the mixture and reacted for 10 min at 25 °C. The absorbance was recorded at 562 nm [27]. The formula was presented as follows:(9) Clearance activity (%) = 1 -As-AcAb×100

Here As represents the absorbance of FMBPHs or ultrasonicated-FMBPHs. The Ac represents the absorbance of the mixture without ferrozine. The Ab represents the absorbance of the blank.

2.8.5 Reduction power

An aliquot of 1.0 mL of sample (0.1 – 5.0 mg/mL) was mixed with equal volumes of deionized water and potassium ferricyanide (10 mg/mL). After 50 °C water bath for 10 min, an equal amount of trichloroacetic acid solution (10 %, w/v) was added to the mixture followed by centrifugation at 1500 × g for 10 min. The supernatant was diluted twice and mixed with FeCl3 (1 mg/mL) at a ratio of 10:1 (v/v) for 10 min in the dark. The absorbance was recorded at 700 nm [18].

2.9 Peptidomics

2.9.1 EASY-nLC1200 Q Exactive plus

After desalination using a C18 stage tips, the FMBPHs and ultrasonicated-FMBPHs samples were measured by EASY-nLC1200 Q Exactive plus in accordance with the method described by Sun et al. [28]. Briefly, the samples were sequentially injected into a C18 reverse phase pre-column (100 μm × 2 cm, 5 μm) and a C18 analytical column (75 μm × 100 nm, 3 μm) and eluted through a linear gradient of mobile phase (containing phase A, 0.1 % formic acid and phase B, 80 % acetonitrile) at a flow rate of 300 nL/min. All samples were subsequently identiffed by the Q-Exactive system (Thermo Fisher Scientific) and performed at 325 °C capillary temperature, 350 – 2,000 m/z scanning range, and 45 ms maximum ion injection time.

2.9.2 Peptide identification

The mass data of samples were subjected to analysis via Proteome Discoverer software V. 24 (Thermo Fisher Scientific) following the previous method of Cui et al. [18]. The abundance of proteins and peptides in the hydrolysate was determined using label-free quantification in accordance with the method described by Pi et al. [20].

2.10 Activity prediction of peptides

Active peptides were predicted with peptideranker (http://distilldeep.ucd.ie/PeptideRanker/). Peptideranker was based on an n-1 neural network probabilistic algorithm for the purpose of scoring the identified peptides and peptides were deemed to be potentially active when the score was over 0.5 threshold [19].

2.11 Statistical analysis

Experiments were performed at least in triplicate, and the results were presented as the mean value ± standard deviation (SD). A one-way analysis of variance (ANOVA) and Duncan's multiple range test were used for statistical analysis using SPSS version 25.0 software (SPSS Inc., Chicago, USA). A level of P<0.05 was deemed to indicate statistical significance. The Venn diagram, peptide map, and heat map were created using online tools such as OmicStudio tools (https://www.omicstudio.cn/tool), Peptigram (https://bioware.ucd.ie/peptigram/), and Chiplot (https://www.chiplot.online/circle_heatmap.html).

3 Results and discussion

3.1 Effects of ultrasound power on the protein structure of FMBPHs

3.1.1 Fourier transform infrared (FTIR) spectroscopy

Fourier Transform Infrared (FTIR) spectroscopy (Fig. 1a) was utilized to ascertain the alterations in the functional groups of FMBPHs following various sonication treatments, thereby providing a deeper comprehension of the modifications in their protein secondary structures (Fig. 1b). As shown in Fig. 1a, there were no new peaks in the infrared spectrum of the samples after ultrasonic treatment, indicating that no new chemical bonds were formed. However, the FMBPHs shifted differently on the following characteristic peaks under the influence of different power ultrasound (60 – 600 W): 3276.94 cm−1 (N-H bending), 2920.35 cm−1 (C-H asymmetric stretching), 1644.42 cm−1 (amide I, C=O stretching), 1540 cm−1 (amide II, N-H and C-N bending), and 1048.66 cm−1 (C-O stretching) [23]. These findings suggest that the strong shear and hydrodynamic turbulence generated by ultrasound induce stretching and bending of multiple functional groups, which may lead to secondary structure changes in FMBPHs. Wang et al. [29] also reported that the shear forces induced by ultrasound can disrupt the intermolecular interactions of pecan protein molecules, thereby affecting their internal secondary structure. Therefore, quantitative information on the secondary structure of all samples was obtained by using Peakfit software V. 4.12 (AISN Software Inc.) calculations in the amide I region (1700 – 1600 cm−1). Fig. 1b displayed that with increasing ultrasound power, the secondary structure of FMBPHs changed gradually from a rigid to a flexible conformation, with α-helix and β-sheet decreasing by 1.48 – 10.99 % and 6.27 – 9.77 %, respectively, and β-turn and random coil increasing by 3.54 – 6.64 % and 2.77 – 7.23 %, respectively. Similar results were reported by Yang et al. [30], who found that ultrasound (240 W, 30 min) promoted the transformation of α-helix and β-sheet structures to random coil structures in soybean protein hydrolysate gels. Yolandani et al. [31] also observed an increase in the content of random coil and β-turn in soy protein hydrolysates pretreated with ultrasonication (20 kHz, 10 min, 160 W/L). This phenomenon may be attributed to the strong shear force and hydrodynamic turbulence generated by ultrasound, which facilitate the unfolding and loosening of the sample structure, thereby enhancing its structural flexibility. However, Bing et al. [32] demonstrated that the application of moderate ultrasound treatment to MPH led to an increase in α-helix and β-sheet content, contrasting with a decrease in β-turn content. These inconsistencies might be due to differences in the protein source, ultrasonic conditions and detection methods [33].Fig. 1 Effects of ultrasound power on the protein structure of FMBPHs. Fourier-transform infrared spectra (a), secondary structure content (b), ultraviolet (UV) spectrum (c), second-derivative UV spectrum and local second-derivative UV spectrum of Trp (288 – 294 nm) (d), fluorescence spectrum (e), surface hydrophobicity (f), free sulfhydryl (SH) content (g) of FMBPHs and ultrasonicated-FMBPHs. Different letters (a-d) indicate significant differences (P<0.05).

3.1.2 UV absorption spectra and second-derivative absorption spectra

The UV absorption, primarily due to aromatic amino acids such as tryptophan and tyrosine, is responsive to alterations in protein or peptide at tertiary structures [8]. Meanwhile, FMBPHs contain multiple aromatic residues that often have overlapping contributions at the same wavelength, requiring resolution through calculation of higher-order (e.g., second-derivative) derivatives of the UV absorption spectrum [34]. The change in position of the UV second-order derivative spectral bands of FMBPHs can be used to characterize changes in the polarity of their tyrosine and tryptophan residues in specific microenvironments [35]. Therefore, the effect of ultrasonic power on the tertiary structure of hydrolysates was explored by UV absorption spectroscopy (Fig. 1c) and UV second-derivative absorption spectra (Fig. 1d). With the increase of ultrasonic power from 0 W to 600 W, the UV absorption intensity within the range of 250 – 300 nm of the FMBPHs samples first increased and then decreased (Fig. 1c). This suggests that the tryptophan and tyrosine residues in the hydrolysate undergo a gradual process of exposure to the outside of the structure and subsequent reburying within the structure. The UV absorption intensity of FMBPHs in the range of 250 – 300 nm reached a maximum at an ultrasound power of 450 W, implying that the most tryptophan and tyrosine residues were exposed under this condition. This may be attributed to the cavitation and mechanical effects generated by higher power ultrasound disrupting the intermolecular interactions between the FMBPHs, leading to molecular unfolding and exposing more hydrophobic groups [36]. Similarly, Yu et al. [37] found that high-intensity ultrasound led to an increase in the UV absorption intensity of black bean protein isolate at 270 nm, thereby exposing hydrophobic groups (chromophores). Yang et al. [38] also observed an increase in UV absorption intensity of rice protein hydrolysate with ultrasonic pretreatment. Similarly, as can be seen from the UV second-derivative absorption spectra of the Tyr residue (270 – 285 nm) and Trp residue (290 – 300 nm) regions of FMBPHs in Fig. 1d, the derivative spectral bands in the Tyr residue region and Trp residue region of the samples were gradually blue-shifted when the power was increased from 0 W to 450 W, and the degree of blueshift of several major derivative spectral bands was positively correlated with the power. Thus, a more obvious blue-shift of the derivative band was observed in the higher power (350 W, 450 W) sonicated samples. This suggests that suitable ultrasound treatment enhances the polarity of the microenvironment surrounding Tyr and Trp residues, leading to a migration of some of these residues from the protein or peptide interior to the surface. However, a certain degree of red shift in the derivative spectral bands in the Tyr and Trp residues region of the FMBPHs occurred with increasing the ultrasound power from 450 W to 600 W. In particular, the most notable red shift could be observed in the spectral bands in the local region of the Trp residues (288 – 294 nm). This indicates that the Trp residues are being re-buried within the structure, potentially linked to protein aggregation induced by high power [16]. Chen et al. [39] also reported that excessive ultrasound power density (above 40 W/L) leads to recrosslinking and aggregation of soy protein molecules as well as reburial of exposed groups.

3.1.3 Intrinsic fluorescence spectra

Intrinsic fluorescence spectra can monitor changes in the microenvironmental polarity of aromatic amino acid residues (Tyr and Trp) in protein to reflect changes in protein tertiary structure [40]. As shown in Fig. 1e, the fluorescence intensity of FMBPHs increased and was accompanied by a redshift of the maximum wavelength (λmax) from 358 to 360 nm after ultrasound treatment with different powers. With the increase of ultrasonic power from 60 W to 600 W, the maximum fluorescence intensity of the FMBPHs first increased and then decreased. The maximum fluorescence intensity of FMBPHs increased to the highest at a sonication power of 450 W, which was 54.48 % higher than that of untreated FMBPHs. This implies that higher power ultrasound obviously affects the tertiary structure of FMBPHs. The observed phenomenon may be ascribed to the ultrasound-induced cavitation effect, resulting in the unfolding of peptide structures within FMBPHs and the exposure of additional hydrophobic groups, including aromatic amino acids [16]. The results were similar to the observation of Tian et al. [14], who reported that the application of ultrasound to soy protein hydrolysates could increase their endogenous fluorescence intensities, causing the unfolding of the tertiary structure of the samples. However, a decrease in the fluorescence intensity of FMBPHs was observed after 600 W ultrasonication. Probably, excessive power ultrasound induced protein aggregation in the hydrolysate, allowing the exposed chromophores to be reburied inside the structure [16]. Thus, the option of ultrasonic power is a pivotal factor affecting the structure of FMBPHs.

3.1.4 Surface hydrophobicity (H0)

Surface hydrophobicity is a critical metric for the quantification of hydrophobic groups on the surface of protein samples, as well as for the assessment of changes in their conformation [41]. The ANS fluorescent probe method was employed to measure the H0 of FMBPHs and ultrasonicated-FMBPHs. Fig. 1f demonstrated that the H0 of ultrasonicated-FMBPHs was significantly increased (P<0.05) compared to that of untreated FMBPHs (369.80 ± 9.67 A.U.), except for the 60 W and 600 W treatment groups. Specifically, the H0 of ultrasonicated-FMBPHs increased first and then decreased. At a power of 450 W, the H0 of the samples reached its maximum value and increased significantly (P<0.05) by 35.53 % over the untreated FMBPHs. This suggested that ultrasonication with appropriate power could promote the exposure of hydrophobic groups in FMBPHs, which can be explained by the unfolding of protein structures induced by the cavitation effect, resulting in the exposure of hydrophobic groups inside the FMBPHs to the protein surface [25]. Ashraf et al. [42] also showed similar results when investigating the effect of thermosonication pre-treatment on MPH and white kidney bean protein hydrolysates. Nevertheless, the H0 of the samples significantly decreased (P<0.05) when the ultrasonic power was increased from 450 W to 600 W. These findings might be due to the masking or disruption of hydrophobic groups caused by protein aggregation [43]. Moreover, Liu et al. [17] observed that high ultrasound power (546 W) leads to protein re-polymerization in MPH via hydrophobic binding, thus reducing the surface hydrophobicity.

3.1.5 Free SH content

The free SH content was associated with the stretching and aggregation of the molecular structure within the hydrolysate [39]. Fig. 1g displayed the effects of ultrasonic treatment (at different powers) on the free SH content of FMBPHs. With an increase in power from 0 W to 450 W, the free sulfhydryl content of FMBPHs increased. Subsequently, the free SH content began to gradually decrease. The highest value of free SH content, recorded at 450 W, was 49.20 μmol/g of protein, representing a significant increase (P<0.05) of 58.34 % over untreated FMBPHs (0 W). The increase in free sulfhydryl content is probably caused by the disruption of the S–S bond and subsequent structural unfolding, allowing sulfhydryl groups to be exposed on the protein surface [26], [39]. This indicates that ultrasonication induces the unfolding of the protein structure in FMBPHs, aligning with the findings of intrinsic fluorescence and surface hydrophobicity (Fig. 1e & Fig. 1f). In a similar vein, Yang et al. [16] observed an increase in free SH content in soy protein hydrolysate following ultrasonic treatment.

3.2 Effects of ultrasound power on the microstructure of FMBPHs

Microstructural changes in FMBPHs and ultrasonicated FMBPHs samples were visualized by scanning electron microscopy photographs (Fig. 2). It was clear that the initial FMBPHs presented multiple plate-like aggregates with relatively compact structures and rough surfaces. After ultrasound treatment, the samples were transformed from multiple plate-like aggregates to thinner lamellar structures, and some of the particles were decreasing in size. Subsequently, the microstructure of the hydrolysate changed more obviously and the sample surface became porous and the lamellar structure changed to small loose fragments when the ultrasonic power was increased to 450 – 600 W. These changes indicated that the ultrasonication promoted the unfolding of peptide structures in the hydrolysate as well as the size reduction. This structural transformation was the result of cavitation produced by ultrasound [36]. Similar results were also observed in the sonication of soy protein hydrolysates [14] and red kidney bean proteins [44]. This porous structure can facilitate the release of hydrophobic groups within the hydrolysate and increases the contact area between the hydrolysate and the solvent to provide better functional properties.Fig. 2 The scanning electron microscopy analysis of FMBPHs and ultrasonicated-FMBPHs.

3.3 Effects of ultrasound power on the physicochemical properties of FMBPHs

3.3.1 ζ-potential

The ζ-potential was used to assess the surface charge distribution and stability of FMBPHs and ultrasonicated FMBPHs (Fig. 3a). All samples exhibited negative surface charges, implying that FMBPHs possess more negatively charged amino acids. This finding is consistent with the results observed by Liu et al. [17] in MPH. With increasing ultrasound power, the absolute ζ-potential of the FMBPHs increased. These phenomena demonstrated that ultrasonic treatment, particularly under high-power conditions, increases the negative charge on the surface of the particles and the electrostatic repulsion between the particles, which promotes their dispersion and stabilization in solution [16] and sunflower protein [45] also observed the improvement of the absolute ζ-potential values after sonication. This could likely be attributed to the destruction of protein structures following ultrasonic treatment, which permits the accumulation of more charges on the molecular chains [46]. Notably, the absolute ζ-potential value of FMBPHs reached a maximum at an ultrasound power of 600 W, which was significantly increased by 0.66-fold (P<0.05) compared to untreated FMBPHs. However, excessively high absolute ζ-potential values may reduce the stability of the solution, leading to protein aggregation of the solution [25].Fig. 3 Effects of ultrasound power on the physicochemical and functional properties of FMBPHs.ζ-potentials and average particle sizes (a), particle size distribution (b), solubility (c), emulsifying activity index and emulsifying stability index (d), foaming capacity and foam stability (e), DSC (f) of FMBPHs and ultrasonicated-FMBPHs. Different letters (a-e) indicate significant differences (P<0.05).

3.3.2 Particle size and distribution

The effect of different ultrasonic treatments on the particle size (Fig. 3a & 3b) of hydrolysates was explored since the change in particle size may be closely related to the functional properties of FMBPHs. Fig. 3a illustrated that the average particle size of ultrasonicated-FMBPHs was significantly decreased (P<0.05) compared to that of untreated FMBPHs (206.80 ± 4.26 nm). The average particle size of the FMBPHs decreased significantly when the ultrasonic power was increased to 250 W – 600 W. Meanwhile, the particle size distribution (PSD) results also displayed that the samples shifted toward the smaller particle sizes, and the peaks of the PSD tend to be uniform and narrow with the increasing ultrasonic power (Fig. 3b). This suggested that a certain intensity of ultrasonic treatment could promote the reduction and homogeneity of the hydrolysate particle size, which is consistent with the microstructural results. This phenomenon may be caused by the strong shear force and hydrodynamic turbulence generated by ultrasound that broke non-covalent bonds, leading to the dissociation of protein aggregates in hydrolysate [47]. Similarly, the reduction in particle size after ultrasound treatment was also reported for soy protein hydrolysates [14] and pecan protein [29].

3.4 Effects of ultrasound power on the functional properties of FMBPHs

3.4.1 Solubility

Solubility is a prerequisite for the functional properties of hydrolysates or proteins. The changes in solubility of FMBPHs after different ultrasonic power treatments were shown in Fig. 3c. The solubility of FMBPHs gradually increased as the ultrasonic power was increased from 0 W to 450 W. In particular, the solubility of the samples treated with 350 W and 450 W ultrasound significantly increased by 18.96 % and 20.49 % (P<0.05), respectively, as compared to that of the untreated FMBPHs (0 W). This suggests that appropriate ultrasound treatment promoted the solubilization of FMBPHs. This may be due to the cavitation bubbles generated by ultrasound disrupting the non-covalent interactions within the insoluble peptide aggregates in the samples and promoting the solubilization of the insoluble peptide aggregates [16]. Yeasmin et al. [44] also reported that the cavitation effect produced by ultrasonication (300 W, 10 min) affected the electrostatic and hydrophobic interactions within red kidney bean proteins, facilitating the hydration of peptide chains and amino acid side chains with water molecules, thereby enhancing their solubility. However, the solubility of the samples exhibited a gradual decrease when the power was increased to 600 W, which may be attributed to the reformation of small aggregates in the samples [14].

3.4.2 Emulsifying properties

The emulsifying activity index (EAI) and emulsifying stability index (ESI) are important variables for assessing the emulsifying properties of FMBPHs. As illustrated in Fig. 3d, a significant (P<0.05) enhancement in EAI and ESI was observed in ultrasonicated-FMBPHs, compared to FMBPHs (0.41 ± 0.05 m2/g, 94.52 ± 14.50 min). After ultrasonication, the EAI of the samples showed a gradual but insignificant (P>0.05) increase and reached a maximum at 600 W. Meanwhile, the ESI of ultrasonicated-FMBPHs increased first and then decreased. At a power of 450 W, the ESI of the samples reached its maximum value and increased significantly (P<0.05) by 2.36-fold over the control FMBPHs. This implies that suitable ultrasonic treatment improves the emulsification performance of FMBPHs, possibly due to the cavitation effect of ultrasonic waves that change the structure of the protein and reduce the sizes of protein particles, thereby improving the adsorption capacity of FMBPHs at the oil–water interface [36]. Additionally, the improvement in emulsifying ability is related to the increased exposure of hydrophobic groups [48]. However, it was considered that an excessive ultrasonic power (600 W) would lead to a decrease in ESI. Therefore, the FMBPHs – 450 W exhibited excellent emulsifying properties, which were also attributed to the lowest particle size (Fig. 3a) and highest H0 (Fig. 1f).

3.4.3 Foaming properties

Generally, foaming capacity (FC) and foam stability (FS) were monitored to analyze the foaming properties of FMBPHs and ultrasonicated-FMBPHs. As illustrated in Fig. 3e, a significant increase (P<0.05) in FC was observed in comparison with the control (74.67 ± 13.32 %) when the ultrasound power exceeded 150 W, reaching a maximum at 450 W (83.47 ± 6.90 %). Meanwhile, a significant (P<0.05) increase in FS was also observed in ultrasonicated-FMBPHs, compared to FMBPHs (54.29 ± 10.74 %). After ultrasonication, the FS of the samples gradually increased and reached a maximum at 350 W. However, the difference between treatment groups was no significant (P>0.05). This suggested that ultrasonic treatment enhanced the foaming capacity and foam stability of FMBPHs, and ultrasonic power had a greater effect on foaming capacity but a lesser effect on foam stability. These findings on the foaming properties of FMBPHs may be associated with the improvement of molecular flexibility, exposure of sulfhydryl groups, and more hydrophobic sites induced by ultrasound [20]. Yang et al. [36] also reported that ultrasonic pretreatment enhances the flexibility of peptides and promotes their adsorption at the air-solvent interface, leading to improved foaming capacity in protein hydrolysates from housefly larvae. Additionally, the smaller particle size of the ultrasonicated samples (Fig. 2b) observed in this study may be another reason. However, the slight reduction in both FC and FS of FMBPHs – 600 W was observed, which may be attributed to protein aggregation induced by high ultrasound power [43]. Overall, appropriate ultrasonic power can enhance the foaming properties of FMBPHs to facilitate their application in aerated food products.

3.4.4 Thermal properties

Thermal properties including the denaturation temperature (Td) and enthalpy (ΔH), a pivotal measure of protein and peptide quality and functionality, can be assessed using differential scanning calorimetry (DSC) [49]. Generally, protein thermal stability is typically gauged by the Td, with elevated peak temperatures signifying increased stability [50]. The ΔH refers to the net energy value required to denature proteins, encompassing both hydrogen bond disruption (endothermic) and hydrophobic interactions (exothermic) [50]. As shown in Fig. 3f, all the samples exhibited a broad thermal denaturation peak at 81.75 – 94.55 °C, which was consistent with a single endothermic peak (69.23 – 71.24 °C) of foxtail millet protein and high intensity ultrasonicated foxtail millet protein reported by Sharma et al. [51]. With increasing ultrasonic power, the Td of the samples increased and then reached the highest value at the ultrasonic power of 450 W (94.55 °C), indicating that appropriate sonication improves the thermal stability of FMBPHs. This improvement may be due to the reduction in the rigid structure of the protein induced by ultrasound [29]. Similarly, studies conducted on quinoa proteins [52] and red kidney bean proteins [44] have also observed the enhancement of denaturation temperature after sonication. Additionally, a decrease in the enthalpy (ΔH) of FMBPHs was observed after sonication, suggesting that less energy was required for the unfolding of ultrasonicated FMBPHs. This reduction may be attributed to the destruction of intramolecular bonds of FMBPHs, potentially caused by cavitation effect from shear forces generated by ultrasonication [16].

3.5 Effects of ultrasound power on the antioxidant activity of FMBPHs

The antioxidant capacity of all samples at graded concentrations (0.1 – 5 mg/mL, 0.5 – 5 mg/mL) was shown in Fig. 4 a-i. The antioxidant capacity of all samples increased in a concentration-dependent manner, suggesting that FMBPHs have better antioxidant properties. Additionally, it was observed that the antioxidant activity of the samples showed an increasing trend at different concentrations after ultrasonication. In particular, the samples at different concentrations showed significantly (P<0.05) increased DPPH radical scavenging and ABTS radical scavenging under high power conditions (350 – 600 W). Consequently, a significant (P<0.05) increase in hydroxyl radical scavenging, Fe2+ chelating and reducing power was observed for higher concentration samples especially at 5 mg/mL under 350 W and 450 W.Fig. 4 Effects of ultrasound power on the antioxidant activity of FMBPHs. DPPH radical scavenging rates (a) and IC50 value (b), ABTS radical scavenging rates (c) and IC50 value (d), hydroxyl radical scavenging rates (e) and IC50 value (f), Fe2+ chelating rates (g) and IC50 value (h), reducing power (i) of FMBPHs and ultrasonicated-FMBPHs. Different letters (a-e) indicate significant differences within different groups (P<0.05).

Furthermore, IC50 values were calculated to evaluate the effect of different power (60 – 600 W) ultrasound treatments on the antioxidant capacity of FMBPHs. As shown in Fig. 4b, d, f, h, the IC50 values for DPPH radical scavenging, ABTS radical scavenging, hydroxyl radical scavenging, and Fe2+ chelating assays in FMBPHs were reduced by 27.57 – 46.25 %, 13.19 – 41.76 %, 1.21 – 9.06 %, and 4.46 – 37.38 %, respectively, after different power ultrasonication compared to that of untreated FMBPHs. Particularly, samples sonicated at 350 W and 450 W exhibited lower IC50 values in most in vitro antioxidant tests. This result is similar to improve the antioxidant ability of MPH with increasing ultrasonic power (114 – 546 W) [17]. This phenomenon can be explained by the unfolding of the FMBPHs protein or peptide structure facilitated by sonication, leading to the exposure of amino acid residues and side chains with antioxidant capacity and more free radical reaction sites [20]. Additionally, the molecular weight of the peptides in the hydrolysates as well as the content of aromatic amino acids were closely related to their antioxidant activity [53]. Thus, the promotion of solubilization of some peptides in insoluble aggregates of hydrolysates by ultrasound may be another reason for the enhancement of antioxidant activity. In summary, ultrasound of appropriate power is a promising method to enhance the antioxidant activity of FMBPHs.

3.6 The changes in protein and peptide composition and distribution of FMBPHs

The differences in protein and peptide composition and the number of active peptides in FMBPHs and ultrasonicated-FMBPHs were evaluated by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) analysis. The innermost part of the venn diagram (Fig. 5a & 5b) indicated amount of proteins or peptides common to all sample groups, the more outwardly the sets intersected the less until the outermost side indicates amount of uniquely owned proteins or peptides. There were 219, 232, 236, 272, 290, 280, 252 proteins (Fig. 5a) and 370, 379, 410, 460, 508, 465 peptides (Fig. 5b) were identified respectively. The number of proteins and peptides of the samples increased obviously after ultrasound compared to that of FMBPHs. Additionally, the number of proteins and peptides increased and then decreased with the increasing of ultrasound power. The maximum number of proteins and peptides was observed in the FMBPHs – 350 W. The number of unique peptides and proteins in the samples exhibited the same trends, in which the number of unique proteins and peptides of FMBPHs increased after ultrasound treatment, with a tendency of increasing and then decreasing. Particularly, when the ultrasound power was greater than 150 W, the number of unique proteins and peptides increased markedly from 16 to 26––37 and from 29 to 55–72, respectively. All results suggested that ultrasonic treatment changes the protein and peptide composition of FMBPHs and that appropriate ultrasonic treatment can increase the number of peptides in the hydrolysate. The changes in the number of proteins and peptides may be attributed to the deagglomeration effects of higher intensity ultrasound that degraded the insoluble peptide aggregates, resulting in the release of small soluble peptides by breaking non-covalent interactions [54]. Similarly, Tian et al. [14] reported that ultrasound treatment increased the percentage of peptides with molecular weight < 1 kDa.Fig. 5 Effects of ultrasound power on the protein and peptide composition and distribution of FMBPHs. Protein venn diagram (a), peptide venn diagram (b), highly active peptide number (peptideranker score > 0.5) (c), distribution of highly active peptides (peptideranker score > 0.5) (d) of FMBPHs and ultrasonicated-FMBPHs.

To ascertain whether changes in the number of peptides induced by ultrasound were the primary cause of the increased activity in the hydrolysates, the peptideranker was employed to predict the activity of all peptides and to investigate the impact of ultrasound on the number of active peptides. A score greater than 0.5 indicates potential physiological activity [55]. Fig. 5c and 5d show the number and distribution of predicted active peptides in different samples. As shown in Fig. 5c, the number of active peptides in FMBPHs increased after ultrasound treatment with different powers. With the increase of ultrasonic power from 60 W to 600 W, the number of active peptides in FMBPHs first increased and then decreased. This indicated that appropriate ultrasound power could elevate the number of active peptides in FMBPHs. This may be attributed to the destruction of non-covalent interactions in the insoluble aggregates in FMBPHs by ultrasound, leading to the release of small soluble peptides and an increase in the number of active peptides, whereas excessive ultrasound power leads to the reaggregation of these smaller peptides, resulting in a decrease in the number of active peptides [14]. Notably, a greater number of active peptides were observed in the hydrolysates after 350 W and 450 W ultrasound treatments, with an increase of 39 and 30 peptides, respectively, compared to the FMBPHs. Additionally, more highly active peptides (peptideranker score ≥ 0.8) were observed under high power conditions (FMBPHs – 350 W, FMBPHs – 450 W, FMBPHs – 600 W), with increases of 60.87 %, 47.83 %, and 39.13 %, respectively, compared those of FMBPHs (Fig. 5d). This result was consistent with the in vitro antioxidant results (Fig. 4). This demonstrated that suitable ultrasound power can be used to enhance the physiological activity of the hydrolysate by boosting the number of active peptides. Thus, differences in the number of unique peptides may be a critical factor in the differences in the functionality and activity of FMBPHs induced by ultrasound.

3.7 The abundance of identical peptides and high coverage protein of FMBPHs

In addition to the effect of differences in peptide composition on hydrolysate properties, changes in peptide content (relative abundance) are also important influences. The heat map analysis was used to explore the changes in the abundance of the identical peptides (81) in samples ultrasonicated at different powers. As shown in Fig. 6a, the abundance of peptides in all samples changed significantly after sonication compared with that of FMBPHs. An increase in the relative abundance of most of the peptides was observed at higher power. Particularly, cluster analysis revealed that the FMBPHs – 600 W had the greatest difference in relative abundance of the identical peptides from the FMBPHs. This demonstrated that ultrasound could induce changes in the structure of peptides in the hydrolysis products, leading to changes in their relative abundances. This can be due to the stronger cavitation effect on the peptide structure [20]. Since the content of active peptides may determine changes in hydrolysate activity, it was hypothesized that an increase in the relative abundance of highly active peptides among the identical peptides may be the crucial reason for the ultrasound enhanced activity of FMBPHs. For the purpose of testing this hypothesis, the activity of the identical peptides in all samples was predicted using peptideranker and the impact of ultrasonication on the relative abundance of these active peptides was explored. There were 28 out of 81 peptides predicted to be highly active peptides (Fig. 6b). There were 22 active peptides with increased relative abundance values after ultrasound treatment compared to that of FMBPHs. In particular, an increase in the relative abundance of 17 active peptides was detected in the 450 W sonicated samples. Among them, the relative abundance values of WPLPPFGE and VAPADPELPPPREPVGGAR increased by 2.41 and 1.44-fold, respectively. It suggested that ultrasound may improve the activity of FMBPHs by increasing the relative abundance of active peptides in them.Fig. 6 Effects of ultrasound power on the abundance of identical peptides of FMBPHs. Heatmap analysis of identical peptides (a) and highly active peptides (peptideranker score > 0.5) (b) from FMBPHs and ultrasonicated-FMBPHs. The red dashed line in Fig. a indicated the highly active peptides predicted by peptideranker. In Fig. b, yellow frames indicated peptides with increased abundance after sonication, and red frames indicated peptides with increased abundance after 450 W sonication.

Since FMBP subunits are widely distributed, strongly represented proteins with high coverage were selected for peptide map to clarify the effect of ultrasound on FMBPHs. Sun et al. [28] also explored the effect of processing on proteins in preterm infant formulas by performing peptide profiling of high-coverage milk proteins. In order to distinguish K3ZXN8 from the A0A368SG44 protein, K3ZXN8 was named Oleosin 1. Fig. 7a-e displayed the differences in the peptide map of high-coverage proteins Oleosin (A0A368SG44), Ubiquitin-like domain-containing protein (K3YTX1), Cupin type-1 domain-containing protein (K4A875), Oleosin 1 (K3ZXN8), and 40S ribosomal protein S19 (K4AGC5)) in FMBPHs and ultrasonicated-FMBPHs. The color depth, width, and length of each band in the peptide map indicate relative abundance, amino acid coverage, and overlap ratio, respectively [18]. In comparison with FMBPHs, the relative abundance of SSDTIDNVKAK (AA130 – 138, AA130 – 138, AA282 – 290) in Ubiquitin-like domain-containing protein (K3YTX1) (Fig. 7b), SEGGHGPHWPLPPFGE (AA256 – 271) in Cupin type-1 domain-containing protein (K4A875) (Fig. 7c), RDLDQVAGR (AA132 – 140) in 40S ribosomal protein S19 (K4AGC5) (Fig. 7d), and MGGGGGGYGDLHRGGER (AA9 – 25) in Oleosin 1 (K3ZXN8) (Fig. 7e) were obviously reduced. Meanwhile, the relative abundance of WPLPPFGE (AA264 – 271), SESEEQEQEE (AA369 – 378), and SREEREEEEEHEGRRGPK (AA477 – 494) in Cupin type-1 domain-containing protein (K4A875), as well as DIKDAAQHRIDQA (AA139 – 151) in Oleosin 1 (K3ZXN8) increased in relative abundance from 69.6, 72.4, 80.8, and 84.8 to 237.5, 161.6, 161.9, and 110.3, respectively. The reduced abundance of peptides suggested that ultrasound masked these peptides, while the increase in abundance indicated peptide exposure. This may be attributed to ultrasound-induced structural changes that lead to the exposure and concealment of some peptides [20]. However, the relative abundance of oleosin peptides was not obviously changed after ultrasound treatment. In summary, ultrasonication could lead to changes in the relative abundance of peptides in the hydrolysate, and Ubiquitin-like domain-containing protein, Cupin type-1 domain-containing protein, 40S ribosomal protein S19, and Oleosin 1 are closely associated with the changes in activity and processing characteristics of FMBPHs.Fig. 7 Effects of ultrasound power on the abundance of high coverage protein of FMBPHs. Peptide map analysis of oleosin (A0A368SG44) (a), ubiquitin-like domain-containing protein (K3YTX1) (b), cupin type-1 domain-containing protein (K4A875) (c), 40S ribosomal protein S19 (K4AGC5) (d), and oleosin 1 (K3ZXN8) (e) from FMBPHs and ultrasonicated-FMBPHs.

4 Conclusion

The study investigated the effects of ultrasound at different power levels (0 – 600 W) on the structure, functionality, antioxidant capacity, peptide profile and active peptides of FMBPHs. The 450 W ultrasonic treatment induced an unfolding of the protein structure of FMBPHs, leading to a shift from a rigid to a flexible structure (increase in random coils and β-turn as well as decrease in α-helix and β-sheet) and the exposure of internal hydrophobic groups. Thus, ultrasonicated FMBPHs exhibited smaller average particle size, higher absolute value of ζ-potential, and more small porous particles in the microstructure than FMBPHs. Meanwhile, the ultrasonication further improved the solubility, foaming properties, emulsifying properties, thermal stability, and in vitro antioxidant capacity of FMBPHs by modifying their protein or peptide structure. Additionally, peptidomics revealed that higher power sonication led to an increase in the number of active peptides of FMBPHs as well as an enhancement in the abundance of active peptides. The changes in the peptide profiles of Ubiquitin-like domain-containing protein, 40S ribosomal protein S19, Cupin type-1 domain-containing protein, and Oleosin 1 induced by ultrasound treatment have affected the characterization of FMBPHs. In conclusion, 450 W ultrasound treatment improved the protein structure of FMBPHs, increased the quantity of active peptides within FMBPHs, and enhanced their functional properties and antioxidant capacity. This study promotes the high-value utilization of FMB and offers the possibility of producing highly active functional foods from foxtail millet bran hydrolysate. In the future, cell and animal assays will be required to verify the effect on physiological activity.

CRediT authorship contribution statement

Zeyu Peng: Writing – original draft, Methodology, Investigation, Conceptualization. Fei Wang: Software, Resources. Luming Yu: Software, Investigation. Bo Jiang: Investigation. Jia Cao: Software, Data curation. Zhigang Sun: Resources, Conceptualization. Jianjun Cheng: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank the financial support received from the Major Science and Technology Projects in Heilongjiang Province (Project No. 2021ZX12B06).
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