3.1 Foaming properties
3.1.1 Acidic pH and cinnamic acid-based polyphenol treatment led to higher FA of OVA
Foaming properties of proteins are important functional characteristics that determine their application in several food products, where aeration and overrun are needed, such as beverages, ice cream, and cakes [23]. Figure 1 exhibits the foaming properties, foam volume and foam size of complexes at different pH conditions and Figure S1 presents the foaming properties obtained with each of the OVA-polyphenol systems. Specifically, OVA-GA, OVA-DA, and OVA-PA were three kinds of benzoic acid-based polyphenol-OVA aggregates, while OVA-CA, OVA-T3A and OVA-T2A belonged to three cinnamic acid-based polyphenol-OVA aggregates. A significant (p < 0.05) increase in FA and a further slight but not significant (p > 0.05) increase in FS were observed when each polyphenol was added to OVA solution, implying a change in the microstructure of OVA. According to our previous results, the supplement of polyphenols may contribute to the proper unfolding of the molecular structure, allowing the proteins to be driven to the water-air interface, thus promoting the conformational flexibility and foaming properties of OVA [2]. The promotional effects of polyphenols on FA of protein have been detected in resveratrol-whey protein isolate (RES-WPI, the FA of RES-WPI increased from 92% (WPI: RES = 100:0) to 132% (WPI: RES = 100:2)), procyanidin-lactoferrin (FA of aggregates was 119%, 128%, 159% and 186% at lactoferrin: procyanidin ratios of 64:0, 64:1, 64:2, and 64:4, respectively) [24], and ferulic acid-OVA (FA of aggregates increased from 90–140% when the molar ratio of ferulic acid to OVA changed from 1: 0 to 1:20) [7]. However, there were counter reports as well. Dai, et al. [25] found that the FA of lactoferrin decreased from around 114–32% as the proportion of lactoferrin/tannic acid changed from 64:0 to 64:10. These conflicting results demonstrate that the effects of polyphenols on protein foaming properties are very complex, and therefore, revealing their action mechanism is a valuable subject for investigation.
The FA of all six OVA-polyphenol aggregates revealed that the highest FA was obtained when the aggregates were treated under acid conditions (pH = 3.0), whereas alkaline treatment imparted a slight decline in FA values (-12.78% for OVA-DA, and − 13.89% for OVA-T2A) as compared to untreated aggregates. pH = 3.0 is close to the isoelectric point of OVA [26], which might be one of the causes that contributes to differences in the FA of samples due to pH treatment. The interaction forces, including hydrophobic interactions, electrostatic repulsions and hydrogen bonds, between OVA and polyphenols may alter after the use of hydrochloric acid, which results in changes in the OVA conformation, thus altering adsorption rate and adsorption capacity of protein at the air-liquid interface and ultimately enhancing the FA of the aggregates [2, 27].
Acid or alkali treatment did not significantly (p > 0.05) affect the FS of six OVA-polyphenol aggregate systems, except for OVA-T3A (Figure S1). Similar to the FA results, the highest FS was observed for OVA-T3A-3.0, followed by OVA-T3A-9.0 and finally the OVA-T3A-7.4. pH = 3.0 is close to the isoelectric point of OVA, resulting in lower electrostatic repulsion and increased attractive intermolecular forces (mainly hydrophobic interactions) between nonpolar protein molecules [11, 28]. These alterations might enhance the strength of the interfacial film, which in turn prevents the air bubbles from collapsing, thus forming a system with excellent foaming characteristics [29]. Secondly, the weakened electrostatic repulsion probably leads to larger particle size of the OVA-polyphenol aggregates, which is also positively related to the increased thickness of the interfacial film, finally causing the formation of a more stable foam system. The electrostatic repulsion of the OVA-polyphenol aggregates at pH 9.0 was greater than that at pH 3.0. As a result, fragile interfacial film may be detrimental to the FS of the aggregates. However, further experiment validation of these interpretations regarding FA and FS is needed.
The FA of OVA-GA, OVA-DA, and OVA-PA aggregates at pH 3.0 did not differ (p > 0.05) and was 65.00%, 65.56% and 57.78%, respectively, which were lifted by 67.14%, 37.21%, and 6.13% compared to the untreated samples, revealing that the number of hydroxyl groups of polyphenols did not significantly affect the FA of OVA. Moreover, compared to the OVA samples at pH 3.0 (34.44%), the FA of aggregates increased by 88.73%, 90.36% and 67.77% for OVA-GA, OVA-DA and OVA-PA. In terms of three cinnamic acid-based polyphenol-OVA aggregates, their FA also showed no significant differences (p > 0.05) and were 88.33%, 96.67% and 101.67% for OVA-CA, OVA-T3A and OVA-T2A, respectively, at pH 3.00, indicating that the position of hydroxyl groups of polyphenols did not significantly affect the FA of OVA. Compared to the untreated samples (OVA-CA: 43.89%, OVA-T3A: 51.11%, OVA-T2A: 53.33%), the FA of the OVA-CA, OVA-T3A and OVA-T2A increased by 89.86%, 89.14% and 90.64%, respectively, while their FAs were improved by 156.47%, 180.69% and 195.21% compared to the untreated OVA solutions, demonstrating that acidic treatment greatly altered the FA of the cinnamic acid-based polyphenol-OVA aggregates. Cinnamic acid-based polyphenols (CA, T3A and T2A) are likely to form more covalent bonds with proteins than benzoic acid-based polyphenols (GA, DA, and PA), further causing greater changes in the structural conformation of OVA, and ultimately contributing to increased FA of the aggregates [30]. In summary, OVA-DA had the strongest FA among all benzoic acid-based polyphenol-OVA aggregates, while OVA-T2A exhibited the highest FA among cinnamic acid-based polyphenol-OVA aggregates. Therefore, OVA, OVA-DA and OVA-T2A were selected for different pH treatments to address the effects of pH on FA and FS of the OVA-polyphenol aggregate systems and possible mechanisms.
3.1.2 Acidic pH contributed to smaller and denser bubble formation of OVA
The foaming microstructure of aggregates under different pH conditions was measured using an optical microscope, and the results were given in Fig. 1E and 1F. The foams of aggregates immediately after homogenization (Fig. 1E) were smaller than that of the foams after a period of setting (Fig. 1F). Air-liquid phase separation may occur as a result of the instability of the foams, causing the accelerated agglomeration of small bubbles with other bubbles [7], which eventually results in the accumulation of large bubbles. However, no significant difference was observed in the variation percentage of foam height of the aggregates. Besides, the bubbles of aggregates at pH 3.0 were always smaller and denser than those of the untreated aggregates (pH = 7.4) both at 0 min and 30 min after homogenization, while no obvious differences were observed between the bubbles of aggregates at pH 9.0 and pH 7.4. This result certainly supports the results of FA and FS.
3.2 Acid and polyphenol treatments increased the viscosity and G’ of OVA
The viscosity, storage modulus (G’) and loss modulus (G”) of the protein dispersion system affect the FA and FS. Conventionally, large viscosity of OVA and G’ > G” contribute to the formation of film with high cohesiveness and elasticity, thereby preventing the air droplets from coalescence and resulting in more stable air bubbles [31]. Figure 2 gives the viscosity, G’ and G” curve of aggregates after different pH treatments. All OVA-polyphenol aggregates had reduced viscosity and decreased gap between G’ and G” as the shear rate increased from 1 to 100 s− 1, showing a shear thinning behavior, which further indicated that these aggregates can be regarded as non-Newtonian fluids and exhibited pseudoplastic fluid behavior [32]. In addition, with the increase of scanning frequency, the G’ and G” of OVA-polyphenols gradually increased and G’ followed the order of pH = 3.0 group > pH = 7.4 group > pH = 9.0 group, while G” followed the order of pH = 7.4 group > pH = 3.0 group = pH = 9.0 group. Higher storage modulus G’ correlates positively with more cohesive and elastic film, thus contributing to higher FS, which explains why comparatively larger FS was obtained for the OVA-polyphenol aggregates at pH = 3.0 [33]. The viscosity, G’ and G” of the OVA-DA and OVA-T2A did not differ significantly at the same pH, except for G’ and G” of OVA-DA-7.4 and OVA-T2A-7.4. Both the viscosity and G’ of the OVA-polyphenols were higher than that of the natural OVA, indicating that the addition of DA and T2A increased the viscosity and storage modulus G’ of OVA, which in turn contributed to the formation of stable, elastic, small and dense foams [34]. As suggested by Li, et al. [35], the supplement of polyphenols altered the secondary structure of the protein, thus inducing the extension of the polypeptide chain and resulting in the exposure of the hydrophobic groups. This leads to the probability of protein binding to the air-liquid interface, which decreases the gas-liquid interfacial tension and enhances the stickiness and viscoelasticity of the OVA-polyphenol system, thereby causing increased foaming properties of the aggregates.
The decreased viscosity and shear thinning behavior became more pronounced along with increasing pH. After raising the pH of the solution to 9.0, the aggregates carried more negative charge, which enhanced the electrostatic repulsion between the molecules and hindered the formation of intermolecular conjugates, thus contributing to shear thinning behavior [36]. Several non-denatured proteins have rigid structures, which results in a low rate of protein adsorption and rearrangement at the air-water interface, thus making it difficult to form a highly viscoelastic film [37, 38]. However, the unfolding of OVA caused by acid treatment is commonly accompanied by swelling, and higher intermolecular entanglements, which facilitates enhanced viscosity at acidic conditions [39]. In addition, increased viscosity also contributes to thicker interfacial film, which is conducive to the formation of protein-polyphenol aggregates with high FA and FS [40].
3.3 Polyphenols and Acidic pH promoted the conversion of α-helix to random coil in OVA
The addition of polyphenols and pH treatment led to significant changes in the CD of OVA, demonstrating that both the addition of polyphenols and the pH affected the non-covalent interactions between OVA and polyphenols, which in turn altered the OVA secondary structure (Fig. 3A). Specifically, the fluorescence intensity (absolute value) of OVA-DA was consistently lower than that of the OVA-T2A. Secondly, the CD spectra of OVA in the pH = 7.4 and pH = 9.0 groups with and without polyphenol addition were similar, but significantly different from that of the aggregates in the pH = 3.0 group, which was consistent with the results of FA and FS.
The specific details regarding the level of secondary structure for each OVA-polyphenol aggregate were calculated employing the online software BeStSel (Fig. 3B). It was obvious that the percentage of α-helix and β-sheet was lower, while the level of the random coil was higher for OVA-polyphenol aggregates at pH = 7.4 compared to that of the OVA-polyphenol aggregates at pH = 9.0. The proportion of α-helix and β-sheet was further decreased, while the ratio of the random coil was further improved when aggregates were treated at pH = 3.0. Acid treatment contributes to the unfolding of the OVA structure, which leads to alterations in their protein conformations [41]. These changes positively affect the adsorption and reorganization of OVA at interfaces, resulting in a higher foaming performance [42]. In contrast, alkaline treatment causes an increase in the accumulation of negative charge, which in turn generates a greater repulsive force and thus facilitates the protein molecules to retain small and uniform sizes. Therefore, this phenomenon is not conducive to protein conformational changes, which restrict the increase in FA and FS of proteins [43]. Furthermore, at pH 3.0, the supplement of polyphenols also caused a reduction in the α-helix content and an increase in the proportion of random coil, while the β-turn content remained stable. The changes in the secondary structures of OVA caused by the supplement of polyphenols revealed that part of the α-helix was converted to random coil conformation, implying that OVA was transformed from a rigid and tightly folded structure to a soft and extended structure, thus eventually creating a looser protein conformation with high FA and FS [2]. The most disordered structure was obtained in the OVA-T2A aggregate system at pH 3.0, as a result, this aggregate had the best foaming properties. As predicted above, T2A may form more covalent bonds with proteins, further leading to greater changes in the structural conformation of OVA.
3.4 Hydrogen bonding and hydrophobic forces participated in the OVA and polyphenol interactions
The main characteristic peaks of OVA shifted in the range of 4000 − 1000 cm− 1 under the effect of various pH treatments and various polyphenols, indicating that protein conformation changes induced by different treatments affected the interactions between OVA and polyphenols (Fig. 3C). Compared to those of the untreated aggregates, the specific absorption peaks of the acid or alkaline-treated OVA and OVA-polyphenol aggregates changed markedly. All aggregates exhibited broad typical peaks near 3300 cm− 1 as a result of intermolecular hydrogen bonding and O-H and N-H stretching vibrations [44]. After acid treatment, the bands of OVA, OVA-DA, and OVA-T2A at 3300.20 cm− 1, 3300.20 cm− 1 and 3296.35 cm− 1 were all blue shifted to 3296.35 cm− 1; while after alkaline treatment, the characteristic peaks of OVA, OVA-DA and OVA-T2A varied to 3300.20 cm− 1, 3296.35 cm− 1 and 3298.28 cm− 1, respectively. These results suggested that hydrogen bonding was involved in the binding between OVA and polyphenols, while pH affected their binding.
The peaks at around 2930 cm− 1 were assigned to the C-H stretching vibration proteome of CH3 and CH2, and their variations indicated the presence of hydrophobic interactions during the formation of OVA-polyphenol aggregates [45]. By adjusting the protein solution to acid condition, the bands of OVA, OVA-DA, and OVA-T2A red shifted from 2933.73 cm− 1, 2929.87 cm− 1 and 2929.87 cm− 1 to 2935.66 cm− 1, 2931.80 cm− 1, and 2933.73 cm− 1, respectively. However, their absorption peaks remained stable at pH 9.0, corresponding to the fact that the interaction forces in OVA-polyphenols involved hydrophobic interactions and this binding force was stronger at pH 3.0. The higher binding force at pH 3.0 can be explained by the following reasons. The acid environment promotes more unfolding of protein structure and more exposure of hydrophobic groups, which leads to greater changes in the hydrophobic interaction forces of the OVA-polyphenol aggregate system. Finally, T2A induced more changes in hydrophobic interactions than DA. This may be due to the fact that cinnamic acid-based polyphenol has a longer C-chain on the branched chain and more C = C bonds than benzoic acid-based polyphenol, resulting in stronger hydrophobic interactions between the polyphenol and OVA, which facilitates the formation of an elastic film at the air-water interface and ultimately leads to higher foaming capacity of OVA-T2A aggregates compared to that of the OVA-DA aggregates. The study of Wen, Zhang, Ning, Li, Zhang, Liu and Zhang [2] also found that polyphenols with different numbers and positions of hydroxyl groups can form aggregates with OVA and these aggregates exhibited different surface hydrophobicity and number of hydrogen bonds, thus showing variable foaming properties.
The amide I band (1600–1700 cm− 1) was assigned to the C = O and N-H tensile vibration [46]. These two characteristic bands in the OVA and OVA-polyphenol aggregate curves significantly shifted under different pH treatments due to the hydrophilic interactions between hydroxyl group of polyphenols and C = O and C-N groups of OVA subunit [47]. Furthermore, the intensity and position of the OVA characteristic peaks also changed, indicating that the secondary structure of OVA was altered after the addition of polyphenols (DA or T2A) or after different pH treatments. The results of CD and FT-IR were consistent.
3.5 Acid conditions increased the surface hydrophobicity of OVA
Surface hydrophobicity is an important indicator for evaluating changes in protein conformation [48]. Figure 4A, 4B, 4C, and 4D present the surface hydrophobicity, sulfydryl group, particle size and zeta-potential of aggregates under different pH treatments. The surface hydrophobicity of OVA and OVA-DA increased after acid treatment, but decreased under alkaline conditions. In terms of OVA-T2A aggregates, they all exhibited higher surface hydrophobicity (p < 0.05) at pH 3.0 than at pH = 7.4; however, the surface hydrophobicity of the OVA-T2A-9.0 was not significantly different from that of the OVA-T2A-7.4 (p > 0.05). pH 3.0 is close to the isoelectric point of OVA, leading to greater exposure of hydrophobic amino acids in OVA and OVA-polyphenols, ultimately inducing increased surface hydrophobicity [2]. This finding confirmed the results of FT-IR.
In terms of alkaline or neutral conditions, the addition of DA and T2A enhanced the surface hydrophobicity of OVA, except for DA at pH 9.0. The non-covalent interactions induced by the addition of polyphenols converted OVA into a loose structure, thus exposing its previously hidden hydrophobic groups, which ultimately enhanced the surface hydrophobicity of OVA-polyphenol aggregates [2]. Furthermore, the enhanced surface hydrophobicity promoted the adsorption rate and adsorption capacity of OVA at the air-liquid interface, which in turn enabled the rapid production of bubbles in large quantities. Results similar to ours were reported in WPI-proanthocyanidin aggregates (WPI-PA). Li and Girard [11] found that the highest surface hydrophobicity was observed in WPI-PA at pH 3.0. However, in the acid-treated group, the addition of polyphenols led to a decrease in the surface hydrophobicity of OVA, which may be due to the fact that the binding of polyphenols to OVA masked the hydrophobic binding sites of OVA, thus resulting in a decrease in the surface hydrophobicity of aggregates.
3.6 OVA-T2A obtained maximum free sulfhydryl group at pH = 3.0
The content of free -SH group represents alterations in the cleavage and accumulation of intramolecular and intermolecular disulfide bonds in the protein molecule [49]. OVA, the only egg white protein that contains free sulfhydryl groups, hides four free -SH groups in its hydrophobic core and a disulfide bond between CYS 74 and CYS 121, allowing for the formation of more stable protein structure [50]. As indicated in Fig. 4B, the content of free -SH groups was in the order of pH 3.0 group > pH 7.4 group ≥ pH 9.0 group. There are two possible reasons for the above phenomenon. Firstly, acid environment induces protein unfolding and degradation, which exposes a higher rate of internal sulfhydryl groups [50, 51]. Secondly, both transformations of thiolate to thiol and thiol to disulfide are suppressed at pH 3.0, thus inhibiting sulfhydryl groups in OVA from forming disulfide bonds [52].
The free sulfhydryl group content of OVA and OVA-polyphenols followed the order of OVA-T2A > OVA-DA = OVA. The use of polyphenols also induced the unfolding of protein structure and promoted the exposure of internal sulfhydryl groups, and the hydroxyl groups of polyphenols could also hinder and reduce the formation of disulfide bonds through redox reactions, ultimately leading to an increase in free -SH group content [53]. Specifically, cinnamic acid-based polyphenols (T2A) formed more covalent bonds with proteins than benzoic acid-based polyphenols (DA), which can lead to a greater degree of protein conformational changes and greater exposure of internal sulfhydryl groups [54]. Additionally, cinnamic acid-based polyphenols exhibited higher antioxidant activity than benzoic acid-based polyphenols [55], thereby preventing the formation of disulfide bonds. These two factors contributed to the increased free sulfhydryl group content in the aggregate systems, causing OVA-T2A aggregates to exhibit the highest free -SH group content among OVA and OVA-polyphenol complexes. The increase in the free -SH group content positively correlates with the unfolding of internal structure and the disruption of the rigid structure of OVA, thus increasing the protein adsorption and rearrangement at the interface, which ultimately enhances the foaming properties of OVA [56].
3.7 Acid treatment and polyphenols led to larger particle size
As shown in Fig. 4C, acid treatment led to larger particle size, except for OVA group, while alkaline treatment did not cause a substantial change in particle size. There exists a low electrostatic repulsion between protein molecules at pH 3.0. Therefore, these proteins exhibit the lowest solubility, possibly causing the self-aggregation of OVA, and the highest swelling, both of which induce larger particle size [57]. However, under neutral and alkaline conditions, OVA and OVA aggregates became charged particles, with large number of negative charges residing on their surface. Therefore, greater negative charges led to higher electrostatic repulsion between the complexes, which prevented the particles from aggregating and contributed to maintaining a small and uniform size. Large particle size caused weaker binding force between OVA-polyphenol groups, which promoted the adsorption, swelling and rearrangement of OVA molecules at the air-liquid interface, thereby improving protein foaming properties [56]. Besides, large particle size also induced an increase in the thickness of interfacial film, which further reduced the possibility of shrinking, growing, coalescing, and moving of air bubbles, finally promoting foam stability.
The addition of DA and T2A led to an increase in the particle size of OVA. On the one hand, polyphenols induce protein aggregation, further resulting in an enhancement in particle size [58]. On the other hand, the interactions between OVA and polyphenols are considered to be the main contributing factor [58]. This result corroborates with previous findings of Chang, et al. [59] and von Staszewski, et al. [60]. These results confirm the conjecture proposed in the FA and FS results.
It was worth noting that peak shapes were also varied between OVA-polyphenols at different pH treatments. The most significant variation was observed in the OVA-T2A aggregates at pH 3.0. Compared to OVA-DA, OVA-T2A-7.4 and OVA-T2A-3.0 had two distinct broad peaks, probably due to the oxidation and cross-linking of polyphenols in the OVA-polyphenol network, thus leading to an increase in particle size of the aggregates [59, 61]. T2A has a longer C-chain and C = C bond on the branched chain, which enables it more unstable and susceptible to oxidizing reactions, thus creating two distinct sizes of particles via binding of OVA with T2A or its oxidation products.
3.8 Acidic and neutral environments weakened the electrostatic repulsion of aggregates
The zeta-potential is a measure of the surface charge density of a protein or protein complex, and it is also a useful indicator for assessing the stability of a dispersion [35]. The treatment of pH can influence the surface charge (number and type of charges) of OVA, which in turn affects its non-covalent interactions with polyphenols [62]. Obviously, all negative charges surrounding the OVA, OVA-DA and OVA-T2A aggregates were neutralized and only a small number of positive charges were present (Fig. 4D), implying weaker electrostatic repulsion and enhanced attractive intermolecular forces. In contrast, more negative charges were observed on the surface of these aggregates when treated at pH 9.0, indicating the existence of strong electrostatic repulsion between aggregates. Such alterations in zeta-potential cause intermolecular force changes between the OVA-polyphenol aggregates, which further result in altered protein size and conformation, ultimately influencing the FA and FS of OVA-polyphenol aggregates. Similar results were reported by Thongkaew, et al. [63].
3.9 OVA-T2A aggregates had the lowest intrinsic fluorescence intensity at pH = 3.0
To investigate the structural changes of proteins after the addition of polyphenols or acid-base treatment, IFS spectroscopy was applied in the present study [64]. The introduction of polyphenols led to a significant reduction in the IFS of OVA (Fig. 5), indicating the presence of non-covalent interactions between OVA-polyphenols. Among them, T2A strongly quenched the IFS of OVA, implying a greater non-covalent binding in OVA-T2A aggregate. These results corroborated the findings observed in FT-IR and surface hydrophobicity.
Both acidic and alkaline treatments caused a decrease in fluorescence intensity of OVA and OVA-polyphenols, and they can be ranked as follows: pH = 3.00 group > pH = 9.00 group, except for OVA-T2A-9.0. Conventionally, non-covalent interactions between polyphenols and OVA are strongest in a pH environment slightly below the protein isoelectric point, which may result from the greatest exposure of hydrophobic groups and conformational changes in OVA-polyphenol aggregates [65]. OVA at pH 9.0 carries a large number of negative charges on its surface, which causes increased electrostatic repulsion between OVA and polyphenols. However, the OVA maintains a small and homogeneous size at this point, leading to protein structure that can barely unfold, thus weakening the hydrophobic forces between OVA-polyphenol complexes. As a result, the non-covalent interactions between the aggregates at pH 9.0 are higher than those of the aggregates at pH 7.4, but lower than those of the aggregates at pH 3.0, corresponding to the fact that lower fluorescence quenching was observed at pH 9.0 compared to that at pH 3.0. However, since the non-covalent interactions are a very complex system [66], the changing pattern of non-covalent interactions of OVA-T2A under different pH treatments is slightly different from that of the other groups. This may be due to the predominance of intermolecular hydrophobic interactions in OVA-T2A, where the intermolecular hydrophobic interactions of OVA-T2A-7.4 are greater than those of OVA-T2A-9.0, thereby inducing a more severe fluorescence quenching.
3.10 T2A strongly quenched the fluorescence intensity of OVA
3-D fluorescence spectroscopy is an essential method for evaluating conformational changes in proteins [67]. Peak A (λex = 275 nm) is the characteristic band of OVA, reflecting changes of protein tertiary structure, while peak B (λex = 230 nm) is the characteristic band of the polypeptide backbone structure (C = O) of OVA, which reveals changes of secondary structure of protein [68]. The 3-D fluorescence spectra of aggregates under different pH treatments is given in Fig. 5D-L, and the related parameters are shown in Table S1. After conjugating with polyphenols, the fluorescence intensity of OVA at peak A and peak B was slightly reduced due to the interaction between OVA and polyphenols. The decrease in fluorescence intensity of peak A indicated that the fluorescence intensity was partially quenched by polyphenols, whereas the reduction in fluorescence intensity of peak B may be attributed to the extension of the polypeptide backbone structure altering the secondary structure of OVA. The degree of quenching of DA and T2A was similar at pH 3.0 and pH 7.4, but T2A obtained higher fluorescence quenching ability than DA at pH 9.0, indicating that greater affinity between OVA and T2A could be the primary reason for greater secondary and tertiary structure changes in the OVA. These observations were consistent with the results of the IFS spectra, CD, FT-IR, and surface hydrophobicity.
Furthermore, the fluorescence intensity of peak A of OVA followed the following order: pH 3.0 groups > pH 7.4 groups > pH 9.0 groups. This is because OVA structure is more expanded in the acidic environment, which contributes to the fluorescence intensity of aromatic amino acids being more easily detected, whereas in the alkaline environment, the structure of OVA is more compact and the fluorescence intensity of aromatic amino acids is significantly masked. The fluorescence intensity of peak B changed significantly under various pH treatments, indicating that pH treatment causes several changes in the secondary structure of OVA. These observations confirmed the results of CD and FT-IR, which further confirmed the results of foaming.
3.11 Mechanism of foam formation
The mechanism diagrams of foam formation of OVA-DA and OVA-T2A at pH 3.0, 7.4 and 9.0 were presented in Fig. 6. On encountering benzoic acid-based polyphenols and cinnamic acid-based polyphenols, the structure of OVA unfolds under strong hydrogen bonding and hydrophobic forces, thus resulting in the exposure of the hydrophobic groups. In addition, the formation of hydrogen bonds between polyphenols and OVA further induces a change in the structure of OVA from a rigid and tightly folded structure to a soft and extended structure, ultimately leading to alterations in the secondary and tertiary structure of protein. Comparatively, cinnamic acid-based polyphenols generally lead to greater changes in the conformation of OVA due to higher affinity between polyphenols and OVA. In the extremely acidic environment, only a small amount of positive charge exists in the OVA-polyphenol system, implying weaker electrostatic repulsion and enhanced intermolecular attraction, which further induces self-aggregation and solubilization of OVA and results in better FA and FS. In an extremely alkaline environment, the presence of a large number of negative charges on the surface of the OVA-polyphenols resulted in high electrostatic repulsion, which prevented the particles from aggregating and contributed to maintaining a small and uniform size. These behaviors weakened the FA of the OVA-polyphenol aggregates, while inducing a decrease in the thickness of the interfacial film. Our study advances the understanding of the effects of pH and polyphenol structure on the interaction forces acting on OVA-polyphenol aggregates. In addition, the present study also contributes to the precise utilization of different pH and polyphenols with different structures to regulate the foaming of OVA, thereby producing high-quality products in the baking industry.
