3.1 Effects of ultrasound on trypsin inhibitory activity of ovomucoid
The changes in trypsin inhibitory activity of OVM under ultrasound treatment at various processing time (5–40 min) and ultrasonic power (100–400 W) were shown in Fig. 1. There was no significant change in trypsin inhibitory activity of OVM between control group and ultrasound treated group at 5 min, both of which were about 90%. However, the trypsin inhibitory activity of OVM showed a decreasing trend with the increase of processing time and power. The inhibitory activity of ovomucoid could be reduced to 30% when the ultrasonic power was 400 W at 40 min. The changes of trypsin inhibitory activity of OVM samples treated with different ultrasonic power were linearly fitted (Scheme S1). Results showed that the slopes of enzyme inhibitor activity with time in 100W, 200W and 400W samples were 0.66, 0.97 and 1.57 respectively, which represented the value of decreasing trypsin inhibitory activity per minute. It was found that high-intensity ultrasound significantly reduced the trypsin inhibitory activity of OVM.
Huang et al (2008) investigated the effect of ultrasound (with 25%-65% amplitude of ultrasonic fields and 5–20 min processing time) on trypsin inhibitory activity of the KTI and BBTI in soybean protein. Results indicated that about 45% of the KTI inhibitory activity was found lost with ultrasound amplitude fixed at 65% for 20 min. However, the trypsin inhibitory activity of BBTI was not significantly influenced by ultrasound treatment. It could be speculated that the effect of ultrasound on trypsin inhibitory activity was related to the type and structure of the protein. Thus, in order to analyze the mechanism of the effect of ultrasound on the activity of OVM, the structural changes were characterized in following experiments. Because OVM had the lowest trypsin inhibitory activity at 40 min with different ultrasonic powers, we set the ultrasonic time at 40 min and focused on the structural changes of proteins with different ultrasonic powers (100, 200, 400 W) in the following experiments.
3.2 Changes in zeta potential and particle size of OVM induced by ultrasound
Zeta potential was used to evaluate the effect of ultrasound on the surface charge of OVM. Since the isoelectric point (pI) of OVM is around 4.1, the total surface charge of the protein is negative under neutral conditions. After ultrasound treatment, OVM solutions showed lower zeta potential (-31.47 mV, -32.77 mV and − 35.20 mV under 100, 200, 400 W treatment respectively) than that of untreated group (-14.67 mV), indicating that the negative charges on the surface of protein showed an increasing trend after ultrasound treatment (Fig. 2A). When zeta potential changed, the electrostatic repulsion of the protein would also change, which may lead to the unfolding and rearrangement of protein molecules (Hou et al., 2019). Correspondingly, it can be seen that the average particle size of control group was 696.33 nm and decreased with the increase of ultrasonic power until reached the minimum at 400 W (219.73 nm) (Fig. 2B). As the relative molecular weight of OVM is 28 kDa, it could be calculated that the average particle size per OVM molecule decreased from 0.024 nm to 0.013, 0.009 and 0.008 nm after 100, 200 and 400 W ultrasound treatment (Kato et al., 1987). Similar studies were found by Nazari et al (2018) that the particle size of millet protein concentrate also showed a decreasing trend after ultrasound treatment. It could be assumed that the electrostatic repulsion between protein molecules gradually increased with the increase of ultrasonic power, which reduced the average particle size.
As shown in Fig. 2C, the particle size distribution of control group showed a polarized trend, mainly concentrated at 5 µm and 900 µm. After ultrasound treatment, the particle size distribution was mainly concentrated between 100 and 400 nm, which was completely consistent with the decreasing trend of the average particle size and Polymer dispersity index (PDI) value in Figure.2B. PDI is a polymer dispersibility index that describes the molecular weight distribution of a polymer. As the molecular weight of a polymer is usually not uniform, the average molecular weight is used to describe the molecular weight of a polymer. The average molecular weight can be divided into number average molecular weight, heavy average molecular weight and viscosity average molecular weight. The ratio of the heavy average molecular weight to the number average molecular weight is called PDI. The smaller the PDI, the more uniform the molecular weight distribution (Pomon et al., 2022). The untreated group exhibited the largest PDI (0.87). After ultrasound treatment, the PDI of OVM solutions reached the minimum at 400 W (0.44). This indicated that ultrasound treatment could not only reduce the overall particle size of protein molecules, but also make the particle size of protein molecules in solution more uniform. Although the average particle size of the protein molecules reached the minimum at 400 W ultrasound (219.73 nm), it could be seen that its maximum peak was larger compared with the samples treated with 200 W ultrasound treatment. This may be due to the aggregation of OVM molecules caused by high power ultrasound. Similar result was found by of Yao et al (2018). Although ultrasound significantly reduced the particle size of walnut protein molecules, long treatment time and high-power ultrasound also caused the protein to aggregate due to denaturation and resulted in in an increase in particle size. Arzeni et al (2012) proved that ultrasound treatment affected the particle size and functional properties differently depending on the nature of the protein. Mechanism of ultrasound on zeta potential and particle size of OVM might depend on mechanical shearing force, which caused the unfolded and re-aggregation of protein molecules and changed the second structure of protein. In order to test this conjecture, we next determined the changes in the secondary structure of OVM under ultrasound treatment by circular dichroism.
3.3 Changes in the secondary conformations of OVM induced by ultrasound
The CD spectra is mainly used to study the secondary structure of proteins based on the circular dichroism of peptide bonds. The contents of α-helix, β-sheet, β-turn and random-coil in control group were 7.67%, 52.80%, 7.93% and 31.6%, respectively (Fig. 2D). After ultrasound treatment, the contents of α-helix, β-turn and random-coil structure gradually increased, while the β-sheet structure showed a decreasing trend and reached the minimum (33.97%) at 400 W ultrasound treatment. This suggested that ultrasound may promote the transformation of β-sheet conformation of OVM to other three secondary structures, which was similar to the research result of Qu et al (2018) that ultrasound treatment had the greatest impact on the decrease in the β-sheet and the increase in the random-coil of rapeseed protein. These changes in secondary structure resulting from ultrasould treatment could be attributed to the pressure alterations, turbulence, and the induced structural transformations (Liu et al.,2022). Among the four secondary structures, α-helix and β-sheet need more hydrogen bonds to maintain the structure, while β-turn and random-coil need no hydrogen bond (Tan et al., 2021). Therefore, the reduction of total content of α-helix and β-sheet meant that the number of hydrogen bonds in the protein decreased and the structure of protein became more disordered and flexible.
Previous studies have demonstrated that β-sheet was the most predominant component of the secondary structure of OVM, which accounted for approximately 46% (Sasan et al., 2020). Weber et al (1981) showed that the structural domains with trypsin inhibitory activity of OVM in Japanese crane quail egg serum exhibited a spherical spatial configuration containing two reverse parallel β-sheet (proline residues at position 22 to glycine residues at position 32) and an α-helix. Therefore, it can be inferred that β -sheet was the main structure to maintain the stability and inhibitory activity of OVM protein. Ultrasound inactivated the trypsin inhibitory activity of OVM by disrupting the β-sheet structure and increased the content of disordered structural.
3.4 Changes in hydrophobicity of OVM induced by ultrasound
Hydrophobicity represents the repulsive force between a non-polar substance and a polar environment (e.g., an aqueous solution). Thermodynamically, it represents the energy required to dissolve a non-polar substance in water, or the tendency of the substance to self-aggregate in the aqueous phase (Jiang et al., 2014). As ANS can be non-covalently bound to the non-polar part of protein, it can sensitively reflect the hydrophobicity change of biological macromolecules. Researches have proved that the maximum emission wavelength of ANS alone was around 480–500 nm, and the fluorescence intensity value was extremely small. After the addition of protein, the maximum emission wavelength was blue-shifted and the fluorescence intensity value increased significantly (Zhang et al., 2023). It can be seen from Fig. 3A that there are two characteristic absorption peaks in the OVM solution samples, which are 450 nm and 470 nm, respectively. The former was the absorption peak of ANS and non-polar protein conjugates and the latter was the absorption peak of ANS alone. The fluorescence intensity was continuously increasing with the increase of ultrasonic power, indicating that hydrophobicity of OVM increased with ultrasonic power (Fig. 3A).
It has been proved that the activity of serine protease inhibitor derives from a series of intramolecular interactions, including a disulfide bond, hydrophobic bond and a network of hydrogen bonds (Huang et al., 2010). According to the amino acid sequence of OVM found by Kato et al (1987), OVM consists of 186 amino acids and its second structural domain (residues 65–130) with trypsin inhibitory activity contains a large number of hydrophobic amino acids, which plays an important role in maintaining the structural stability of proteins, including 2 phenylalanine (Phe), 5 valine (Val), 3 leucine (Leu), 1 isoleucine (Ile), 2 alanine (Ala), 2 proline (Pro) and 6 glycine (Gly). In particular, ,the major reactive site for trypsin is the Arg89-Ala peptide bond in the second domain, with seven hydrophobic groups in its vicinity. The hydrophobicity of protein molecules was due to its non-polar amino acid residues. These amino acid residues would not interact with the polarity water molecules (with the exception of van der Waals force), which resulted in the hydrophobic groups tend to be combined in the interior of the natural globulin hydrophobic region (Kovacs et al., 2010). During ultrasound treatment, the aggregation between protein molecular was disrupted by mechanical shearing force of ultrasound. Therefore, the non-polar amino acid residues in second structural domain of OVM, were gradually exposed, which also disrupted the second structural domain and decreased the trypsin inhibitory activity of OVM.
3.5 Changes in UV-Vis absorption of OVM induced by ultrasound
Changes in the UV-Vis absorption of OVM due to the presence of aromatic residues, such as tyrosine (Tyr), Tryptophan (Trp) and phenylalanine (Phe), were used to monitor the folding and unfolding transitions of protein molecules after ultrasound treatment (Roychaudhuri et al., 2003). The UV-Vis absorption difference spectra of OVM under 100–400 W ultrasound treatment could be observed in Fig. 3B. According to Kato et al (1987), OVM contains 10 Phe and 5 Tyr, while the second structural domain (residues 65–130) with trypsin inhibitory activity of OVM contains contains 2 Phe and 3 Tyr. Tyr usually absorb light in the range of 280–288 nm, whereas Phe absorbs from 255 to 265 nm. It is speculated that peaks at 260–290 nm were the absorption peaks of Tyr and Phe partially overlapping. Compared with control group, the UV-Vis absorption intensity of OVM around 280 nm showed an increasing trend with the increase of ultrasonic power and basically stabilized after the ultrasonic condition reached 200 W. It could be concluded that the residues of aromatic amino acids (Tyr and Phe) were exposed after the disulfide bond was broken by ultrasound treatment.
Studies by Roychaudhuri et al (2004) have showed that the aromatic amino acid residues within the structure play a critical role in determining the unfolding and refolding characteristics of protein molecule. Roychaudhuri et al (2003) investigated the thermal denaturation and renaturation of soybean Kunitz trypsin inhibitor. Changes in UV difference spectra showed increased absorbance at 292 and 297 nm upon heating to 70°C, while the trypsin inhibitory activity decreased, which was consistent with our result. Combined with the previous results, it can be speculated that the exposure of aromatic residues (Tyr and Phe) and hydrophobic amino acids (Phe, Val, Leu, Ile, Ala, Pro and Gly) under ultrasound treatment denatured the second structural domain in OVM with trypsin inhibitory activity, which was responsible for the change in the secondary structure of OVM and the decrease in trypsin inhibitory activity.
3.6 Changes in the simulated digestion characteristics of OVM induced by ultrasound
In order to investigate the changes in the resistance of OVM to digestive enzymes after the structure of OVM was changed by ultrasound, four groups of different ultrasonic power treated samples were digested with trypsin and pepsin, respectively. The digestion of OVM by trypsin and pepsin after ultrasound treatment was assessed using SDS-PAGE (Fig. 4). As shown in lanes 2–5, compared to the control group (lane 2), the molecular weight range of OVM did not change significantly after ultrasound treatment after ultrasound treatment (lane 3 and 5). This indicated that although ultrasound changed the secondary structure and physicochemical properties (zeta potential, particle size, etc.) of OVM, it did not cause protein degradation of OVM (Fig. 3A). This result was consistent with previous studies (Liu et al., 2022; Xiong et al., 2018). As shown in lane 6, the native OVM underwent only a small portion of hydrolysis due to its high hydrolysis resistance to trypsin. Ultrasound under low power (100 W and 200 W) had less effect on the trypsin resistance of ovomucoid (lane 7 and 8). However, OVM began to undergo significant degradation when the ultrasonic power was increased to 400W, while the number of 13 kDa and 17 kDa peptide fragments increased significantly (lane 9). Kovacs et al. (2000) found that large OVM fragments remained and may act as allergens under normal pepsin digestive conditions for untreated OVM, which was consistent with the results of lane 10. However, the hydrolysis resistance to pepsin of OVM reduced under ultrasound treatment over 100 W (lane 10–13). Moreover, the degradation degree of OVM gradually increased with the increase of ultrasonic power from 100 W to 400W, which was reflected in the increase of the number of peptide fragments at 10–15 kDa (lane 13). In general, our results indicated that the structure of OVM changed after ultrasound, which made it more digestible. This suggests that ultrasound could be applied to facilitate digestion and reduce allergic substances of OVM.
3.7 Changes in thermal stability of OVM induced by ultrasound
DSC was used to determine the thermal stability of OVM under different power ultrasound treatment (Fig. 5A and Table. 1). ∆H represents the energy that leads to the unfolding and destruction of the quaternary structure of protein, while Tmax, Tonset and Tend represent the maximum peak temperature, start temperature and end temperature, respectively (Haug et al., 2009). As shown in Fig. 5, all samples showed a broad endothermic peak, while the peak temperature corresponding to native OVM was about 91.91°C, which was slightly higher than about 79°C in previous studies (Julia, et al., 2007), which may due to different breeds of laying hens or differences in protein extraction methods. Tmax, Tonset and Tend of OVM all increased slightly with the increase of ultrasonic power during 100 and 200 W(Table. 1). However, When the ultrasonic power reached 400W, Tmax decreased significantly to 84.32°C, while Tonset and Tend also decreased. ∆H decreased sequentially with the increase of ultrasonic power, indicating that the energy required for thermal denaturation of OVM reduced after ultrasound. This also showed that ultrasound could reduce the thermal stability of protein structures. Similar researches showed that ultrasound could also reduce the thermal stability of rice protein isolate and date palm pollen protein (Nazari et al., 2018; Sebii et al., 2019). Our previous experimental results showed that ultrasound treatment resulted in a decrease in the content of rigid structures (β-sheet) and an increase in the content of protein disordered structures (β-turn, random coil) by mechanical shearing force. In our conjecture, this may be because ultrasound made the protein structure looser and more disordered, thus reduced the thermal stability.
In order to observe the relationship between the secondary structure and thermal stability of OVM, we investigated the secondary structure changes of OVM after ultrasound treatment with different powers and heating at 45°C and 85°C for 10 min (Fig. 5B). It could be seen that changes in the secondary structure of four ultrasound treated OVM samples (0, 100, 200, 400 W) during different temperature had occurred. Among them, there was no significant difference in the content of random coil in all samples. Interestingly, the secondary structure of control group (especially α-helix, β-folding and β-turn) was more affected by temperature, while the secondary structure of OVM sample after ultrasonic treatment of 100–400 W had a relatively small change range, which was contrary to our initial prediction. At the same time, an unexpected conclusion was drawn that although the secondary structure of OVM had greatly changed compared with the control group after ultrasonic treatment, it become more stable and insensitive to temperature. According to our previous studies, we speculated that ultrasound made the hydrophobic amino acids and aromatic residues of OVM unfolded by mechanical shearing force, resulting in a decrease in the content of rigid structures (β-sheet) and an increase in the content of protein disordered structures (β-turn, random coil), which led to the reduction of trypsin inhibitory activity. It’s well known that the allergenicity and anti-digestive factors of OVM remain stable under traditional thermal treatment and high-pressure processing because of its unique property (Ma et al., 2020). Results of our experiment showed that ultrasound treatment could make OVM more susceptible to inactivation. Meanwhile, OVM with low anti-digestive factors also had a stable secondary structure, which also provided ideas for it to be used as a new food processing material for people allergic to eggs.