Effect of ultrasound-assisted pH-shifting treatment on the physicochemical properties of melon seed protein

Abstract

Melon seeds have received considerable attention in recent years because of their high protein content, but they have not yet been fully used. The modification of melon seed protein (MSP) using ultrasound-assisted pH-shifting treatment was investigated in this study by analyzing structural characteristics and functional properties. The particle size, free sulfhydryl content, surface hydrophobicity, solubility, secondary structure, water-holding capacity, oil-holding capacity, emulsification activity index, and emulsification stability index of MSP were determined. MSP treated with ultrasound-assisted, pH-shifting had a smaller particle size, lower free sulfhydryl content, higher surface hydrophobicity, and solubility increased from 43.67 % to 89.12 %. The secondary structure of MSP was affected by ultrasonic treatment, manifesting as an α-helix increase and β-helix, β-turn, and random coil content decrease, which may be the reason why the protein structure became more compact after treatment. The water and oil holding capacities of MSP increased from 2.74 g/g and 3.14 g/g in untreated samples to 3.19 g/g and 3.97 g/g for ultrasound-treated samples, and further increased to 3.97 g/g and 5.02 g/g for ultrasound-assisted, pH-shifting treatment at pH 9.0, respectively. The emulsification activity index of MSP was 21.11 m²/g before treatment and reached a maximum of 32.34 m²/g after ultrasound-assisted, pH-shifting treatment at pH 9.0. The emulsification stability of MSP was maximized by ultrasonic treatment at pH 7.0. Ultrasound-assisted, pH-shifting treatment can effectively improve the functional properties of MSP by modifying the protein structure, which improves the potential application of melon seed protein in the food industry.

1. Introduction

Recently, fruit seeds have received considerable attention because they contain a rich variety of nutrients of excellent nutritional value. Fruit seeds are important sources of plant protein and have irreplaceable attributes compared to animal proteins, such as good reproducibility and a low correlation with chronic disease risk [1]. Melons belong to the Cucurbitaceae family and are known for their sweet flavor. Currently, melons are grown in numerous tropical regions globally, mainly in Asia, and account for 68.9 % of total cultivation. China is the world's largest producer, producing over 1.7 million tons per year (FAO, 2021). Melon pulp accounts for 52–65 % of the total, whereas the skin and seeds account for 25–44 % and 3.4–7 % of the total [2].

The inedible portion of the melon, such as seeds, is usually removed and discarded but is rich in protein, lipids, fiber, and minerals [3], [4]. Currently, melon seeds are mainly used for oil extraction because of their high unsaturated fatty acid content, which leads to lower cardiovascular disease risks[5]. The residues after oil extraction are also rich in protein and particularly high in many amino acids, such as glutamate (18–20 % w/w), arginine (12–16 % w/w), aspartate (9–10 % w/w), leucine (7–8 % w/w), and cysteine (5–8 % w/w) [6], [7], [8], which are important plant-based sources for protein supplementation.

However, melon seed protein (MSP) has not been fully utilized owing to its poor functional properties, resulting in a serious waste of protein resources. Numerous studies have shown that moderate structural modifications enhance protein functionality, such as their molecular conformation, which determines their hydrophobicity, solubility, and emulsification properties [9]. Thus, the purpose of this study was to improve the functional qualities of MSP by modifying its structure. Many physical and chemical methods, such as pH shifting [10], ultrasonic treatment [11], enzymatic hydrolysis [12], and ionic induction [13] have been used to improve the structural properties of proteins.

Processing by pH shifting is a convenient and feasible method for protein modification by which the unfolded protein structure formed under extremely acidic or alkaline conditions refolds to form a “molten globule” state when the pH is adjusted back to neutral [14]. Dissociated protein subunits and disrupted hydrophobic interactions caused by pH-shifting can lead to a loose protein structure, therefore enhancing the functional properties of the protein [15]. Although this method has improved the functional properties of whey protein isolates [10], peanut protein isolates [16], and ginkgo seed proteins [17], its negative effects, such as severe protein denaturation and structural damage, still pose challenges for its application in the food industry. Therefore, an alternative method that can be used synergistically with pH treatment is required.

Ultrasonic treatment is a safe, non-polluting, and environmentally friendly method for improving protein function[18]. The modification of proteins using this method relies mainly on the cavitation effect and shear force generated by ultrasound, which break the chemical bonds of proteins and induce dissociation and aggregation of subunits [19]. Many studies have shown that ultrasound combined with pH shifting can enhance the functional properties of proteins [15], [20], [21]. For example, the particle size distribution of whey protein isolate is almost unaffected by individual pH-shifting treatments, whereas it is substantially reduced when combined with ultrasound treatment [22]. After the combined treatment, the particle size of pea protein can be reduced from 206.9 nm to 45.2 nm, and its solubility can be increased seven-fold [21]. The foaming properties of chickpea protein isolates can be maximally improved by combined treatment, as indicated by the minimum particle size, interfacial tension, zeta potential, maximum solubility, and surface hydrophobicity [23].

However, the effect of combined ultrasound-assisted, pH-shifting treatment on MSP has not yet been reported and is investigated in this study with the aim of expanding its application scope in the food industry.

2. Materials and methods

2.1. Materials

Melon seeds were provided by the College of Life Science and Technology, Xinjiang University (Urumqi, Xinjiang, China). All the other chemicals were of analytical grade.

2.2. Preparation of melon seed protein

Melon seed protein (MSP) was extracted by alkaline extraction and acid precipitation method which was slightly modified by Ghribi et al[24]. The peeled melon seeds were powdered in a grinder and defatted using Soxhlet extraction with an appropriate amount of n-hexane to remove the melon oil. The defatted powder was sieved through an 80-mesh sieve, and then the defatted melon seed powder was mixed with deionized water at a ratio of 1:60 (m/v). The pH of the pretreated solution was adjusted to 10 with 1 mol/L NaOH solution, and the solution was extracted at 40 ℃ for 2 h. The supernatant was centrifuged at 4000 rpm for 20 min and collected. The pH of the supernatant was adjusted to 4.3 with 1 mol/L HCl, left for 20 min, and centrifuged at 8000 rpm for 20 min to obtain the MSP precipitate. The pH of the protein precipitate was adjusted to neutral, and the MSP powder was freeze-dried and stored under refrigeration. The protein purity was determined to be 84.35 % using the Kjeldahl method.

2.3. Ultrasound-assisted pH-shifting treatment

The MSP was prepared as a 5 % suspension with deionized water, and the pH was adjusted to 3, 5, 9, and 11 with 1.0 mol/L HCl or 1.0 mol/L NaOH, and the pH was adjusted back to 7 after standing at 25℃ for 1 h to obtain pH shifting-treated MSPs, named pH 3, pH 5, pH 9, and pH 11, respectively. A blank control group, pH 7, was set up.

For the ultrasound-assisted, pH-shifting treatment MSPs, the pH value was first adjusted according to the same procedure, and samples were then placed in an ice-water bath at 450 W for 15 min (with a working time of 2 s and an intermittent time of 2 s), which were named UpH 3, UpH 5, UpH 9, and UpH 11, respectively, whereas the ultrasonic-only treatment MSP was named UpH 7.

2.4. Particle size and zeta potential

First, MSP (1 mg/mL) was prepared in deionized water, and the particle size, zeta potential, and polymer dispersibility index (PDI) were determined using a Zetasizer (Nano ZS90, Malvern Instrument Limited, UK) at an equilibrium time of 120 s. The scans were repeated three times.

2.5. SDS-PAGE

SDS-PAGE was performed according to the method described by Zhao et al. with slight modifications [25]. A 1 mg/mL solution of MSP was mixed with 500 µL of sample dissolution buffer containing 1 mol/L Tris-HCL buffer, 50 % glycerin, 10 % sodium dodecyl sulfate (SDS), 1 % bromophenol blue, and 1 % β-mercaptoethanol and boiled for 5 min at 100 °C. The gel was then centrifuged at 10 000 × g for 5 min to remove the proteins precipitated by heating. Then, 20 µL of the supernatant was added to the gel wells, which had a 10 % separating gel and an 8 % stacking gel. The gels were run at 75 V for 30 min and 150 V for 1.5 h. The proteins were then immobilized by soaking in a 20 % trichloroacetic acid solution for 30 min. Finally, the gels were stained with a Caumas Brilliant Blue R-250 solution for 60 min.

2.6. Free sulfhydryl (SH) content

Phosphate-buffered saline (pH 8) was used to prepare 1 mM EDTA as a buffer. Sample solutions of 20 mg/mL and 1.0 mM DTNB reagent (Ellman's reagent) were prepared using a buffer. An amount of 500 µL of sample solution, 2.45 mL of buffer solution, and 50 µL of Ellman's reagent were mixed and stored at 25℃ and protected from light for 20 min. The absorbance value at 412 nm was determined using an ultraviolet spectrophotometer, and the buffer solution was used as a blank instead of the sample solution. The free sulfhydryl content of the MSP was calculated according to Equation (1):

$$\text{SH content}\left(\text{μmol/g}\right)=\frac{\text{1}{\text{0}}^{\text{6}}\times \text{A}\times \text{D}}{\text{13600}\times \text{C}}$$ (1)

where A is the absorbance at 412 nm for the different samples, D is the dilution factor, and C is the MSP concentration (mg/mL).

2.7. Surface hydrophobicity

First, the samples were dissolved in deionized water and diluted to 0.05, 0.1, 0.2, 0.5, and 1 mg/mL. Next, 20 μL of 8 mM ANS (8-anilino-1-naphthalenesulfonic acid) solution was added to 4 mL of the sample solution separately and mixed immediately. The fluorescence intensity was measured at a wavelength of 390/470 nm (excitation/emission). The fluorescence intensity was plotted against the protein concentration (mg/mL). The slope obtained from the linear regression indicated the surface hydrophobicity index of MSP.

2.8. Scanning electron microscopy

An appropriate amount of freeze-dried powder was fixed with a conductive adhesive on a sample disk for gold spraying, and the micromorphological structure of the powder was observed using a scanning electron microscope (5.0 kV) at 200, 500, 1,000, and 2,000 magnifications.

2.9. Circular dichrogram (CD) spectrum measurements

MSP (0.1 mg/mL) was prepared using deionized water. The bandwidth was set to 1.0 nm at 25 °C with a single step-up. The spectral measurement range was 180–260 nm, and the scan was repeated three times (Chirascan, Applied Photophysics Limited, UK). The secondary structure of MSP was analyzed from the CD spectra using the CDNN software program.

2.10. Fluorescence measurements

An MSP solution (1 mg/mL) was prepared using deionized water. The excitation wavelength was 285 nm, the emission wavelength was 300–500 nm, the excitation and emission slit widths were 10.0 nm, and the scanning speed was 300 nm/min (F-2700, Hitachi Corp, Japan).

2.11. Fourier transform infrared (FTIR) spectroscopy

The MSP was pressed into 1–2 mm pellets/slices using KBr. Scanning wavelengths were 4000–500 cm⁻¹ with a resolution of 2 cm⁻¹, and scans were repeated at least 64 times per sample (Nicolet IS 10, Thermo Electron Corporation, USA).

2.12. Solubility of MSP

MSP solubility was determined using the biuret method described by Ji et al., with slight modifications [26]. Firstly, 1.50 g of cupric sulfate and 6.0 g of potassium sodium tartrate were dissolved in 500 mL of deionized water, and 300 mL of a 10 % NaOH solution was added with stirring and diluted to 1000 mL with deionized water to make a biuret reagent. A standard curve was generated using bovine serum proteins as the standard. A 10 mg/mL MSP solution was prepared with deionized water and centrifuged at 5000 rpm for 15 min. An amount of 1 mL of the supernatant and 4 mL of biuret reagent were placed in a 37 ℃ constant temperature water bath and heated up for 20 min. The absorbance of the samples was determined at 540 nm using an ultraviolet spectrophotometer. The measured absorbance value was substituted into the standard curve to calculate the protein content. The solubility was calculated using Equation (2):

$$\text{W}\left(\%\right)=\frac{{\text{A}}_{\text{1}}}{{\text{A}}_{\text{0}}}\times \text{100}$$ (2)

where A₁ is the protein concentration in the supernatant (mg/mL), and A₀ is the initial protein concentration (mg/mL).

2.13. Water and oil holding capacity

After weighing 0.025 g of MSP in a 5-mL centrifuge tube, it was weighed as W₁; 2 mL of deionized water/soybean oil was added, vortexed, and vibrated for 5 min to make a homogeneous mixture, allowed to stand for 1.0 h, and then centrifuged at 4000 rpm for 20 min. The supernatant was removed, and the residual water and oil droplets were removed from the wall of the centrifuge tube with filter paper, and this was weighed as W₂. The water- holding capacities (WHC) and oil-holding capacities (OHC) of the MSP were calculated according to Equation (3):

$$\text{W}\text{H}\text{C/O}\text{H}\text{C (g/g)}=\frac{{\text{W}}_{\text{2}}\text{-}{\text{W}}_{\text{1}}}{{\text{W}}_{\text{0}}}$$ (3)

where W₀ is the MSP mass/g, W₁ is the centrifuge tube and protein amount/g, and W₂ is the centrifuge tube and protein amount/g after the removal of the supernatant.

2.14. Emulsion properties

A 5 mg/mL solution of MSP was prepared, and soybean oil was added at a 3:1 ratio of water to oil. The solution was emulsified using a high-speed homogenizer at 10000 rpm for 1 min. An amount of 20 µL of the liquid at the bottom of the emulsion was aspirated immediately, and 20 µL of the liquid at the bottom of the emulsion was aspirated after 10 min. The solution was then reacted with 2 mL of 0.1 % SDS solution, and the absorbance values at 500 nm were determined and recorded as A₀ and A₁₀, respectively. The samples were replaced with distilled water as a blank control. The emulsification activity (EAI) and emulsification stability (ESI) of MSP were calculated using Eq. (4) and Eq. (5), respectively:

$${\text{EAI(m}}^{\text{2}}\text{/g) =}\frac{\text{4.606}\times {\text{A}}_{\text{0}}\times \text{N}}{\text{C}\times \mathrm{\Phi }\times \text{L}\times {\text{10}}^{\text{4}}}$$ (4)

$$\text{ESI(min) =}\frac{{\text{A}}_{\text{0}}}{{\text{A}}_{\text{0}}{\text{- A}}_{\text{10}}}\times \text{10}$$ (5)

where N is the dilution factor, C is the protein concentration, Φ is the volume fraction of the oil phase in the emulsion, and L is the optical length of the cuvette (1 cm).

2.15. Statistical analysis

All measurements were performed in triplicate, and the experimental data were analyzed using SPSS Statistics 27 unless otherwise noted. Results are represented as the mean ± standard deviation, and changes in the data were assessed using Duncan’s Multiple Range Test at a significance level of 0.05.

3. Results and discussion

3.1. Physicochemical properties

3.1.1. Particle size and zeta potential

The particle size of the MSP after the ultrasound-assisted pH-shifting treatment is shown in Fig. 1A. The particle size of the control group was 269.50 nm, which decreased after ultrasound treatment. This change is due to the hydrodynamic shear forces associated with ultrasonic cavitation, which disrupt inter- or intramolecular noncovalent bonds (such as hydrogen bonding and electrostatic interactions), leading to the depolymerization of large protein aggregates into smaller fragments [11]. The particle size of the pH-shifted proteins decreased substantially after ultrasonication. At pH 11, the particle size of the proteins treated by ultrasound decreased drastically from 211.97 nm to 152.30 nm, which may arise because of the unfolding of the protein molecular structure during pH-shifting treatment of the proteins, thereby making them more susceptible to modification by ultrasound [27]. Similarly, perilla protein isolate [25], rapeseed protein isolate [28], soy protein hydrolysate [29], and pea protein isolate [30], which were treated with pH-shifting followed by ultrasonic treatment, showed similar changes in particle size.

Fig. 1.

Effect of different treatment on particle size (A), zeta potential (B), free sulfhydryl content (C), and surface hydrophobicity (D) of MSP.

The zeta potential reflects the net surface charge of solution particles and is often used to characterize the electrostatic stability of particles. The absolute value of the zeta potential increased after ultrasonic treatment and was greater than 25 mV except for the pH3 shift alone (Fig. 1B). This indicates that ultrasonic treatment enhanced the stability of the protein system. This may be a result of the mechanical force provided by ultrasound dispersing the protein aggregates, leading to the exposure of more polar groups [31]. This is consistent with the fact that a decrease in the particle size after ultrasonic treatment increases the stability and dispersion of the system.

3.1.2. SDS-PAGE analysis

The SDS-PAGE profiles of natural and ultrasound-assisted, pH-shifted MSP are shown in Fig. 2. For natural and treated MSP, four bands from 17 to 52 kDa were observed. The intensity of the 52 kDa band was weakened in the pH3 and UpH3 groups compared to that in the other samples. This may be due to the cavitation effect of ultrasound and the strong acidic pH shift affecting the molecular structure of the proteins, leading to disulfide bond breakage, which dissociated the 52 kDa subunit to form a smaller subunit [32]. The composition of the subunits in the remaining samples was similar, indicating that the combined treatment of ultrasound-assisted pH-shifting did not cause substantial changes in the molecular weight of the MSPs. Similar conclusions were obtained for milk protein and micellar casein concentrates, in which ultrasound-assisted, alkaline pH-shifting did not change the molecular weight composition [33].

Fig. 2.

SDS-PAGE patterns of MSP with different treatment conditions.

3.1.3. Free sulfhydryl (SH) content

The −SH group is significant in proteins because it reflects changes in their tertiary and quaternary structures [34]. The −SH content of the MSP after the ultrasound-assisted, pH-shifting treatment is shown in Fig. 1C. The −SH content of natural MSP was 44.60 μmol/g and increased to 46.85 μmol/g after ultrasonic treatment; the MSP after pH shifting that continued to undergo ultrasonic treatment showed the same phenomenon. This is a result of the cavitation effect of ultrasound and mechanical shear (which allows the sulfhydryl groups buried inside the molecule to be exposed), leading to the formation of sulfhydryl groups by the breaking of disulfide bonds. The breaking of disulfide bonds leads to a decrease in the particle size of the protein molecule, which is consistent with the change in particle size above [35]. With or without ultrasonic treatment, the free sulfhydryl content of MSP under acidic or basic (pH3, pH9, and pH11) conditions was substantially lower than that under neutral conditions, because sulfhydryl groups are more likely to bind to disulfide bonds under strongly acidic and basic polar conditions, resulting in a decrease in the free sulfhydryl content [28].

3.1.4. Surface hydrophobicity (H₀)

Surface hydrophobicity reflects the quantity of hydrophobic groups on the surface of a protein and is closely related to its foaming, emulsifying, and solubilizing abilities [36]. The effect of the ultrasound-assisted, pH-shifting treatment on the H₀ of MSP is shown in Fig. 1D. The H₀ of the natural MSP was the lowest but increased substantially after ultrasonic treatment. This may be because the hydrophobic groups of MSP are buried inside the protein, and the cavitation effect produced by ultrasonication weakened the intermolecular forces, causing the protein structure to unfold, ultimately resulting in the exposure of the originally buried hydrophobic groups [37]. When acid- and base-shifted protein solutions were subjected to ultrasonic treatment, the surface hydrophobicity increased further. The highest values were observed at pH 9 for ultrasonic treatment, suggesting that the combined effect of acid–base polarity and ultrasonic cavitation intensified the stimulation of protein molecules and the disruption of the hydrophobic forces increased, which led to further exposure of hydrophobic moieties [38].

3.1.5. Scanning electron microscopy

Scanning electron microscopy is typically used to observe protein microstructures. Fig. 3 shows the microstructures of MSP at 200×, 500×, 1000×, and 2000 × under different treatment conditions. The surface of MSP became rougher and fluffier after pH shifting, especially in the pH3 treatment, which may be due to the property of pH shifting, forming the state of a “molten ball,” which plays a role in loosening the structure of the proteins. After ultrasonic treatment, the MSP particles were smaller and more uniformly distributed, and the surface was smooth and dense; the UpH5 and UpH11 groups showed a clear boundary layer structure. This phenomenon may be the result of the shear force generated by ultrasound, which has a dispersing effect on the MSP structure. In other words, the MSP dispersions after ultrasonic treatment had smaller particle sizes and were more evenly distributed, which is consistent with the measured particle sizes and PDI. In addition, smaller dispersions may yield better solubility because of the exposure of the hydrophobic and polar groups within the protein, resulting in an increased contact area with water molecules.

Fig. 3.

Microstructure of MSP under 200x, 500x, 1000x, and 2000x scanning electron microscopy with different treatment conditions.

3.2. Structural properties

3.2.1. Circular dichroism (CD) spectra

The effect of the ultrasound-assisted, pH-shifting treatment on the CD spectrum of MSP is shown in Fig. 4A. The untreated MSP had a clear positive peak near 192 nm and a negative peak near 206 nm, indicating that an α-helix is a major component in the protein composition. The positions of the peaks shifted after the ultrasound-assisted, pH-shifting treatment, indicating that the secondary structure of MSP changed in different degrees after treatment.

Fig. 4.

Effect of different treatment on circular dichroism (A), endogenous fluorescence (B), FTIR spectra (C&D) of MSP.

The secondary structural composition of the protein obtained using analysis of the CD spectra is shown in Table 1. The α-helix, β-sheet, β-turn, and random coil of natural MSPs accounted for 32.67 %, 21.07 %, 18.61 %, and 27.06 %, respectively. After ultrasonic treatment, the percentage of α-helix increased dramatically to 79.8 %, which was 2.4 times higher than that of untreated; β-sheet decreased sharply to 2.37 %; and β-turn and random coil decreased slightly.

Table 1.

Effects of different treatments on secondary structure of MSP.

Sample	α-Helix (%)	β-Sheet (%)	β-Turn (%)	Random coil (%)
pH3	60.90 ± 1.80c	13.70 ± 2.45e	20.52 ± 1.10a	3.65 ± 0.29h
UpH3	50.41 ± 1.67e	19.30 ± 1.47d	21.45 ± 1.21a	6.84 ± 0.18g
pH5	26.60 ± 0.20i	31.17 ± 0.60b	19.07 ± 0.38b	24.25 ± 0.25c
UpH5	64.92 ± 1.47b	6.57 ± 0.40g	17.58 ± 0.79c	10.03 ± 0.30f
pH7	32.67 ± 0.55g	21.07 ± 0.55d	18.61 ± 0.23bc	27.06 ± 0.18b
UpH7	79.80 ± 0.37a	2.37 ± 0.06h	10.96 ± 0.26d	6.86 ± 0.19g
pH9	28.77 ± 0.72h	25.83 ± 0.78c	18.71 ± 0.22bc	26.96 ± 0.21b
UpH9	55.15 ± 1.53d	9.20 ± 0.62f	18.21 ± 0.61bc	16.08 ± 0.40e
pH11	21.42 ± 0.24j	35.63 ± 0.06a	17.79 ± 0.21c	28.87 ± 0.27a
UpH11	37.48 ± 0.76f	18.50 ± 0.60d	19.22 ± 0.30b	23.43 ± 0.15d

The composition of the protein's secondary structure was altered to different degrees after the pH-shifting treatment. The α-helix presented the highest value at pH 3 and the lowest value at pH 11, whereas the β-sheet was opposite to this. The α-helix percentage increased after ultrasonic treatment with or without pH-shifting. The secondary structure of peanut protein isolate underwent similar changes after ultrasonic treatment [39]. The α-helix is the closest-connected structure in protein molecules, and ultrasonication increased the proportion of α-helix and decreased the proportion of β-sheet in the proteins. This may be due to the increased collision of protein molecules caused by mechanical vibrations and intense microjet flow resulting from the cavitation effect of ultrasound, which leads to the formation of denser structures [31].

3.2.2. Fluorescence spectroscopy

Fluorescence spectroscopy, which determines the presence of tryptophan, phenylalanine, and tyrosine residues, is a common method for determining the tertiary structures of proteins [40]. The effect of the ultrasound-assisted, pH-shifting treatment on the fluorescence spectra of MSP is shown in Fig. 4B. The maximum emission wavelength of natural MSP was 332 nm, and the maximum emission wavelength increased (red-shifted) after pH shifting alone, ultrasound treatment alone, and acid–base shifting combined with ultrasound treatment, suggesting that the treatments affected the conformation of the proteins and the tryptophan residues caused by exposure to a polar microenvironment [41]. The change in the fluorescence intensity of proteins after pH shifting treatment (pH3 and UpH11) may be the result of changes in the number of chromophore groups due to their structural alterations, which were further exacerbated by the combined treatment with ultrasound [42]. The UpH11 group had the highest fluorescence intensity, probably due to the greatest change in MSP. Yang et al. reported a similar trend in pH-shifting combined with ultrasonication of perilla protein isolate, suggesting that pH-shifting and ultrasonication can alter the tertiary structure of proteins [42].

3.2.3. Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of MSP after pH shifting combined with ultrasound treatment are shown in Fig. 4C and 4D. The characteristic peaks at 3290 and 2947 cm⁻¹ correspond to the symmetric and asymmetric vibrations of the amide A group, –NH, stretching, and amide B groups, –CH₂ and –CH₃, respectively [43]. The 1640 cm⁻¹ infrared absorption peak corresponding to the amide I region (1700–1600 cm⁻¹) is the result of stretching vibration of the C=O bond, is sensitive to changes in the secondary structure of proteins, and can reflect changes in the secondary structure of proteins [44].

The absorbance of proteins in the amide I band changed after pH-shifting and ultrasonication of MSP, and the infrared absorption peak of the amide I band shifted slightly with pH-shifting and ultrasonication (Fig. 4C, D). This shift indicates that pH shifting and ultrasonic treatment can substantially affect the secondary structure of MSP. The 1529 cm⁻¹ infrared absorption peak corresponding to the amide II region (1550–1500 cm⁻¹) is the result of the N–H bending vibration, which can reflect intramolecular or intermolecular hydrogen bonding changes [45]. The absorption peak shifted from 1529 cm⁻¹ to higher wavelengths after ultrasound treatment, indicating that the cavitation effect of ultrasound broke the hydrogen bonds [28].

3.3. Functional properties

3.3.1. Solubility

Fig. 5A shows the solubility of MSP after the ultrasound-assisted pH-shifting treatment. The solubility of untreated MSP was 43.67 %, and ultrasound treatment increased the solubility to 89.12 %. This is attributed to the fact that the intense mechanical forces generated by ultrasound disrupt hydrogen bonding and hydrophobic interactions between MSPs, leading to a marked increase in the solubility of the proteins [11]. The reduction in particle size caused by ultrasonic treatment may be another reason for the increase in solubility, because a reduction in particle size enhances the interaction between proteins and water [46].

Fig. 5.

Effect of different treatment on solubility (A), water holding capacity and oil holding capacity (B), Emulsifying activity index and emulsion stability index (C) of MSP.

When the pH was increased to 9, the solubility of MSP increased 1.2-fold, probably because of the increase in the ionic strength of charged proteins and water under alkaline conditions, leading to an increase in solubility [44]. However, under strongly alkaline conditions (pH 11), MSP solubility was reduced to 70 % of its original value, which may be related to the formation of protein aggregates under strong alkaline polarity conditions [47]. The solubility of MSP in acidic treatments (pH 3 and pH 5) showed the opposite result to that of alkaline treatments, mainly because of the weaker interaction of proteins with water molecules near the isoelectric point of MSP (pI=4.3), which made protein molecules to lean on each other or even form macromolecular aggregates [43].

3.3.2. Water and oil holding capacity

Water-holding capacity (WHC) and oil-holding capacity (OHC) reflect the ability of proteins to retain water and oil and are important indicators for evaluating the emulsification capacity of proteins [48]. Fig. 5B shows the effect of the ultrasound-assisted pH shifting treatment on the WHC and OHC of MSP. The WHC of natural MSP was 2.74 g/g, and that of the ultrasonic treatment was 3.19 g/g, an increase of 16.4 %. The WHC of MSP increased after ultrasonic treatment and showed the highest WHC (3.97 g/g) at pH 9 after ultrasonic treatment. This can be attributed to the increase in solubility during ultrasonic treatment and the dissociation of proteins, resulting in enhanced interaction with water, which increased the WHC [49]. All OHCs increased after ultrasonic treatment compared to the natural MSPs, which was due to the exposure of hydrophobic residues hidden in the MSPs after the ultrasonic treatment. Zhao et al. reported similar results during ultrasonic extraction of Dolichos lablab L. protein [50]. The MSP treated with ultrasound for 15 min at pH 9 had the highest WHC and OHC values.

3.3.3. Emulsifying activity and emulsion stability indices

Fig. 5C shows the emulsification activity index (EAI) and emulsion stability index (ESI) of MSP after the ultrasound-assisted, pH-shifting treatment, which indicates the ability of the protein to stabilize the oil–water interface. The natural MSP had an EAI of 21.11 m²/g, the UpH9 group showed the best EAI (32.34 m²/g) and a better ESI (46.72 min), and the UpH7 group exhibited the best ESI (57.48 min). Moreover, there was enhancement of both EAI and ESI after ultrasonic treatment, which concurs with published studies on sunflower protein [38], black gram protein [49], and tamarind seed protein [51]. This may be due to the increase in the surface hydrophobicity and solubility of MSP and the decrease in particle size [52]. Ultrasonic treatment enhances the surface hydrophobicity of the protein, resulting in a good balance between the hydrophobicity and hydrophilicity required for emulsification, which leads to an increase in EAI and ESI [53]. The increased solubility accelerated the diffusion rate of MSP at the oil–water interface [54]. Smaller particles are more easily adsorbed at the oil–water interface, which further improves the emulsification ability of MSP [55].

4. Conclusion

In this study, ultrasound-assisted pH-shifting treatment was effective in altering the physicochemical properties of MSP. The surface hydrophobicity of pH9-shifted, ultrasound-treated MSPs was substantially higher than that of untreated MSPs. The pH-shifting treatments, especially the alkaline-shifted treatments, substantially improved the emulsification activity of MSP, and the combined ultrasonic treatment exacerbated this change. The emulsification activity of MSP is highly dependent on particle size, surface hydrophobicity, solubility, and changes in protein conformation. The pH-shifting treatment further unfolds the protein structure and makes it more susceptible to the physical forces of ultrasound. Thus, the combined use of ultrasound and pH-shifting can maximize the improvement of the structural and functional properties of MSP.

CRediT authorship contribution statement

Guojun Fu: Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation. Man Zhao: Validation, Methodology, Investigation, Data curation. Xinmiao Wang: Formal analysis. Zehao Zheng: Formal analysis. Shiyu Shen: Formal analysis. Jiawen Yan: Formal analysis. Qun Li: Resources. Chao Gao: Formal analysis. Xuyan Dong: Supervision, Funding acquisition, Formal analysis. Junxia Xiao: Supervision, Formal analysis. Liang Liu: Writing – review & editing, Project administration, Methodology, 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.