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

S1350-4177(24)00273-6
10.1016/j.ultsonch.2024.107025
107025
Original Research Article
Effects of ultrasound pretreatment on the structure, IgE binding capacity, functional properties and bioactivity of whey protein hydrolysates via multispectroscopy and peptidomics revealed
Pang Lidong a
Liu Ming a
Chen Chen a
Huang Zhen a
Liu Shiyu a
Man Chaoxin a
Jiang Yujun ab
Zhang Wei 50834927@qq.com
a⁎
Yang Xinyan yangxinyan@neau.edu.cn
a⁎
a Key Laboratory of Dairy Science, Ministry of Education, College of Food Science, Northeast Agricultural University, Harbin 150030, China
b Food Laboratory of Zhongyuan, Luohe 462300, Henan, China
⁎ Corresponding authors. 50834927@qq.comyangxinyan@neau.edu.cn
17 8 2024
11 2024
17 8 2024
110 10702520 5 2024
30 7 2024
10 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

Whey protein is an important food ingredient, but it is also considered a major food allergen. The aim of this study was to investigate the effect of ultrasound pretreatment on the structure, IgE binding capacity, functional properties and biological activity of whey protein isolate (WPI) hydrolysates (WPH), including WPI hydrolyzed by a combination of enzymes from Bromelain and ProteAXH (BA-WPI) and WPI hydrolyzed by a combination of enzymes from Papain W-40 and ProteAXH (PA-WPI). The IgE binding capacity of BA-WPI and PA-WPI was reduced to 40.28% and 30.17%, respectively, due to disruption/exposure/shielding of conformational and linear epitopes. The IgE binding capacity of sonicated WPI was increased, but ultrasound pretreatment further reduced the IgE binding capacity of the hydrolysates to 32.89% and 28.04%. This is due to the fact that ultrasound pretreatment leads to conformational changes including increased α-helix and β-sheet structure, exposure of aromatic amino acids, surface hydrophobicity, and increased sulfhydryl content, which increases the accessibility of allergenic epitopes to WPI by the enzyme. Multispectral and LC-MS/MS results further indicated that ultrasound pretreatment altered the conformational and primary structural changes of the hydrolysates. The thermograms showed that ultrasound pretreatment mainly altered the epitope spectra of β-lactoglobulin hydrolysates, while it had less effect on the epitope spectra of α-lactalbumin hydrolysates. Additionally, ultrasound pretreatment significantly improved the foaming properties, antioxidant activity, and α-glucosidase inhibition of the hydrolysates without impairing the solubility and emulsification properties of the hydrolysates. Therefore, ultrasound pretreatment is a feasible method to reduce the allergenicity of WPH and to improve their functional properties and bioactivity. Notably, ultrasonic pretreatment improved the effectiveness and efficiency of WPI hydrolysis, which is a feasible method to produce high-quality protein feedstock in a green, efficient, and economical way.

Keywords

Whey protein
Ultrasound
Enzymatic hydrolysis
IgE binding capacity
Functional properties
Bioactivity
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pmc1 Introduction

Food allergy is a worldwide food safety problem [1]. It affects about 4 % of adults and 10 % of children, and its incidence is increasing [2]. Eight allergenic foods including milk, crustacea, eggs, sesame, fish, peanuts, specific tree nuts, and cereals containing gluten cause more than 90 % of food allergies, and proteins are their main allergens [3]. Whey protein is the main component of milk protein, accounting for about 20 %, and is widely used in the development of functional foods with potential health benefits due to its excellent functional properties and biological activity [4]. However, it is also one of the top allergens in milk [5]. The binding of allergic epitopes of whey proteins to allergen-specific antibodies on mast cells causes an IgE-mediated adverse reaction, resulting in skin, intestinal, and respiratory symptoms that may be life-threatening in severe cases [6]. Therefore, Therefore, the development of methods to reduce whey proteins allergenicity is critical.

Numerous studies have shown that the functional properties or allergenicity of whey proteins are improved after treatments including ultrasound, enzymatic hydrolysis, high hydrostatic pressure, glycation, and covalent binding to chlorogenic acid [6], [7], [8], [9], [10]. However, single treatments rarely improve the functional properties of whey proteins while reducing their allergenicity. Therefore, the researchers tried to solve this problem through a combination of physical and biochemical treatments.

Ultrasound is a proven, safe and green processing technology and has been widely utilized [11], [12], [13]. It produces mechanical and chemical effects that alter modify protein structure, influencing their allergenicity and functional properties [14]. Although ultrasound improves whey protein functional properties, it does not reduce its allergenicity [15], [16], [17]. Notably, ultrasound as a pre-treatment method can increase the effectiveness and efficiency of other processes. Bu et al. found that ultrasound pre-treatment allowed glycation to further reduce the allergenicity of α-lactalbumin [18]. Similarly, Yang et al. found that ultrasound pre-treatment promotes ovalbumin glycation by increasing glycation sites and efficiency, which reduced its allergenicity and improved its functional properties [19]. In addition, ultrasound pretreatment induces structural changes in whey proteins, resulting in whey proteins that are more sensitive to enzymatic hydrolysis and thus produce more bioactive peptides [20]. However, the effect of ultrasound pretreatment on whey protein hydrolysates (WPH) allergenicity has not been reported, let alone the improvement of functional properties while reducing allergenicity.

Enzymatic hydrolysis is the most well-established and effective method for reducing the allergenicity of food proteins and has been widely used in commercialized products [21], [22]. However, enzymatic hydrolysis treatment alone may not completely eliminate proteins allergenicity and may impair their functional properties [3]. It was reported that physical processing pre-treatment could improve the efficiency and hydrolysis site by improving enzyme accessibility, which further reduces protein allergenicity and improves functional properties. For example, Lorenzetti et al. found that ultrasound pre-treatment could shorten the hydrolysis process of whey proteins by 6 h [23]. Liu et al. found that sequential treatment with microwave and enzymatic hydrolysis reduced the allergenicity of ovalbumin more significantly [24]. In addition, Shao et al. reported that compared to non-ultrasound-pretreated β-lactoglobulin (β-Lg), ultrasound-pretreated β-Lg was less allergenic after digestion [25]. This suggests that sequential treatment with ultrasound and enzymatic hydrolysis has the potential to further reduce protein allergenicity. However, in order to reduce allergenicity, low-specificity enzymes must be explored to reduce allergenicity. This is because low-specificity proteases cleave proteins at more sites than specific proteases, thereby destroying more allergic epitopes [26]. In addition, it has been shown that the combined use of endopeptidases and exopeptidases may be effective in reducing allergenicity and improving functional properties [27], [28]. Therefore, this is a promising approach for developing proteins that reduce their allergenicity while improving their functional properties. However, studies using combinations of endopeptidases and exopeptidases to improve whey proteins functional properties and reduce their allergenicity have not been reported.

This study investigated the effect of ultrasound pre-treatment on the allergenicity, functional properties and bioactivity of whey protein isolate (WPI) hydrolyzed by the combination of endopeptidases and exopeptidases. Changes in protein structure were measured by multispectroscopy and high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Changes in peptide profiles, epitope profiles and bioactive peptides were identified by peptidomics and bioinformatics. These results will give us new insights into the role of successive treatments of ultrasound and enzymatic hydrolysis in reducing whey proteins allergenicity and improving their functional properties and bioactivity.

2 Materials and methods

2.1 Materials

WPI (9410, 90 %) from Hilmar Ingredients (California, USA). Papain W-40 (≥400 U/mg, endopeptidase from Carica papaya L.) and ProteAXH (≥1400 u/g, exopeptidase produced by fermentation of Aspergillus oryzae) were from Amano Enzyme Inc. (Nagoya, Japan). Bromelain (≥3 U/mg, endopeptidase from the stem of pineapple) was from Sigma-Aldrich (St. Louis, MO, USA). Human sera and goat anti-human immunoglobulin E (IgE) horseradish peroxidase (HRP) conjugate from 10 milk-allergic patients were obtained from Wolcavi Biotechnology Co., Ltd. (Chongqing, China). Human sera information is shown in Table S1. 10 sera were pooled together for the IgE binding capacity assay. All other chemical reagents were at or better than analytical grade.

2.2 Sample preparation

One hundred millilitre of 1 mg/mL WPI solution was placed in the beaker and processed with a probe sonicator for 10 min (XM-650 T All-in-One Ultrasonic Cell Pulverizer from Xiaomei Ultrasonic Instrument Co., Ltd, China) at 25 kHz, 650 W in the ice water bath. The untreated and ultrasound WPI were hydrolyzed with a combination of bromelain and ProteAXH or a combination of Papain W-40 and ProteAXH. The ratios of both bromelain and Papain W-40 to ProteAXH were 1:1 (w/w). Hydrolysis conditions: pH 7.0, 55 °C, 1 h, protease to WPI ratio of 1:50 (w/w). Enzyme inactivation is achieved by heating the hydrolysate at 95 °C for 5 min. The supernatant was obtained by centrifugation at 4000 × g with 15 min. All samples were dried by vacuum drying overnight to obtain lyophilized powder for subsequent experiments. Untreated and ultrasounded WPI were named WPI and US-WPI, respectively. WPI treated with the combination of bromelain and ProteAXH or Papain W-40 and ProteAXH were named BA-WPI, PA-WPI, respectively. US-WPI treated with the combination of bromelain and ProteAXH (BA enzyme) or the combination of Papain W-40 and ProteAXH (PA enzyme) were named US-BA-WPI, US-PA-WPI, respectively.

2.3 Degree of hydrolysis (DH)

Mix 400 μL of WPH with 3 mL of o-phthalaldehyde (OPA) reagent. Incubate for 5 min at room temperature and measure the absorbance at 340 nm [29].

2.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Concentration gels (4.5 %) and separation gels (15 %) for SDS-PAGE were prepared (Shanghai Yase Biomedical Technology Co., Ltd.). 1 mg/mL of sample was mixed with SDS uploading buffer and boiled. The sample volume was 10 μL, and the voltage was set to 80 V at the beginning of electrophoresis, and 120 V when the samples were put into the separation gels. BeyoBlue™ Caulmers Brilliant Blue Ultrafast Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd., China) was used to stain proteins for 1 h. Then, decolorization was performed using ultrapure water.

2.5 Particle size and zeta potential

Measurements of particle size and zeta potential of the 0.2 mg/mL samples were taken using the Nano Zetasizer (Malvern Instruments Ltd, UK) [30].

2.6 Structural analysis

2.6.1 Fourier transform infrared (FTIR) spectroscopy

The samples powders were blended and pressed with potassium bromide and their absorbance was measured at 400–4000 cm−1 using the FTIR spectrometer (Nicolet iS5, Thermo Fisher, USA) [31].

2.6.2 Circular dichroism (CD) spectroscopy

Measurements of the 0.1 mg/mL sample were performed using the CD spectrometer (Chirascan, Applied Photophysics, UK) at 195–260 nm [32].

2.6.3 UV absorption spectroscopy

Measurement of sample absorbance at 280–400 nm by UV–Vis spectrophotometer (UV-2600, Shimadzu Instruments Co., Ltd.).

2.6.4 Intrinsic fluorescence spectroscopy

The samples were measured using a fluorescence spectrometer (RF6000, Shimadzu, Japan) at 600 nm/min. The wavelength of excitation and emission were 280 nm and 300–400 nm, respectively.

2.6.5 Surface hydrophobicity (H0)

Mix 4 mL of sample solution and 20 μL 8-Anilino-1-naphthalenesulfonic acid (ANS) solution (8 mmol/L, pH 7.4). Incubate at room temperature away from light for 20 min, and then measured the fluorescence intensity. Wavelength of excitation and emission are 390 nm and 470 nm, respectively [33].

2.6.6 Total sulfhydryl (SH) group content

To 1 mL of 1 mg/mL of sample dissolved in Tris-glycine buffer (pH 8.0) containing 8 M urea was added 10 μL 4 mg/mL Ellman's reagent. Incubate at room temperature for 15 min away from light, and then measure absorbance at 412 nm (A412) using a SpectraMax i3x multifunctional microplate reader (Molecular Devices Co., Ltd., CA, USA) [34]. SH (μM/g) = 73.53 × A412 × D/C.

2.7 IgE binding capacity

First, one hundred microliter of 5 μg/mL sample was incubated in a 96-well plate at 4 °C for 12 hrs. However, the wells were sealed with 3 % gelatin solution. Add milk allergy patient serum (1:20 dilution). Add goat anti-human IgE HRP conjugate (1:3000 dilution). After each of the above steps, the wells are washed three times with 0.05 % Tween-20 in PBS buffer (PBST buffer). Finally, TMB one-component substrate solution (Beijing Sola Biotechnology Co., Ltd.) was used for color development, and the reaction was then terminated by the addition of H2SO4 solution [35]. Measure the absorbance at 450 nm (OD450) and take the IgE binding capacity of WPI as 100 %.IgE-binding capacity (%) =OD450(WPH)OD450(WPI)×100

2.8 Functional properties

2.8.1 Solubility

The samples were shaken at 150 rpm at 30 min to dissolve completely in ultra-pure water at a concentration of 1 mg/mL. 15 min centrifugation at 7500 × g was performed, and measured for soluble proteins in the supernatant by bicinchoninic acid (BCA) method [36].Solubility%=SolubleproteincontentTotalproteinconcentration×samplevolume×100

2.8.2 Foaming capacity (FC) and foaming stability (FS)

Ten millilitre (V0) 1 mg/mL sample was homogenized at 10,000 rpm at room temperature with 1 min (T18 digital ULTRA-TURRAX, IKA Instrumente & Ausrüstung Co.). Leave them to stand at room temperature. Record the volume of each sample at 0 min (VT) and 10 min (Vt) [37].FC(%)=VT-V0V0×100

FS(%)=Vt-V0VT-V0×100

2.8.3 Emulsifying capacity (EAI) and emulsion stability (ESI)

Two mL of olive oil was used as the oil phase, and 6 mL of 0.5 mg/mL hydrolysate solution (diluted in PBS) was homogenized (T18 digital ULTRA-TURRAX, IKA Instrumente & Ausrüstung Co.) at 10,000 rpm for 1 min. At 0 and 10 min, 50 μL emulsion was taken from the bottom of the vessel and diluted 100 times with 0.1 % SDS solution (w/v). The mixture was stirred for 10 s, 200 uL of the mixture was taken and the absorbance of the diluted solution was measured at 500 nm using an enzyme meter. The absorbance of the diluted samples measured at 0 min (A0) and 10 min of emulsification (A10) was used to calculate the EAI and ESI [38].EAI(m2g)=2×2.303×DF×A0c×0.25×10000

ESI(min)=A0A0-A10×10

Note: DF is the dilution factor, t is the resting time after homogenization, c is the concentration of the hydrolysate (g/mL), 0.25 is the amount of olive oil fraction, and A0 and A10 are the sample absorbance at 0 min and 10 min, respectively.

2.9 Bioactivities

2.9.1 Total antioxidant capacity

Total antioxidant capacity was determined according to the method provided by the reagent supplier (Nanjing Jianjian Bioengineering Institute Co., Ltd., China). 10 μL of 10 mg/mL sample was added to 20 μL of peroxidase application solution and 170 μL of ABTS working solution and reacted for 6 min at room temperature. After 6 min reactions in room temperature, read the absorbance at 405 nm of the mixtures.

2.9.2 Ferric ion reducing antioxidant power

One hundred microliter of 5 mg/mL sample, 250 μL of water and 250 μL of K3[Fe(CN)6] (1 %, w/v) were blended and incubated in a 50 °C water bath for 20 min [39]. Then 250 μL of TCA (10 %, w/v) was added and centrifuged 10 min at 1500 × g. Mix 500 μL supernatant with 500 μL water and 100 μL FeCl3 (0.1 %, w/v). Incubate 10 min at room temperature and read the absorbance in 700 nm [39].

2.9.3 DPPH radical scavenging capacity

DPPH radical scavenging capacity was measured based on the reagent supplier's method (Nanjing Jianjian Bioengineering Institute Co., Ltd., China). Specifically, mix 400 μL of 1 mg/mL sample with 600 μL of DPPH working solution. Keep away from light for the reaction at room temperature for 30 min. Measure supernatant absorbance at 517 nm after centrifugation of 5 min at 4000 rpm.

2.9.4 α-Glucosidase inhibitory ability

Mix 50 μL of 2 mg/mL sample with 50 μL of α-glucosidase solution and incubate at 37 °C at 10 min. Add 50 μL pNPG solution and incubate at 37 °C for 30 min. Finally, add 100 uL of 0.5 mol/L Na2CO3 solution and read the absorbance at 405 nm [40]. The lower the absorbance, the higher the α-glucosidase inhibition of the sample.

2.10 LC-MS/MS

LC-MS/MS was performed according to the method of Cui et al. [27]. The enzymatically hydrolyzed samples were filtered through a 10 kDa cut-off filter and desalted using a C18 column head. Separate the peptides in the sample with EASY-nLC 1200 (Thermo Fisher Scientific, Massachusetts, USA). The peptides were finally identified using a Q-Exactive system (Thermo Fisher Scientific, Massachusetts, USA).

2.11 Peptide identification

Data were analyzed using Proteome Discoverer 2.2 software (Thermo Fisher Scientific). Allergenic peptides in the samples were identified with the Biopep database (https://www.uwm.edu.pl/biochemia). The bioactive activity of the peptides was predicted for all samples using the PeptideRanker platform (https://distilldeep.ucd.ie/PeptideRanker). Biologically active peptides in WPH were identified using the Biopep database and the MBPDB database (https://mbpdb.nws.oregonstate.edu/).

2.12 Statistical analyses

All trials were conducted in triplicate. SPSS 26 is used to assess the results, which are expressed as mean ± SD. Significant differences between groups are represented by different letters (P<0.05).

3 Results and discussion

3.1 DH

DH is closely related to the specificity of protease on WPI and reflects the hydrolysis effect of protease on WPI. In this study, ultrasound pre-treatment was used for enhancing the hydrolysis efficiency of protease on WPI. Meanwhile, the combination of endopeptidase and exopeptidase was employed to further improve the hydrolysis efficiency, thus destroying as many allergic epitopes as possible. The protein hydrolysates of this study may have lower bitter taste and superior functional properties due to higher levels of exopeptidases. Meinlschmidt et al. found that the combined use of endopeptidases and exopeptidases resulted in hydrolysates with low levels of intact allergenic proteins, low bitterness, and superior technical-functional properties [28]. Shown in Fig. 1A, the DH of the products of both ultrasound-enzyme hydrolysis combined treatments were significantly increased compared to the products of enzymatic hydrolysis alone. US-BA-WPI and US-PA-WPI showed an increase of 1.54 % and 3.32 % compared to BA-WPI and PA-WPI, respectively, which suggests that ultrasound pre-treatment can increase the hydrolysis efficiency of proteins by proteolytic enzymes. This finding is agreeable with Liang et al. who showed that β-Lg pretreated with short ultrasound time (20 min and 30 min) showed a significant increase in DH after hydrolysis by Alcalase [41]. This is caused by ultrasound de-folding the protein, thereby exposing more enzymatic sites. Moreover, the inconsistent degree of DH enhancement may be related to the specificity of the protease.Fig. 1 DH (A), SDS-PAGE (B), Particle size (C), and Zeta potential (D).

3.2 SDS-PAGE

The results of SDS-PAGE are shown in Fig. 1B, where α-La (about 14 kDa), β-Lg (about 18 kDa), and BSA (about 66.4 kDa) were the major proteins in the WPI. The similarity of the bands for WPI and US-WPI shows that ultrasound has no significant effect on WPI's molecular weight. Previous studies found the same result [42]. Meanwhile, there was no significant difference in the bands of US-BA-WPI versus BA-WPI, but there was a decrease in β-Lg in US-PA-WPI compared to PA-WPI. This may be due to the fact that sonication of WPI, although exposing more cleavage sites, had little effect on the ability of bromelain and ProteAXH to degrade WPI, whereas Papain W-40 and ProteAXH were sensitive to the sites exposed on β-Lg.

3.3 Particle size and zeta potential

The particle size of protein ii intimately related with its functional properties [43]. As shown in Fig. 1C, the particle size of WPI (264.0 nm) was significantly reduced after either ultrasound or enzyme hydrolysis treatments, to 198.5 nm (US-WPI), 178.5 nm (BA-WPI) and 191 nm (PA-WPI), respectively. This is due to the fact that shear force, impact force or vortex generated by ultrasound disperses the protein aggregates, and enzymatic hydrolysis disrupts the aggregation and structure of WPI by enzymatic cleavage of WPI by proteolytic enzymes. Similar results were obtained in studies of enzymatic hydrolysis of goat milk whey protein [40]. In addition, the particle sizes of both US-BA-WPI (155.0 nm) and US-PA-WPI (159.3 nm) were also significantly reduced compared to the corresponding WPH without ultrasound treatment. This may be attributed to the fact that ultrasound treatment not only made WPI more dispersed, but also loosened the structure of WPI, which allowed further enzymatic hydrolysis to disrupt the structure of WPI more effectively.

Zeta potential reflects the intensity of repulsion or attraction between the particles, and its high absolute value implies a stable system [41]. Our results (Fig. 1D) showed that the zeta potential of US-WPI, BA-WPI's were not statistically significant (P>0.05) compared to WPI's, but PA-WPI was significantly increased. US-BA-WPI was significantly decreased compared to BA-WPI, but US-PA-WPI was not statistically significant (P>0.05) compared to PA-WPI. This may be related to the specificity of the protease.

3.4 Structural characterization

3.4.1 FTIR spectroscopy

Fig. 2A shows the FTIR spectral region of WPI and its hydrolyzates in the range of 4000 to 400 cm−1, reflecting the variation of protein secondary structure. In the FTIR spectrum of WPI, the amide A band (3700–3000 cm−1) represents NH stretching and hydrogen bonding with a major absorption peak of 3224.29 cm−1; the amide I band (1700–1600 cm−1) represents C=O stretching with a major absorption peak of 1650.07 cm−1; the amide II band (≈1540 cm−1) represents CN stretching and NH bending modes, with a major absorption peak of 1541.25 cm−1 [44]. For the amide A band, the peaks of US-WPI, BA-WPI, PA-WPI, US-BA-WPI, and US-PA-WPI are shifted toward 400 cm−1 and their intensities are all significantly increased compared to WPI, suggesting that the hydrogen bonding of WPI has changed [45]. Compared to BA-WPI or PA-WPI, the absorption intensity of US-BA-WPI or US-PA-WPI increased and the peak was shifted. This suggests that ultrasound pre-treatment not only destroys or generates new hydrogen bonds, but that the hydrogen bonds of the pre-treated proteins also undergo changes during enzymatic hydrolysis that are different from those of enzymatic hydrolysis alone, and that such changes are related to the specificity of the enzyme. The amide I band was similarly shifted from its peak and hydrolysis or ultrasound combined with enzymatic hydrolysis had a greater effect on WPI, whereas ultrasound alone had a small effect. Notably, ultrasound pretreatment did not have a significant effect on the amide I band wave number of PA-WPI, but it did have a significant effect on BA-WPI. This shows that the influence of ultrasound pretreatment on WPH secondary structure varies depending on the protein species. The peaks and intensities in the amide II region also changed, with trends similar to those in the amide A region. The amide III band also changed. These are further evidences of changes in WPI secondary structure [46]. It also shows that the WPI was treated with changes in CN stretching and NH bending modes. In addition, it can also be concluded from the results of amide II and amide III bands that US-BA-WPI and US-PA-WPI were changed compared to both BA-WPI and PA-WPI. This indicates that the ultrasound pretreatment further affected the changes in WPI secondary structure. The secondary structural changes were similar with the results of Meng et al. for ultrasound-modified WPI [42], Abadía-García et al. for the hydrolysis of whey proteins with ultrasound in combination with bromelain [47], and Kaur et al. for the induced hydrolysis of concentrated whey proteins with kiwiin [48]. The reason for the incomplete agreement may be due to the fact that the complex enzyme used in this study contained exopeptidase [30].Fig. 2 FTIR (A), CD (B), UV absorption spectrum (C), Intrinsic fluorescence spectrum (D), H0 (E), Total SH content (F).

3.4.2 CD spectrum

CD spectroscopy is considered to make one of the most effective tools for assessing changes in protein secondary structure [49]. To further assess the secondary structure changes, we employed CD spectroscopy to characterize samples. The results are shown in Fig. 2B, where WPI and US-WPI have negative peaks at 208 nm, and the strength of US-WPI is below that of WPI, which suggests that ultrasound decreases the α-helix content of WPI [50]. The peak of the product of enzymatic hydrolysis is shifted to an offset around 200 nm, which indicates a more stretched secondary structure of WPI [51]. Meanwhile, compared to BA-WPI, US-BA-WPI has a smaller peak shift despite its higher intensity, suggesting that its degree of unfolding may be smaller. In addition, the difference in CD spectral changes between US-PA-WPI and PA-WPI was not significant, suggesting that ultrasound pretreatment had less effect on the secondary structure of WPI hydrolyzed by PA enzymes. This is in agreement to the secondary structure changes observed in the FTIR spectra. Meanwhile, a broad peak around 220 nm is characteristic of the β-sheet [52]. Compared to WPI, the peak intensities of BA-WPI, PA-WPI, US-BA-WPI, and US-PA-WPI showed enhancement, which indicated the increase of β-sheet. The combined FTIR results reveal that enzymatic hydrolysis is responsible for the transformation of α-helices into β-sheets by altering hydrogen bonding, and that ultrasound pretreatment influences the changes in WPI secondary structure. In summary, enzymatic hydrolysis can stretch the structure of WPI and ultrasound pretreatment can affect this change.

3.4.3 UV absorption spectroscopy

UV absorption spectroscopy is an important method used to assess conformational changes in proteins, reflecting changes in tyrosine and tryptophan residues in protein side-chain molecules [53]. Compared to WPI, the UV-absorbed intensities of the treatment groups were increased (Fig. 2C), indicating that ultrasound and/or enzymatic hydrolysis caused the molecules to be unfolded and the buried hydrophobic groups to be exposed [54]. Similar results to the present study were obtained in a study on the effect of ultrasound pretreatment on the structure of rice protein hydrolysate [55]. Meanwhile, Compared to BA-WPI, US-BA-WPI was significantly stronger, which indicated that ultrasound pretreatment could promote the destruction of WPI tertiary structure by BA enzyme. However, the intensity of PA-WPI and US-PA-WPI did not differ significantly, but the peaks were slightly shifted, which indicated that ultrasound pretreatment did not have much effect on the tertiary structure destruction of WPI by PA enzyme. This is related to the specificity of the enzyme. In addition, both US-BA-WPI and US-PA-WPI showed reduced intensity and shifted peaks compared to US-WPI, which may be due to the destruction of the chromophore by the protease or the masking of the chromophore by structural changes. Similarly, Cui et al. found that the 60-min hydrolyzed products of milk proteins from both Flavourzyme and Alcalase had reduced UV absorption intensities compared to the 30-min hydrolyzed products [36].

3.4.4 Intrinsic fluorescence spectroscopy

Intrinsic fluorescence spectroscopy is also an important tool for characterizing changes in proteins tertiary structure, which reflect changes in the aromatic amino acids (tryptophan, tyrosine, and phenylalanine) of proteins [56]. Compared to WPI, the fluorescence intensity of the treated group underwent an increase and peak shift, indicating a conformational change of WPI after treatment (Fig. 2D), which is in agreement with the results of UV absorption spectroscopy. In particular, US-WPI showed an increase in intensity and a small peak shift, which could be attributed to the structural de-folding of WPI by ultrasound, resulting in the exposure of hydrophobic regions and an increase in H0 [57]. WPH not only underwent an increase in intensity, but also a large shift in the peak, suggesting an increase in Trp polarity owing to the defolding of the protein molecule [58]. Similarly, the study of β-Lg after ultrasound and enzyme treatment revealed a significant increase in fluorescence intensity and a redshift of the peak [59]. Compared to BA-WPI, the intensity of US-BA-WPI underwent enhancement and the peak was blue-shifted, suggesting that ultrasound treatment facilitated the disruption of the WPI conformation by the BA enzyme, which is in agreement with the results of UV absorption spectroscopy. In addition, the intensity of US-PA-WPI decreased and sent a red shift compared to US-WPI and PA-WPI, suggesting that the combined treatment with ultrasound and PA enzyme resulted in the exposure of amino acid residues of WPI with aromatic properties to a less hydrophobic environment [60].

3.4.5 H0

H0 is commonly utilized for characterize protein conformational changes, which is a key contributor to protein stability, function and microstructural properties [56]. As a result, as shown in Fig. 2E, the H0 of US-WPI, BA-WPI, PA-WPI, and US-BA-WPI were significantly increased compared to WPI, which may be caused by hydrophobic region exposure of WPI. This is in agreement with the results of Yang et al. [61]. However, there was no significant change in the H0 of US-PA-WPI, which was probably caused by the hydrolysis of PA enzyme after ultrasound treatment exposing more hydrophilic groups of WPI to the outside [62]. This corroborates with the results of intrinsic fluorescence spectroscopy.

3.4.6 Total SH content

SH is an important component of the protein structure and has a great influence on WPI properties [63]. The results, as shown in Fig. 2F, the SH content of US-WPI was significantly increased compared to WPI, which was due to the exposure of buried SH by forces such as shear and vortex generated by ultrasound. However, the SH content was reduced in WPH, which may due to sulfhydryl groups oxidation to disulfide groups during enzymatic hydrolysis and further induced cross-linking between protein molecules [64]. It is noteworthy that the enzymatic hydrolysis products after ultrasound pretreatment had higher total SH content compared to enzymatic hydrolysis alone. This is related to the increased accessibility of the protease by ultrasound pretreatment. An interesting phenomenon was that the increase in DH increased the total SH content, but the extent of the increase depended on the type of enzyme. Zhang et al. found that the increase in DH could enhance the exposure and formation of −S-S- bonds in hydrolysis [62].

3.5 IgE binding capacity

WPI allergy is caused by linear and conformational epitopes, which can trigger IgE type I hypersensitivity reactions leading to the development of severe allergic symptoms. Therefore, IgE binding capacity is a key indicator for assessing the allergenicity of WPI.

The allergenicity of WPI and WPH was evaluated by iELISA and the results are shown in Fig. 3A. Compared to WPI (100 %), US-WPI (105.37 %) showed a significant increase in IgE binding capacity, which was caused by ultrasound opening up the structure of WPI and exposing its internal allergic epitopes. Similarly, a study by Shao et al. found an increase in the IgE binding capacity of ultrasound-treated β-Lg [65]. However, the IgE-binding capacity of WPH was clearly reduced, which was associated with protease destroying the allergic epitope of WPI. Notably, the IgE binding capacity of US-BA-WPI (32.89 %) and US-PA-WPI (28.04 %) was significantly reduced compared to BA-WPI (40.28 %) and PA-WPI (30.17 %). This was caused by ultrasound increasing the accessibility of proteases to allergic epitopes [25]. Also, the variation in IgE binding capacity of different hydrolysates varied because of different specificity of proteases.Fig. 3 IgE binding capacity (A), solubility (B), FC (C), FS (D), EAI(E), ESI(F).

3.6 Functional properties

3.6.1 Solubility

Solubility is considered to be among the most important indicators of protein functional properties, and the emulsification and foaming properties of proteins can be improved by increasing solubility [66]. Fig. 3B shows the solubility changes of WPI, US-WPI and their hydrolysates. The solubility of US-WPI, BA-WPI, and US-BA-WPI was significantly improved compared to WPI. The main reason for the increase in solubility could be that the unfolding of the spatial structure of WPI related to the enhanced binding of WPI to water. However, the solubility of PA-WPI and US-PA-WPI was not improved. This may be due to the enzyme specificity and the fact that proteases cause WPI to form different insoluble aggregates during the hydrolysis of WPI [46]. However, the limited improvement in WPI solubility was unsatisfactory. Similarly, Ding et al. found that both trypsinized and alkaline protease hydrolysates of soy protein showed reduced solubility compared to untreated soy protein at pH 2.0–7.5 [67]. Meanwhile, ultrasound pretreatment did not significantly improve the solubility of enzymatically hydrolyzed WPI. This is similar to the findings of Yolandani et al. that ultrasound pretreatment did not significantly affect the solubility of soybean isolate protein hydrolysates [68]. Previous studies show that solubility increases with increasing DH [69]. However, in this study, ultrasound pretreatment had little effect on the WPH. Therefore, this may be the reason why ultrasound pretreatment did not significantly improve WPI solubility.

3.6.2 Foaming properties

Foaming properties perform an essential role in the processing of foods such as cake and ice cream [32]. Fig. 3C shows the FC of WPI, US-WPI and their hydrolysates, compared to WPI, US-WPI, BA-WPI, PA-WPI, US-BA-WPI, US-PA-WPI showed an increase in foaming characteristics by 24 %, 39 %, 38.67 %, 80.33 % and 70.67 %, respectively. Enzymatic hydrolysis, especially after ultrasound pretreatment, greatly increased the foaming properties of the enzymatic hydrolysis products because of the hydrolysis of the proteins into small fragments with smaller molecular morphology and faster migration rate, which allowed them to be adsorbed to the interface more quickly [60]. Meanwhile, the ability of ultrasound and/or enzymatic hydrolysis treatments to enhance the foaming properties of WPI is related to their ability to cause an increase in the flexibility of WPI as well as structural de-folding thereby increasing the ability of the WPI product to diffuse across the air–water interface to encapsulate the air. The results of the FTIR and CD spectroscopies further support this conclusion. Similarly, the FS of ultrasound and/or WPH was significantly increased (Fig. 3D). However, the FS of PA-WPI and US-PA-WPI was lower at 26.74 % and 28.09 %, whereas BA-WPI and US-BA-WPI showed higher FS. This is related to the specificity of the protease. Based on the analysis of the zeta potential results, PA-WPI and US-PA-WPI had higher absolute values of zeta potential, which suggests that their strong electrostatic intramolecular repulsion gives them low molecular flexibility, resulting in films with lower viscoelasticity [70]. WPI and US-WPI or BA-WPI and US-BA-WPI's FS are also consistent with this result. Notably, this is also related to the method of treatment/type of enzyme, which could be the reason why the FS changes of WPI, US-WPI and BA-WPI are not in line with this finding.

3.6.3 Emulsifying properties

Fig. 3E shows the EAI of WPI, US-WPI, BA-WPI, PA-WPI, US-BA-WPI, and US-PA-WPI, which were 41.15 %, 33.29 %, 49.99 %, 53.55 %, 51.34 %, and 55.03 %, respectively. The significant increase in EAI of the enzymatic hydrolysis products was attributed to their higher protein flexibility [71]. For the same protease, ultrasound pretreatment did not result in no significant change in the emulsification properties of the WPH. This was related to the fact that there was not a significant difference between their solubility. EAI is related in protein stability, molecular flexibility, solubility, H0, and protein structure [72]. ESI is the ability of the emulsion to remain stable for a specific period of time and the results are shown in Fig. 3F. The ESI tends to correlate negatively with EAI, and the enzyme hydrolysis did not significantly improve WPI's ESI. This may be due to the high hydrolysis of WPI by using endo/exo peptide complex enzymes in this study [73].

3.6.4 Antioxidant capacity

Fig. 4A shows that the total antioxidant capacity of WPI, US-WPI, BA-WPI, PA-WPI, US-BA-WPI, and US-PA-WPI were 12.37 %, 10.64 %, 45.20 %, 50.20 %, 49.05 %, and 51.76 %, respectively. Enzymatic hydrolysis treatment significantly increased the total antioxidant capacity of WPI, and the combined ultrasound and enzymatic hydrolysis treatment was significantly higher than that of enzymatically hydrolyzed WPI. This may be attributed to the fact that protease hydrolysis produced antioxidant active peptides, while ultrasound increased enzyme accessibility and thus produced more antioxidant active peptides [29], [9]. This result was further supported by the ferric ion reducing capacity (Fig. 4B). However, Fig. 4C shows that PA-WPI had the highest DPPH removal capacity, and the differences between the other groups and WPI were not statistically significant (P>0.05). Similarly, Cui et al. found that the results of different research methods for testing antioxidant capacity varied [29]. Therefore, antioxidant capacity must be characterized by multiple methods. In conclusion, enzymatic hydrolysis, especially ultrasound combined enzymatic hydrolysis, can significantly improve the antioxidant capacity of WPI.Fig. 4 Total antioxidant capacity (A), Ferric ion reducing capacity (B), DPPH clearance capacity (C) and α-Glucosidase inhibitory capacity (D).

3.6.5 α-Glucosidase inhibitory ability

Diabetes has become one of the most serious chronic diseases worldwide, and superior α-Glucosidase inhibitors are essential for lowering postprandial glucose to alleviate type 2 diabetes [74]. α-Glucosidase reacts with pNPG to produce the yellow color substance p-nitrophenol. Thus, lower absorbance represents higher α-Glucosidase inhibitory ability. food-borne bioactive peptides are considered as potential solutions as inhibitors of α-Glucosidase. The α-Glucosidase inhibitory abilityof WPI, US-WPI and WPH is depicted in Fig. 4D. Enzymatic hydrolysis significantly enhanced the α-Glucosidase inhibitory ability of WPI and US-WPI, and ultrasound pretreatment significantly increased the α-Glucosidase inhibitory ability. This may be due to the fact that ultrasound pretreatment increased DH of WPH, thereby releasing more bioactive peptides that interact with α-Glucosidase. In addition, the α-Glucosidase inhibitory ability of different enzymatic hydrolyzates varied because of differences in enzyme specificity and substrate affinity. Thus, sequential treatment with ultrasound and enzymatic hydrolysis is an effective method for producing bioactive peptides with superior α-glucosidase inhibition.

3.7 Peptide profiles and allergic epitopes

LC-MS/MS can provide detailed information of peptides in WPH. Peptide profiles of β-Lg (P02754), β-Lg-1/B (P67976), α-La (Q9TSN6), β-Lg-3 (P33688), β-Lg-1A/1C (P04119), and lactotransferrin (P14632) are shown in Fig. 5 [75]. Interestingly, there was no significant difference in the peptide profile distribution of the products hydrolyzed by different proteases, nor was there a significant difference in the peptide profile distribution of the hydrolysates after ultrasound pre-treatment and without ultrasound pre-treatment. According to our previous study (unpublished), this may result from the similar peptide profile distributions of pineapple protease and papain and the presence of the same exopeptidase in the two combinatorial enzymes used in this study. Notably, differences in the cleavage sites of endopeptidases resulted in different peptide intensities at different sites. In addition, sonication pretreatment alters the number of overlapping peptides in the WPH. In P67976, the number of overlapping peptides was higher in US-PA-WPI than in PA-WPI. However, no major effect of ultrasound pretreatment on the number of overlapping peptides in WPH was found in other peptide profiles. Meanwhile, the number of overlapping peptides in the four WPHs did not differ much. Thus, the differences in properties between WPHs have little to do with the number of overlapping peptides or the distribution of peptide spectra, and are mainly influenced by the intensity of the peptides.Fig. 5 Peptide profiles of β-Lg (P02754) (A), β-Lg-1/B (P67976) (B), α-La (Q9TSN6) (C), β-Lg-3 (P33688) (D), β-Lg-1A/1C (P04119) (E), and lactotransferrin (P14632) (F).

The abundance of US-PA-WPI and PA-WPI and the abundance of US-BA-WPI and BA-WPI in β-Lg varied differently in abundance at different amino acid sites. β-Lg-1/B, α-La, and β-Lg-3 had lower abundance of US-PA-WPI than PA-WPI, and higher abundance of US-BA-WPI than BA-WPI. β-Lg-1A/1C was completely opposite. Meanwhile, both US-BA-WPI and US-PA-WPI were more abundant in lactotransferrin than in the corresponding enzyme-only hydrolyzed treatment group. Thus, the changes in the abundance of WPI hydrolyzed products after ultrasound pretreatment varied due to differences in endopeptidases. In addition, a totally 456 peptides have been identified from the four hydrolyzed products in this study (Fig. 6A), and these were derived from 14 Master Protein species (Fig. 6B). The hydrolysis product with the least number of peptides was BA-WPI, which contained 423 different peptides. Cui et al. [76] found that milk protein concentrates that were extensively hydrolyzed (4 h) by different enzymes yielded a maximum of 472 different peptides in the samples. Compared to the above studies, the present study targeted WPIs in milk proteins for hydrolysis and obtained a similar number of peptide species in a much shorter period of time. This is due to two reasons, firstly, the further hydrolysis of the hydrolysis products of the endopeptidase by the exopeptidase, which produces a large number of peptides, and secondly, the short time of hydrolysis, which results in the peptides not being completely hydrolyzed into amino acids. Compared with BA-WPI and PA-WPI, the number of peptide species increased by 6 and 12 for US-BA-WPI and US-PA-WPI, respectively, suggesting that ultrasound pretreatment opened the structure of WPI, leading to the enzymatic hydrolysis to produce more peptides. This also shows that the decrease in allergenicity of WPI is due to the ultrasound pretreatment increasing the accessibility of the enzyme and allowing more allergenic epitopes to be hydrolyzed.Fig. 6 Venn diagram of peptides (A) and master Proteins (B).

Conformational epitopes are mostly destroyed during ultrasound and hydrolysis, so linear epitopes are more important [77]. Fragments containing allergenic epitopes can be recognized as allergic epitopes [78]. Therefore, the linear epitopes in WPH were further identified in this study through the biopep database (Table S2 and Table S3). The results showed that for 2 major allergens (α-La and β-Lg) in WPI, 24 and 314 epitope peptides were identified, respectively. This suggests that WPH also retained some allergic epitopes. In addition, eight unique epitopes were identified in different hydrolysates, all of them derived from β-Lg and distributed 1, 0, 0 and 6 in BA-WPI, PA-WPI, US-BA-WPI and US-PA-WPI, respectively. This is further evidence that the variability of protease cleavage sites and ultrasound pretreatment promoted the hydrolysis of WPI. We also performed a correlation analysis of these epitopes (Figure S1 and Figure S2). Among the α-La epitopes, PA-WPI and US-PA-WPI were grouped together, but PA-WPI and US-PA-WPI were not. Similarly, the sensitizing epitopes of β-Lg hydrolyzed by the same enzyme showed variability. Although the epitope characteristics of α-La hydrolyzed by the PA enzyme showed similarity before and after ultrasound, this similarity was not shown in the other hydrolysates. This is a result of ultrasound opens the structure of WPI and exposes the hydrolysis site of the enzyme, resulting in differences in the epitope profiles. This is further confirmed by the changes in the exclusive epitopes distribution in WPHs of the same enzyme in Table S3. Meanwhile, PA-WPI and US-BA-WPI showed similarity, which may be due to the fact that both enzymes contain the same exopeptidase.

The allergic epitopes in Tables S2 and S3 were labeled according to the specific allergenic fragments they contained. Changes in allergenicity are largely dependent on changes in the abundance of certain key epitopes that strongly influence allergenicity [71]. Based on the changes in allergenicity (Fig. 3A), we mapped the epitopes with corresponding decreases in abundance to allergens (Fig. 7) because they imply a greater likelihood of causing allergenic changes. Most of these key epitopes are located on β-sheet and β-turn structures, and the decrease in the abundance of these epitopes represents the disruption of these structures, providing further evidence that protease disruption of allergenic structures as well as ultrasound pretreatment increases protease disruption of protein structures. However, there were also epitopes in Tables S2 and S3 that showed an increase in abundance, which may be due to the fact that ultrasound opened up the WPI structure, resulting in the exposure of more epitopes that can be preferentially hydrolyzed by proteases. Notably, the extent of change in the abundance of certain epitopes therefore does not fully determine the change in allergenicity. The extent to which these epitopes influence allergenicity should be investigated more thoroughly in the future.Fig. 7 Location of allergic epitopes on α-La allergen (A-B) and β-Lg allergen (C-O).

3.8 Bioactive peptide

The bioactivity of peptides in WPH was predicted by the PeptideRanker platform, and those with a score greater than 0.5 were considered to have the potential to be used as bioactive peptides, with the higher the score, the greater the potential. The results, as shown in Fig. 8A-D, did not change the percentage of bioactive peptide species for hydrolysates with the same enzyme. However, there was an increase in peptide species after ultrasound pretreatment (Fig. 6A), which suggests that ultrasound pretreatment can the number of bioactive peptides in the enzymatic hydrolysate. This is consistent with the results of Fig. 4, which showed that the bioactivity of the hydrolysates improved after ultrasound pretreatment. Therefore, ultrasound pretreatment is an effective means to increase the bioactivity of WPH and a promising method for screening new bioactive peptides. In addition, we analyzed the abundance of bioactive peptides with scores above 0.8. The results are shown in Fig. 8E, and there were four bioactive peptides with scores above 0.8. The total abundance of these bioactive peptides in the corresponding hydrolysates increased after ultrasound pretreatment. This further indicates that ultrasound pretreatment can improve the bioactivity of protein hydrolysates. The bioactive peptides were further partitioned using Biopep database and MBPDB database. The results were shown in Table 1, and the abundances of BA-WPI, PA-WPI, US-BA-WPI, and US-PA-WPI were 1224.2, 1505.2, 1502.5, and 1768, respectively. The abundances of bioactive peptides of WPH were all increased after ultrasound pretreatment, which was due to the fact that ultrasound pretreatment opened up the structure of the WPI and increased the accessibility to proteases, thus releasing more bioactive peptides.Fig. 8 Percentage of predicted bioactive peptides. (A) BA-WPI; (B) PA-WPI; (C) US-BA-WPI; (D) US-PA-WPI; (E) Total abundance of bioactive peptides with scores above 0.8.

Table 1 Information on bioactive peptides in WPH.

Sequence	Predicted scores in the PeptideRanker platform	Biological activities identified in the BIOPEP database	Biological activities identified by the MBPDB platform	Abundances (Grouped)	
BA-WPI	PA-WPI	US-BA-WPI	US-PA-WPI	
KILDKVGIN	0.120838	Antimicrobial	Zinc binding peptide	97.3	111.3	103.5	87.9	
FHTSGYDTQA	0.158998		DPP-IV Inhibitory	104.5	100.7	88.5	106.2	
AASDISLLDAQSAPLR	0.542074		Antimicrobial	63.8	78.3	116	141.8	
TPEVDDEALEK	0.0891689	Antimicrobial & DPP-IV Inhibitory	DPP-IV Inhibitory	115.9	84.5	90.1	109.5	
VEELKPTPEGDLEIL	0.250818		Zinc binding peptide	70.6	62.4	124.3	142.7	
DAQSAPLRVY	0.476482	ACE-inhibitory	ACE-inhibitory	90.1	87.1	118.7	104.1	
ELKPTPEGDLEIL	0.309788		Zinc binding peptide	68.3	107.3	110.8	113.6	
LDAQSAPLR	0.533652	ACE-inhibitory	Antimicrobial	86.2	132.1	66.8	114.9	
IPAVFKIDAL	0.504166		DPP-IV Inhibitory	61.8	109.5	78.6	150	
KTKIPAVF	0.241382		Zinc binding peptide	64.2	94.9	102.4	138.5	
LKPTPEGDLEIL	0.327915	DPP-IV Inhibitory	DPP-IV Inhibitory	92.3	95.3	91.1	121.4	
DTDYKKY	0.194891		Zinc binding peptide	72.9	130.1	108.3	88.7	
ALPMHIR	0.626675	ACE-inhibitory & antihypertensive & increase cellular growth	ACE-inhibitory	66	115.6	90.6	127.8	
IDALNENK	0.110754	Antimicrobial & increase cellular growth		72.4	71.2	118.7	137.7	
DAQSAPLR	0.592074	Antimicrobial		97.9	124.9	94.1	83.2	


	
Total abundance	1224.2	1505.2	1502.5	1768	

4 Conclusion

The allergenicity of WPH was successfully reduced by ultrasound pretreatment, which improved their functional properties and bioactivity. Meanwhile, this study revealed the potential mechanism of ultrasound pretreatment to reduce the allergenicity of WPH by multispectral and high-resolution mass spectrometry. This study showed that ultrasound pretreatment could disrupt the spatial conformation of WPI to increase protease accessibility, and then disrupt the conformational epitopes and linear epitopes to reduce allergenicity. We also found by peptidomics combined with bioinformatics that the combined hydrolysis of endopeptidases and exopeptidases greatly increased the peptide content of the hydrolysates and that ultrasound pretreatment increased the number of bioactive peptides in the hydrolysates. This be an important reason for the improved bioactivity of the hydrolysates. Notably, the peptide profiles of the different allergen hydrolysates were different due to the variability of the protease hydrolysis sites. However, the distribution of the peptide profiles of the four hydrolyzates was similar, which be due to the presence of the same exopeptidase in both enzymes. Impressively, epitope peptides remained in all four hydrolysates, which could be the reason why the hydrolysates were still allergenic. Therefore, elimination of these linear epitopes by combining them with other methods is a promising approach to further reduce allergenicity. In addition, further optimization of the reaction conditions should be considered and the degree of reduction of the allergenicity of the hydrolysates should be verified by animal or human experiments.

Funding

This work was supported by Heilongjiang Provincial Key R&D Program (GA22C009).

CRediT authorship contribution statement

Lidong Pang: Writing – original draft, Visualization, Methodology, Data curation. Ming Liu: Writing – review & editing, Formal analysis. Chen Chen: Writing – review & editing, Methodology. Zhen Huang: Formal analysis. Shiyu Liu: Formal analysis. Chaoxin Man: Resources. Yujun Jiang: Resources. Wei Zhang: Writing – review & editing, Resources, Project administration. Xinyan Yang: Writing – review & editing, Resources, Methodology.

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.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplementary Data 1

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ultsonch.2024.107025.
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