Effect of benzoic acid-based and cinnamic acid-based polyphenols on foaming properties of ovalbumin at acidic, neutral and alkaline pH conditions

Effect of benzoic acid-based and cinnamic acid-based polyphenols on foaming properties of ovalbumin at acidic, neutral and alkaline pH conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of benzoic acid-based and cinnamic acid-based polyphenols on foaming properties of ovalbumin at acidic, neutral and alkaline pH conditions Hedi Wen, Deju Zhang, Zhenzhen Ning, Zihao Li, Yan Zhang, Jingbo Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4011113/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Mar, 2024 Read the published version in Food Hydrocolloids → Version 1 posted You are reading this latest preprint version Abstract To broaden the application of ovalbumin (OVA) in food formulations, it is meaningful to improve its foaming characteristics. This study aimed to investigate the effect of benzoic acid-based (3,4-dihydroxybenzoic acid, DA) and cinnamic acid-based polyphenols (trans-2-hydroxycinnamic acid, T2A) on the foaming properties of OVA at acidic (pH 3.0), neutral (pH 7.4) and alkaline (pH 9.0) pH conditions. Both the addition of polyphenols and acid treatment enhanced the foaming properties of OVA. Surface hydrophobicity, circular dichroism, free sulfhydryl groups, and Fourier transform infrared spectroscopy results indicated that after acidic workup, the presence of stronger hydrophobic interactions in OVA-polyphenol aggregates induced more disordered protein conformation and conversion or breakage of disulfide bonds. Particle size and zeta potential indicated that acidic treatment neutralized protein surface charges, further inducing self-aggregation and swelling of OVA, ultimately enhancing foaming properties. Comparatively, T2A exhibited better foam-inducing capacity due to its stronger interaction with OVA, leading to the unfolding of the OVA structure and the exposure of more hydrophobic groups. The intrinsic and 3-D fluorescence spectra experiments also confirmed that OVA-T2A aggregates at pH 3.0 had greater altered non-covalent interaction forces and protein secondary and tertiary structures compared to other complexes. This study provides a theoretical basis for designing protein formulations with excellent foaming properties. Food Science & Technology Polyphenols pH Ovalbumin Protein-polyphenol interactions Foaming properties Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Egg white protein (EWP) is a popular ingredient in many food formulations, such as cake, mousse, and ice cream [ 1 ], due to their excellent functional properties, including gelling, foaming, and emulsifying capacity [ 2 ]. The major components of EWP are ovalbumin (OVA), ovotransferrin, ovomucoid and lysozyme, which account for 54%, 12%, 3.5% and 3.4% of the total EWP, respectively [ 3 ]. Among them, OVA occupies the main component and also contributes most to the functionality of EWP. When used in the food industry, EWP can encapsulate and hold air, thereby enhancing the volume of foam and further creating a food system with smooth and soft texture [ 4 ]. However, natural OVA has a rigid structure, resulting in limited foaming ability (FA) and unstable foams [ 5 ], which makes improving the foaming properties of OVA a valuable issue. Recently, various methods, such as protein-saccharide graft [ 6 ], protein-polyphenol conjugate [ 7 ], heating treatment [ 8 ], pH treatment and ultrasonic processing [ 9 ], have been explored to enhance foaming properties of proteins. Comparatively, polyphenol-protein conjugates not only improve the functionality of protein, but also contribute to the nutritional value and sensory properties of food [ 2 ]. When proteins are conjugated with polyphenols, the hydrogen bonds and hydrophobic interactions between them are altered, thereby causing changes in the secondary and tertiary structure of proteins, ultimately inducing different FA and foaming stability (FS) [ 2 ]. pH treatment is a highly efficient and low-cost method for improving the foaming characteristics of OVA. It can change the protein conformation by affecting the electrostatic interaction forces, which ultimately influence the protein foaming properties [ 10 ]. Therefore, the combination of pH treatment and the addition of polyphenols may improve the foaming characteristics of OVA to the largest extent because of the combined effects on electrostatic forces, hydrogen bonding and hydrophobic forces of protein-polyphenol aggregates. Li and Girard [ 11 ] enhanced the foaming performance of whey protein using sorghum proanthocyanidins, with the formed aggregates achieving the FA at pH 5.0 and the highest FS at pH 7.0. However, this study focused only on protein and polyphenol binding under acidic and neutral conditions, while the effects of alkaline conditions on the interaction forces between aggregates were lacking. Additionally, it neglected to explore the relationship between polyphenol types and the foaming properties of whey protein, causing its results insufficient to guide the selection of the most appropriate polyphenols for enhancing FA. Polyphenols are secondary plant metabolites with aromatic rings containing one or more hydroxyl or methoxy groups, and include types such as phenolic acids, flavonoids, tannins, and lignans [ 12 , 13 ]. Among them, phenolic acids are one of the most common types of polyphenols, known for their simple structure, strong antioxidant activity, and well-defined biological activities. According to the constitutive carbon frameworks, they can be categorized into benzoic acid-based (C6–C1 structures) and cinnamic acid-based phenolic acids (C6–C3 structures) [ 12 ]. Both two phenolic acids can bind to proteins, but their affinities differ, further leading to different protein foaming performances. Li, Li, Dai, Hu, Niu, Liu and Chen [ 12 ] demonstrated that ferulic acid (cinnamic acid-based phenolic acids) had a greater binding affinity with β-casein compared to syringic acid (benzoic acid-based phenolic acids). However, the limitation of this study is that it only examined the binding force changes between β-casein and polyphenols, neglecting the alterations in FA and protein interfacial behavior caused by protein-polyphenol interactions. As a result, the findings cannot directly guide the formulation development of high-foaming products such as baked goods and ice cream. Meanwhile, the number and position of the hydroxy group in polyphenols also affect the foaming performance of proteins, which was also not explored in this study. Another study by Yuan, et al. [ 14 ] reported that phenolic acid with hydroxy group at the 2-position induced higher binding affinity to bovine serum albumin, whereas a negative effect was observed at the 4-position. Nevertheless, whether different carbon frameworks of polyphenols also influence protein conformation is unclear. Therefore, in order to determine the patterns by which the hydroxy group and carbon framework of polyphenols and pH affect protein foamability, three benzoic acid-based polyphenols with different hydroxyl group numbers (P-hydroxybenzoic acid (PA, 1 hydroxyl group), 3,4-dihydroxybenzoic acid (DA, 2 hydroxyl groups) and gallic acid (GA, 3 hydroxyl groups)) and three cinnamic acid-based polyphenols with various hydroxyl group positions ( trans -3-hydroxycinnamic acid (T3A, meta-position), trans -2-hydroxycinnamic acid (T2A, ortho-position) and 4-coumaric acid (CA, para-position)) were combined with OVA at pH 3.0, 7.4 and 9.0, respectively, and their foaming properties were compared to screen the OVA-polyphenol complexes with the highest foaming properties. Subsequently, to interrogate the reasons behind the varying foaming of OVA, two OVA-polyphenol aggregates with comparatively higher FA, OVA-DA and OVA-T2A, were selected from six aggregates, and further exploration was conducted on the microscopic changes occurring in OVA due to the addition of polyphenols and the use of acidic pH. Our study will provide a theoretical foundation for the precise application of polyphenols to enhance the foaming properties of OVA. 2. Materials and Methods 2.1 Materials OVA with different purity (> 98% or 62–88%) was supplied by Sigma Chemical Co. (St. Louis, Mo, USA). GA (98%), DA (≥ 97%), PA (99%), CA (98%), T3A (99%), T2A (99%), phosphate buffer solution (PBS, pH 7.4), 8-anilino-1-naphthalene sulfonic acid (96%) and 5, 5'-dithio-bis-2-nitrobenzoic acid (98%) were procured from the Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). Tris (99%), glycine (≥ 99%), and hydrochloric acid (HCl, 36.0–38.0%) were procured from Solarbio (Beijing, China), Beyotime Institute of Biotechnology (Shanghai, China) and Sinopharm Chemical Reagent Co., Ltd. (Beijing, China), respectively. All other chemicals exploited in this research were of analytical grade. 2.2 Sample preparation The OVA powders (purity, 62–88%) and polyphenols were blended in distilled water to yield a final concentration of 0.4 mmol/L and 4 mmol/L, respectively, and then stirred for 1 h using a magnetic stirrer. All protein solutions were stored at 4 ℃ overnight to facilitate further solubilization. After that, the OVA solution was thoroughly mixed with an equal volume of each polyphenol solution to obtain the OVA-GA, OVA-DA, OVA-PA, OVA-CA, OVA-T3A and OVA-T2A aggregates (molar ratio of 1:10). In the control group, the polyphenol samples were substituted with distilled water. Next, the OVA-polyphenol solutions were split into thirds: one aliquot was adjusted to pH 9.0 using NaOH (OVA-GA-9.0, OVA-DA-9.0, OVA-PA-9.0, OVA-CA-9.0, OVA-T3A-9.0 and OVA-T2A-9.0), one aliquot to pH 3.0 using HCl (OVA-GA-3.0, OVA-DA-3.0, OVA-PA-3.0, OVA-CA-3.0, OVA-T3A-3.0 and OVA-T2A-3.0) and the other remained untreated (pH = 7.4, OVA-GA-7.4, OVA-DA-7.4, OVA-PA-7.4, OVA-CA-7.4, OVA-T3A-7.4 and OVA-T2A-7.4). All prepared OVA-polyphenol solutions were further magnetically stirred for 1 h before storing in the dark in a refrigerator at 4 ℃. 2.3 Foaming properties 2.3.1 Determination of foaming ability and foaming stability The foaming properties of OVA-polyphenol complexes treated by different pH were characterized at 25 ℃ according to Zhang, et al. [ 15 ]. Briefly, 30 mL of sample placed in a graduated glass cylinder with an internal diameter of 22 mm was subjected to a homogenizer (T25, IKA, Germany, 10000 r/min, 1 min) to generate foam. The appearance of the bubbles was photographed using a camera, while the final volume of each solution after stirring was determined at 0 and 30 min and their foaming properties were calculated using the following relationships: FA = (V 1 - V 0 )/V 0 × 100% (1) FS = (V 2 - V 0 )/(V 1 - V 0 ) × 100% (2) Here, V 0 , V 1 and V 2 represented the volume of the foam before blending, after blending, and after 30 min of standing. 2.3.2 Foaming microstructure measurement Based on the FA and FS results, OVA, OVA-DA, due to their highest foaming among the benzoic acid-based polyphenol-OVA aggregates, and OVA-T2A, due to their highest foaming among the cinnamic acid-based polyphenol-OVA aggregates, were selected for subsequent studies. The microstructure of the bubbles was observed using a Nikon Instrument (Eclipse TS100, Nikon, Japan) according to Wen, Zhang, Ning, Li, Zhang, Liu and Zhang [ 2 ]. In detail, the air bubbles of these solutions were gently spread on a glass plate and covered with a coverslip, before observing on the microscope at a magnification of × 40 (4 × eyepiece and 10 × objective lens). 2.4 Measurement of rheological properties The rheological properties (viscosity and storage modulus G′) of OVA and OVA-polyphenol solutions at various pH treatments were determined by HR-1 rheometer (RS6000, Haake, Germany) following the procedure of Liu, et al. [ 16 ]. In terms of steady-state rheology, 1.5 mL of the solutions was carefully added to the plate and the range of shear rate was kept from 0.1 to 100 s − 1 during the steady shear measurement at 25 ℃. For dynamic rheology, the samples were replated onto plates and were measured at a strain of 3% and a shear frequency ranging from 0.1 to 10 Hz. Subsequently, the viscosity, shear stress and storage modulus G′ of the solution system were recorded as a function of frequency. 2.5 Measurement of CD The OVA solutions (purity > 98%, similarly hereinafter) with or without polyphenols were prepared and measured as described by Wen, Zhang, Ning, Li, Zhang, Liu and Zhang [ 2 ]. Then the aggregates were diluted to 0.025 mmol/L and their CD spectra were recorded with a MOS-500 Circular Dichroism Spectrometer (Bio-Logic, France). Data of the samples were collected from 190 to 260 nm and imported into the Bestsel website ( http://bestsel.elte.hu/index.php ) to calculate the secondary structure content of the OVA. 2.6 Measurement of FT-IR The aggregates under different pH treatments were freeze-dried and analyzed using FT-IR spectroscopy as described by Jiang, et al. [ 17 ], with minor modifications. KBr transparent flakes were prepared by blending 2 mg of each OVA-polyphenol aggregate with 200 mg of KBr and pressing the mixture into a 13-mm disk using a die press. Thereafter, the sample flakes were subjected to the IRPrestige-21 Spectrometer (Shimadzu, Japan) and the spectra was accumulated over the wavenumber range of 400 to 4000 cm − 1 with a nominal resolution of 4 cm − 1 for 64 scans. 2.7 Surface hydrophobicity determination The surface hydrophobicity of the samples under different pH treatment conditions was measured using an F-7100 fluorescence spectrophotometer (Hitachi High-Tech Science, Japan) as per described by Wen, et al. [ 18 ]. Briefly, 1 mL of OVA or OVA-polyphenol aggregates (0.025 mmol/L) was mixed with 10 µL of ANS (8 mmol/L, suspended in PBS) and was further incubated in the dark at room temperature for 30 min. Subsequently, the resulting samples were recorded using the fluorescence spectrophotometer, with the following measurement settings: excitation wavelength of 390 nm, emission wavelengths of 400–600 nm, and constant slit width of 5 nm. 2.8 Determination of free -SH group The -SH group content in OVA and OVA-polyphenol aggregates at different pH conditions was analyzed according to Lyu, et al. [ 19 ]. The content of the sulfhydryl group was calculated as follows: -SH (µmol/g) = 75.53 × D × A 412 /C (3) Here, A 412 denoted the absorbance of samples measured at 412 nm, C referred to the concentration of OVA (0.025 mmol/L), and D was the dilution factor (2). 2.9 Particle size and zeta-potential The diluted OVA and OVA-polyphenol aggregate solutions used in Section 2.5 were placed into a Zetasizer Nano ZS90 instrument (Malvern Co., UK) for analyzing particle size and zeta-potential using the procedure described by Wen, et al. [ 20 ]. 2.10 Measurement of intrinsic fluorescence spectra Aggregate solutions were diluted to 0.05 mmol/L with deionized water and then heated in a water bath maintained at 303 K for 1 h, before intrinsic fluorescence spectra (IFS) intensity measurement. Changes in IFS spectra of different samples were recorded by an F-7100 fluorescence spectrophotometer as per described by Yu, et al. [ 21 ]. 2.11 Measurement of three-dimensional fluorescence As suggested by Ren, et al. [ 22 ], the 3-D fluorescence spectra of OVA and OVA-polyphenol aggregates diluted to 0.0025 mmol/L was determined by fluorescence spectrophotometer instrument using excitation wavelength of 200 nm to 400 nm, emission wavelength of 200 nm to 400 nm, scanning speed of 12000 nm/min, and slit width of 5 nm. 2.12 Statistical analysis Each experiment was repeated three times and the mean and standard deviation were obtained from these results. All analyses were subjected to one-way analysis of variance (ANOVA) (Tukey’s test) using SPSS 20.0 (IBM SPSS Statistics, IBM Corp., Somers, NY), and the level of significance used was p < 0.05. 3. Results and discussion 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 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 CH 3 and CH 2 , 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). 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. 4. Conclusion DA and T2A, one of the benzoic acid-based and cinnamic acid-based polyphenols, respectively, can induce protein conformational changes in OVA at different pH conditions, thus influencing the FA and FS of the formed OVA-polyphenol aggregates. Both the application of polyphenols and the acid treatment improved the FA and FS of OVA, which was due to the strong hydrophobic interactions between aggregates leading to greater protein conformational disorder and conversion or breakage of disulfide bonds, further creating an interfacial film with high cohesiveness and elasticity, ultimately contributing to the formation of stable and small density foams. The results of particle size and zeta-potential indicated that acidic treatment led to neutralization of protein surface charges, further inducing swelling and self-aggregation and promoting protein adsorption and rearrangement at the air-liquid interface. Notably, T2A, compared to DA, and acidic environment, compared to neutral and alkaline conditions, induced more alterations in disulfide bonds, protein conformation and non-covalent interaction forces, which in turn increased the positive effect on foaming properties of OVA. The IFS and 3-D fluorescence spectra experiments also confirmed that OVA-T2A aggregates at pH 3.0 had large altered non-covalent interaction forces and protein secondary and tertiary structures, which explained their high foaming properties and allowed them to serve as promising functional ingredients in food systems. This study may provide guidance on the use of polyphenols to regulate the foaming properties of proteins in the baking industry. Declarations Conflict of interest All authors have no conflict of interest. Author contributions Hedi Wen: Investigation; Methodology; Validation. Zhenzhen Ning: Methodology; Formal analysis; Data curation; Validation. 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Lwt-Food Sci Technol 146. 10.1016/j.lwt.2021.111383 von Staszewski M, Jara FL, Ruiz A, Jagus RJ, Carvalho JE, Pilosof AMR (2012) Nanocomplex formation between β-lactoglobulin or caseinomacropeptide and green tea polyphenols: Impact on protein gelation and polyphenols antiproliferative activity. J Funct Foods 4:800–809. 10.1016/j.jff.2012.05.008 Zhou SD, Lin YF, Xu X, Meng L, Dong MS (2020) Effect of non-covalent and covalent complexation of (-)-epigallocatechin gallate with soybean protein isolate on protein structure and in vitro digestion characteristics. Food Chem 309:125718. 10.1016/j.foodchem.2019.125718 Wang Y, Zhang J, Zhang LF (2022) Study on the mechanism of non-covalent interaction between rose anthocyanin extracts and whey protein isolate under different pH conditions. Food Chem 384:132492. 10.1016/j.foodchem.2022.132492 Thongkaew C, Gibis M, Hinrichs J, Weiss J (2014) Polyphenol interactions with whey protein isolate and whey protein isolate-pectin coacervates. Food Hydrocolloids 41:103–112. 10.1016/j.foodhyd.2014.02.006 Tian R, Feng JR, Huang G, Tian B, Zhang Y, Jiang LZ, Sui XN (2020) Ultrasound driven conformational and physicochemical changes of soy protein hydrolysates. Ultrason Sonochem 68:105202. 10.1016/j.ultsonch.2020.105202 Hagerman AE, Butler LG (1981) The specificity of proanthocyandin-protein interactions. J Biol Chem 256:4494–4497 Han JR, Du YN, Yan JN, Jiang XY, Wu HT, Zhu BW (2021) Effect of non-covalent binding of phenolic derivatives with scallop (Patinopecten yessoensis) gonad protein isolates on protein structure and in vitro digestion characteristics. Food Chem 357:129690. 10.1016/j.foodchem.2021.129690 Jian LZ, Liu YJ, Li L, Qi BK, Ju MN, Xu Y, Zhang Y, Sui XN (2019) Covalent conjugates of anthocyanins to soy protein: Unravelling their structure features and in vitro gastrointestinal digestion fate. Food Res Int 120:603–609. 10.1016/j.foodres.2018.11.011 Hasan Z, Islam A, Khan LA (2023) Spectroscopic investigations on fungal aspartic protease as target of gallic acid. Int J Biol Macromol 228:333–345. 10.1016/j.ijbiomac.2022.12.218 Additional Declarations The authors declare no competing interests. Supplementary Files Supplement.doc GA.png Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 01 Mar, 2024 Read the published version in Food Hydrocolloids → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4011113","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276166217,"identity":"1cf297c8-cda9-48d4-a499-88ab5c559d05","order_by":0,"name":"Hedi Wen","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Hedi","middleName":"","lastName":"Wen","suffix":""},{"id":276166218,"identity":"9feec878-8e02-4fa7-807e-debab5c61577","order_by":1,"name":"Deju Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Deju","middleName":"","lastName":"Zhang","suffix":""},{"id":276166219,"identity":"a77c431a-c847-418a-8300-113e594b0013","order_by":2,"name":"Zhenzhen Ning","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhenzhen","middleName":"","lastName":"Ning","suffix":""},{"id":276166220,"identity":"bf67b555-19cc-454a-822b-9bd76f73cb5b","order_by":3,"name":"Zihao Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zihao","middleName":"","lastName":"Li","suffix":""},{"id":276166221,"identity":"7152594f-7679-4013-b58f-e8bc7512976a","order_by":4,"name":"Yan Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":276166222,"identity":"44765928-7d47-4411-ad59-4cc3fc47d24e","order_by":5,"name":"Jingbo Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jingbo","middleName":"","lastName":"Liu","suffix":""},{"id":276166223,"identity":"50e16976-96f9-4640-bcda-eedd1f420953","order_by":6,"name":"Ting Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIie3PIWsDMRTA8YSDU9nOvpJ9iBsHYWL0s2QEXk1FZcXYUpOZwez1W0xOpgRSE6g9UVMK85Uzxy4wZkrazk3kLwIJ/Mh7hORy/7Fqod1X3z+//b5ASQjVMk3ArfaktHSpf+7nCUHVRPJuLyW1DYLP2LZo1s7vZ/PxE+EGgX5gmrhXwVv4LEXASdMGBeTGe6BhevIXYHXBRMcEvzJ2GGxigJr5CTIdiCygaSPpLyLD+sy6uoZIdCQ4DGbSg406t9otNUoIiJx5NTKA6u7BpNe/3iy0Peh7Wb04z9njuKoAb7uDUUlyXBkP+QeQy+VyueO+ARaoUvnMvL/nAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Ting","middleName":"","lastName":"Yu","suffix":""},{"id":276166224,"identity":"ac19f3e1-42fa-45e1-96ef-553847132737","order_by":7,"name":"Ting Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-04 08:28:29","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4011113/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4011113/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.foodhyd.2024.109998","type":"published","date":"2024-03-01T22:02:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51992199,"identity":"44de58a2-d000-4d42-9066-5fda5130d695","added_by":"auto","created_at":"2024-03-05 04:48:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":448688,"visible":true,"origin":"","legend":"\u003cp\u003eFoaming properties of OVA-polyphenol aggregates at different pH conditions. Foaming ability of OVA, OVA-DA and OVA-T2A (A), foaming stability of OVA, OVA-DA and OVA-T2A (B), foam volume of OVA, OVA-DA and OVA-T2A at 0 min after homogenization (C), foam volume of OVA, OVA-DA and OVA-T2A at 30 min after homogenization (D), foam size of OVA, OVA-DA and OVA-T2A at 0 min after homogenization (E), and foam size of OVA, OVA-DA and OVA-T2A at 30 min after homogenization (F). Ovalbumin, OVA; 3,4-dihydroxybenzoic acid, DA; trans-2-hydroxycinnamic acid, T2A. A-C means for the same sample at different pH values without common letters are significantly different (p \u0026lt; 0.05). a-b means for the different samples with same pH values without common letters are significantly different (p \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/9455ce3f8d1db22093bf4ceb.png"},{"id":51992171,"identity":"a8dac06e-d90f-41ae-bb19-aafc76ea3e78","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187028,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity (A) and storage modulus G’ (B) of OVA-polyphenol interaction systems at different pH conditions\u003c/p\u003e","description":"","filename":"Fig2.GG.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/3b6ee8d16066ff8b5fad6e0f.png"},{"id":51992165,"identity":"ecdf91b1-adaa-4275-929a-3215450328f6","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292115,"visible":true,"origin":"","legend":"\u003cp\u003eThe circular dichroism (CD) spectra, protein secondary structure content and fourier transform infrared (FT-IR) spectra of OVA-polyphenol interaction systems at different pH conditions. CD spectra of OVA-polyphenol aggregates (A), protein secondary structure content of OVA-polyphenol aggregates (B), and FT-IR spectra of OVA-polyphenol aggregates (C)\u003c/p\u003e","description":"","filename":"Fig3.CDandFTIR3.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/d3c50769c3bdb55aef0b141f.png"},{"id":51992167,"identity":"881f32c7-84f7-4b30-940e-55a98daf2715","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":923764,"visible":true,"origin":"","legend":"\u003cp\u003eSurface hydrophobicity (A), sulfydryl group (B), particle size (C) and zeta-potential (D) of OVA, OVA-DA and OVA-T2A\u003c/p\u003e","description":"","filename":"Fig4.particlesize5.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/f61898ad33ae1a9675d6dd04.png"},{"id":51992166,"identity":"4188cbd1-3b47-44cc-8ea4-90d14569413e","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325465,"visible":true,"origin":"","legend":"\u003cp\u003eIntrinsic fluorescence and three-dimensional fluorescence spectra of OVA, OVA-DA, and OVA-T2A aggregates. Intrinsic fluorescence spectra of OVA (A), OVA-DA (B), and OVA-T2A (C) and three-dimensional fluorescence spectra of OVA at pH 3.0 (D), OVA at pH 7.4 (E), OVA at pH 9.0 (F), OVA-DA at pH 3.0 (G), OVA-DA at pH 7.4 (H), OVA-DA at pH 9.0 (I), OVA-T2A at pH 3.0 (J), OVA-T2A at pH 7.4 (K), and OVA-T2A at pH 9.0 (L)\u003c/p\u003e","description":"","filename":"Fig5intensity2.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/4380895796ba2fd99a3dd495.png"},{"id":51992172,"identity":"29a51900-e1e1-4a68-9f45-31153b5de07b","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2146715,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism diagrams of foam formation of OVA-DA and OVA-T2A at pH 3.0, 7.4 and 9.0\u003c/p\u003e","description":"","filename":"Fig6.overall2.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/5bb7e9bc76b0827370e4a6b5.png"},{"id":52796114,"identity":"c4ad5913-9ce9-4864-ab94-f05d257a1e88","added_by":"auto","created_at":"2024-03-15 22:02:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2443636,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/65edf330-fee6-47c0-baf6-70799840d012.pdf"},{"id":51992170,"identity":"6fe18549-e53f-4ca2-be98-00ed00246f45","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4207104,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.doc","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/b8bf9e4336c9b9519431f0ff.doc"},{"id":51992169,"identity":"0fe4a8a8-447c-4826-bd93-70d31bd24eab","added_by":"auto","created_at":"2024-03-05 04:40:53","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":534522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-4011113/v1/ba7362572eb615614c6db9aa.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEffect of benzoic acid-based and cinnamic acid-based polyphenols on foaming properties of ovalbumin at acidic, neutral and alkaline pH conditions\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEgg white protein (EWP) is a popular ingredient in many food formulations, such as cake, mousse, and ice cream [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], due to their excellent functional properties, including gelling, foaming, and emulsifying capacity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The major components of EWP are ovalbumin (OVA), ovotransferrin, ovomucoid and lysozyme, which account for 54%, 12%, 3.5% and 3.4% of the total EWP, respectively [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among them, OVA occupies the main component and also contributes most to the functionality of EWP. When used in the food industry, EWP can encapsulate and hold air, thereby enhancing the volume of foam and further creating a food system with smooth and soft texture [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, natural OVA has a rigid structure, resulting in limited foaming ability (FA) and unstable foams [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which makes improving the foaming properties of OVA a valuable issue.\u003c/p\u003e \u003cp\u003eRecently, various methods, such as protein-saccharide graft [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], protein-polyphenol conjugate [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], heating treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], pH treatment and ultrasonic processing [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], have been explored to enhance foaming properties of proteins. Comparatively, polyphenol-protein conjugates not only improve the functionality of protein, but also contribute to the nutritional value and sensory properties of food [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When proteins are conjugated with polyphenols, the hydrogen bonds and hydrophobic interactions between them are altered, thereby causing changes in the secondary and tertiary structure of proteins, ultimately inducing different FA and foaming stability (FS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. pH treatment is a highly efficient and low-cost method for improving the foaming characteristics of OVA. It can change the protein conformation by affecting the electrostatic interaction forces, which ultimately influence the protein foaming properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, the combination of pH treatment and the addition of polyphenols may improve the foaming characteristics of OVA to the largest extent because of the combined effects on electrostatic forces, hydrogen bonding and hydrophobic forces of protein-polyphenol aggregates. Li and Girard [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] enhanced the foaming performance of whey protein using sorghum proanthocyanidins, with the formed aggregates achieving the FA at pH 5.0 and the highest FS at pH 7.0. However, this study focused only on protein and polyphenol binding under acidic and neutral conditions, while the effects of alkaline conditions on the interaction forces between aggregates were lacking. Additionally, it neglected to explore the relationship between polyphenol types and the foaming properties of whey protein, causing its results insufficient to guide the selection of the most appropriate polyphenols for enhancing FA.\u003c/p\u003e \u003cp\u003ePolyphenols are secondary plant metabolites with aromatic rings containing one or more hydroxyl or methoxy groups, and include types such as phenolic acids, flavonoids, tannins, and lignans [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among them, phenolic acids are one of the most common types of polyphenols, known for their simple structure, strong antioxidant activity, and well-defined biological activities. According to the constitutive carbon frameworks, they can be categorized into benzoic acid-based (C6\u0026ndash;C1 structures) and cinnamic acid-based phenolic acids (C6\u0026ndash;C3 structures) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Both two phenolic acids can bind to proteins, but their affinities differ, further leading to different protein foaming performances. Li, Li, Dai, Hu, Niu, Liu and Chen [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] demonstrated that ferulic acid (cinnamic acid-based phenolic acids) had a greater binding affinity with β-casein compared to syringic acid (benzoic acid-based phenolic acids). However, the limitation of this study is that it only examined the binding force changes between β-casein and polyphenols, neglecting the alterations in FA and protein interfacial behavior caused by protein-polyphenol interactions. As a result, the findings cannot directly guide the formulation development of high-foaming products such as baked goods and ice cream. Meanwhile, the number and position of the hydroxy group in polyphenols also affect the foaming performance of proteins, which was also not explored in this study. Another study by Yuan, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] reported that phenolic acid with hydroxy group at the 2-position induced higher binding affinity to bovine serum albumin, whereas a negative effect was observed at the 4-position. Nevertheless, whether different carbon frameworks of polyphenols also influence protein conformation is unclear.\u003c/p\u003e \u003cp\u003eTherefore, in order to determine the patterns by which the hydroxy group and carbon framework of polyphenols and pH affect protein foamability, three benzoic acid-based polyphenols with different hydroxyl group numbers (P-hydroxybenzoic acid (PA, 1 hydroxyl group), 3,4-dihydroxybenzoic acid (DA, 2 hydroxyl groups) and gallic acid (GA, 3 hydroxyl groups)) and three cinnamic acid-based polyphenols with various hydroxyl group positions (\u003cem\u003etrans\u003c/em\u003e-3-hydroxycinnamic acid (T3A, meta-position), \u003cem\u003etrans\u003c/em\u003e-2-hydroxycinnamic acid (T2A, ortho-position) and 4-coumaric acid (CA, para-position)) were combined with OVA at pH 3.0, 7.4 and 9.0, respectively, and their foaming properties were compared to screen the OVA-polyphenol complexes with the highest foaming properties. Subsequently, to interrogate the reasons behind the varying foaming of OVA, two OVA-polyphenol aggregates with comparatively higher FA, OVA-DA and OVA-T2A, were selected from six aggregates, and further exploration was conducted on the microscopic changes occurring in OVA due to the addition of polyphenols and the use of acidic pH. Our study will provide a theoretical foundation for the precise application of polyphenols to enhance the foaming properties of OVA.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eOVA with different purity (\u0026gt;\u0026thinsp;98% or 62\u0026ndash;88%) was supplied by Sigma Chemical Co. (St. Louis, Mo, USA). GA (98%), DA (\u0026ge;\u0026thinsp;97%), PA (99%), CA (98%), T3A (99%), T2A (99%), phosphate buffer solution (PBS, pH 7.4), 8-anilino-1-naphthalene sulfonic acid (96%) and 5, 5'-dithio-bis-2-nitrobenzoic acid (98%) were procured from the Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). Tris (99%), glycine (\u0026ge;\u0026thinsp;99%), and hydrochloric acid (HCl, 36.0\u0026ndash;38.0%) were procured from Solarbio (Beijing, China), Beyotime Institute of Biotechnology (Shanghai, China) and Sinopharm Chemical Reagent Co., Ltd. (Beijing, China), respectively. All other chemicals exploited in this research were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample preparation\u003c/h2\u003e \u003cp\u003eThe OVA powders (purity, 62\u0026ndash;88%) and polyphenols were blended in distilled water to yield a final concentration of 0.4 mmol/L and 4 mmol/L, respectively, and then stirred for 1 h using a magnetic stirrer. All protein solutions were stored at 4 ℃ overnight to facilitate further solubilization. After that, the OVA solution was thoroughly mixed with an equal volume of each polyphenol solution to obtain the OVA-GA, OVA-DA, OVA-PA, OVA-CA, OVA-T3A and OVA-T2A aggregates (molar ratio of 1:10). In the control group, the polyphenol samples were substituted with distilled water. Next, the OVA-polyphenol solutions were split into thirds: one aliquot was adjusted to pH 9.0 using NaOH (OVA-GA-9.0, OVA-DA-9.0, OVA-PA-9.0, OVA-CA-9.0, OVA-T3A-9.0 and OVA-T2A-9.0), one aliquot to pH 3.0 using HCl (OVA-GA-3.0, OVA-DA-3.0, OVA-PA-3.0, OVA-CA-3.0, OVA-T3A-3.0 and OVA-T2A-3.0) and the other remained untreated (pH\u0026thinsp;=\u0026thinsp;7.4, OVA-GA-7.4, OVA-DA-7.4, OVA-PA-7.4, OVA-CA-7.4, OVA-T3A-7.4 and OVA-T2A-7.4). All prepared OVA-polyphenol solutions were further magnetically stirred for 1 h before storing in the dark in a refrigerator at 4 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Foaming properties\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Determination of foaming ability and foaming stability\u003c/h2\u003e \u003cp\u003eThe foaming properties of OVA-polyphenol complexes treated by different pH were characterized at 25 ℃ according to Zhang, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Briefly, 30 mL of sample placed in a graduated glass cylinder with an internal diameter of 22 mm was subjected to a homogenizer (T25, IKA, Germany, 10000 r/min, 1 min) to generate foam. The appearance of the bubbles was photographed using a camera, while the final volume of each solution after stirring was determined at 0 and 30 min and their foaming properties were calculated using the following relationships:\u003c/p\u003e \u003cp\u003eFA = (V\u003csub\u003e1\u003c/sub\u003e - V\u003csub\u003e0\u003c/sub\u003e)/V\u003csub\u003e0\u003c/sub\u003e \u0026times; 100% (1)\u003c/p\u003e \u003cp\u003eFS = (V\u003csub\u003e2\u003c/sub\u003e - V\u003csub\u003e0\u003c/sub\u003e)/(V\u003csub\u003e1\u003c/sub\u003e - V\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100% (2)\u003c/p\u003e \u003cp\u003eHere, V\u003csub\u003e0\u003c/sub\u003e, V\u003csub\u003e1\u003c/sub\u003e and V\u003csub\u003e2\u003c/sub\u003e represented the volume of the foam before blending, after blending, and after 30 min of standing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Foaming microstructure measurement\u003c/h2\u003e \u003cp\u003eBased on the FA and FS results, OVA, OVA-DA, due to their highest foaming among the benzoic acid-based polyphenol-OVA aggregates, and OVA-T2A, due to their highest foaming among the cinnamic acid-based polyphenol-OVA aggregates, were selected for subsequent studies. The microstructure of the bubbles was observed using a Nikon Instrument (Eclipse TS100, Nikon, Japan) according to Wen, Zhang, Ning, Li, Zhang, Liu and Zhang [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In detail, the air bubbles of these solutions were gently spread on a glass plate and covered with a coverslip, before observing on the microscope at a magnification of \u0026times; 40 (4 \u0026times; eyepiece and 10 \u0026times; objective lens).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Measurement of rheological properties\u003c/h2\u003e \u003cp\u003eThe rheological properties (viscosity and storage modulus G\u0026prime;) of OVA and OVA-polyphenol solutions at various pH treatments were determined by HR-1 rheometer (RS6000, Haake, Germany) following the procedure of Liu, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In terms of steady-state rheology, 1.5 mL of the solutions was carefully added to the plate and the range of shear rate was kept from 0.1 to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during the steady shear measurement at 25 ℃. For dynamic rheology, the samples were replated onto plates and were measured at a strain of 3% and a shear frequency ranging from 0.1 to 10 Hz. Subsequently, the viscosity, shear stress and storage modulus G\u0026prime; of the solution system were recorded as a function of frequency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Measurement of CD\u003c/h2\u003e \u003cp\u003eThe OVA solutions (purity\u0026thinsp;\u0026gt;\u0026thinsp;98%, similarly hereinafter) with or without polyphenols were prepared and measured as described by Wen, Zhang, Ning, Li, Zhang, Liu and Zhang [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Then the aggregates were diluted to 0.025 mmol/L and their CD spectra were recorded with a MOS-500 Circular Dichroism Spectrometer (Bio-Logic, France). Data of the samples were collected from 190 to 260 nm and imported into the Bestsel website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bestsel.elte.hu/index.php\u003c/span\u003e\u003cspan address=\"http://bestsel.elte.hu/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to calculate the secondary structure content of the OVA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Measurement of FT-IR\u003c/h2\u003e \u003cp\u003eThe aggregates under different pH treatments were freeze-dried and analyzed using FT-IR spectroscopy as described by Jiang, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], with minor modifications. KBr transparent flakes were prepared by blending 2 mg of each OVA-polyphenol aggregate with 200 mg of KBr and pressing the mixture into a 13-mm disk using a die press. Thereafter, the sample flakes were subjected to the IRPrestige-21 Spectrometer (Shimadzu, Japan) and the spectra was accumulated over the wavenumber range of 400 to 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a nominal resolution of 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 64 scans.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Surface hydrophobicity determination\u003c/h2\u003e \u003cp\u003eThe surface hydrophobicity of the samples under different pH treatment conditions was measured using an F-7100 fluorescence spectrophotometer (Hitachi High-Tech Science, Japan) as per described by Wen, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, 1 mL of OVA or OVA-polyphenol aggregates (0.025 mmol/L) was mixed with 10 \u0026micro;L of ANS (8 mmol/L, suspended in PBS) and was further incubated in the dark at room temperature for 30 min. Subsequently, the resulting samples were recorded using the fluorescence spectrophotometer, with the following measurement settings: excitation wavelength of 390 nm, emission wavelengths of 400\u0026ndash;600 nm, and constant slit width of 5 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of free -SH group\u003c/h2\u003e \u003cp\u003eThe -SH group content in OVA and OVA-polyphenol aggregates at different pH conditions was analyzed according to Lyu, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The content of the sulfhydryl group was calculated as follows:\u003c/p\u003e \u003cp\u003e-SH (\u0026micro;mol/g)\u0026thinsp;=\u0026thinsp;75.53 \u0026times; D \u0026times; A\u003csub\u003e412\u003c/sub\u003e/C (3)\u003c/p\u003e \u003cp\u003eHere, A\u003csub\u003e412\u003c/sub\u003e denoted the absorbance of samples measured at 412 nm, C referred to the concentration of OVA (0.025 mmol/L), and D was the dilution factor (2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Particle size and zeta-potential\u003c/h2\u003e \u003cp\u003eThe diluted OVA and OVA-polyphenol aggregate solutions used in Section 2.5 were placed into a Zetasizer Nano ZS90 instrument (Malvern Co., UK) for analyzing particle size and zeta-potential using the procedure described by Wen, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Measurement of intrinsic fluorescence spectra\u003c/h2\u003e \u003cp\u003eAggregate solutions were diluted to 0.05 mmol/L with deionized water and then heated in a water bath maintained at 303 K for 1 h, before intrinsic fluorescence spectra (IFS) intensity measurement. Changes in IFS spectra of different samples were recorded by an F-7100 fluorescence spectrophotometer as per described by Yu, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.11 Measurement of three-dimensional fluorescence\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAs suggested by Ren, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the 3-D fluorescence spectra of OVA and OVA-polyphenol aggregates diluted to 0.0025 mmol/L was determined by fluorescence spectrophotometer instrument using excitation wavelength of 200 nm to 400 nm, emission wavelength of 200 nm to 400 nm, scanning speed of 12000 nm/min, and slit width of 5 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.12 Statistical analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eEach experiment was repeated three times and the mean and standard deviation were obtained from these results. All analyses were subjected to one-way analysis of variance (ANOVA) (Tukey\u0026rsquo;s test) using SPSS 20.0 (IBM SPSS Statistics, IBM Corp., Somers, NY), and the level of significance used was \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Foaming properties\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Acidic pH and cinnamic acid-based polyphenol treatment led to higher FA of OVA\u003c/h2\u003e \u003cp\u003eFoaming 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Figure\u0026nbsp;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 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in FA and a further slight but not significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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\u0026thinsp;=\u0026thinsp;100:0) to 132% (WPI: RES\u0026thinsp;=\u0026thinsp;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) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and ferulic acid-OVA (FA of aggregates increased from 90\u0026ndash;140% when the molar ratio of ferulic acid to OVA changed from 1: 0 to 1:20) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, there were counter reports as well. Dai, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] found that the FA of lactoferrin decreased from around 114\u0026ndash;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.\u003c/p\u003e \u003cp\u003eThe FA of all six OVA-polyphenol aggregates revealed that the highest FA was obtained when the aggregates were treated under acid conditions (pH\u0026thinsp;=\u0026thinsp;3.0), whereas alkaline treatment imparted a slight decline in FA values (-12.78% for OVA-DA, and \u0026minus;\u0026thinsp;13.89% for OVA-T2A) as compared to untreated aggregates. pH\u0026thinsp;=\u0026thinsp;3.0 is close to the isoelectric point of OVA [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcid or alkali treatment did not significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;=\u0026thinsp;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 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe FA of OVA-GA, OVA-DA, and OVA-PA aggregates at pH 3.0 did not differ (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;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 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;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 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Acidic pH contributed to smaller and denser bubble formation of OVA\u003c/h2\u003e \u003cp\u003eThe foaming microstructure of aggregates under different pH conditions was measured using an optical microscope, and the results were given in Fig.\u0026nbsp;1E and 1F. The foams of aggregates immediately after homogenization (Fig.\u0026nbsp;1E) were smaller than that of the foams after a period of setting (Fig.\u0026nbsp;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 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], 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\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Acid and polyphenol treatments increased the viscosity and G\u0026rsquo; of OVA\u003c/h2\u003e \u003cp\u003eThe viscosity, storage modulus (G\u0026rsquo;) and loss modulus (G\u0026rdquo;) of the protein dispersion system affect the FA and FS. Conventionally, large viscosity of OVA and G\u0026rsquo; \u0026gt; G\u0026rdquo; 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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Figure\u0026nbsp;2 gives the viscosity, G\u0026rsquo; and G\u0026rdquo; curve of aggregates after different pH treatments. All OVA-polyphenol aggregates had reduced viscosity and decreased gap between G\u0026rsquo; and G\u0026rdquo; as the shear rate increased from 1 to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, showing a shear thinning behavior, which further indicated that these aggregates can be regarded as non-Newtonian fluids and exhibited pseudoplastic fluid behavior [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, with the increase of scanning frequency, the G\u0026rsquo; and G\u0026rdquo; of OVA-polyphenols gradually increased and G\u0026rsquo; followed the order of pH\u0026thinsp;=\u0026thinsp;3.0 group\u0026thinsp;\u0026gt;\u0026thinsp;pH\u0026thinsp;=\u0026thinsp;7.4 group\u0026thinsp;\u0026gt;\u0026thinsp;pH\u0026thinsp;=\u0026thinsp;9.0 group, while G\u0026rdquo; followed the order of pH\u0026thinsp;=\u0026thinsp;7.4 group\u0026thinsp;\u0026gt;\u0026thinsp;pH\u0026thinsp;=\u0026thinsp;3.0 group\u0026thinsp;=\u0026thinsp;pH\u0026thinsp;=\u0026thinsp;9.0 group. Higher storage modulus G\u0026rsquo; 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\u0026thinsp;=\u0026thinsp;3.0 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The viscosity, G\u0026rsquo; and G\u0026rdquo; of the OVA-DA and OVA-T2A did not differ significantly at the same pH, except for G\u0026rsquo; and G\u0026rdquo; of OVA-DA-7.4 and OVA-T2A-7.4. Both the viscosity and G\u0026rsquo; 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\u0026rsquo; of OVA, which in turn contributed to the formation of stable, elastic, small and dense foams [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. As suggested by Li, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], 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.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Polyphenols and Acidic pH promoted the conversion of α-helix to random coil in OVA\u003c/h2\u003e \u003cp\u003eThe 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.\u0026nbsp;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\u0026thinsp;=\u0026thinsp;7.4 and pH\u0026thinsp;=\u0026thinsp;9.0 groups with and without polyphenol addition were similar, but significantly different from that of the aggregates in the pH\u0026thinsp;=\u0026thinsp;3.0 group, which was consistent with the results of FA and FS.\u003c/p\u003e \u003cp\u003eThe specific details regarding the level of secondary structure for each OVA-polyphenol aggregate were calculated employing the online software BeStSel (Fig.\u0026nbsp;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\u0026thinsp;=\u0026thinsp;7.4 compared to that of the OVA-polyphenol aggregates at pH\u0026thinsp;=\u0026thinsp;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\u0026thinsp;=\u0026thinsp;3.0. Acid treatment contributes to the unfolding of the OVA structure, which leads to alterations in their protein conformations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These changes positively affect the adsorption and reorganization of OVA at interfaces, resulting in a higher foaming performance [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Hydrogen bonding and hydrophobic forces participated in the OVA and polyphenol interactions\u003c/h2\u003e \u003cp\u003eThe main characteristic peaks of OVA shifted in the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 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.\u0026nbsp;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\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as a result of intermolecular hydrogen bonding and O-H and N-H stretching vibrations [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. After acid treatment, the bands of OVA, OVA-DA, and OVA-T2A at 3300.20 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 3300.20 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3296.35 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were all blue shifted to 3296.35 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; while after alkaline treatment, the characteristic peaks of OVA, OVA-DA and OVA-T2A varied to 3300.20 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 3296.35 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3298.28 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. These results suggested that hydrogen bonding was involved in the binding between OVA and polyphenols, while pH affected their binding.\u003c/p\u003e \u003cp\u003eThe peaks at around 2930 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the C-H stretching vibration proteome of CH\u003csub\u003e3\u003c/sub\u003e and CH\u003csub\u003e2\u003c/sub\u003e, and their variations indicated the presence of hydrophobic interactions during the formation of OVA-polyphenol aggregates [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. By adjusting the protein solution to acid condition, the bands of OVA, OVA-DA, and OVA-T2A red shifted from 2933.73 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2929.87 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2929.87 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 2935.66 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2931.80 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 2933.73 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 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\u0026thinsp;=\u0026thinsp;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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] 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.\u003c/p\u003e \u003cp\u003eThe amide I band (1600\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was assigned to the C\u0026thinsp;=\u0026thinsp;O and N-H tensile vibration [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. 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\u0026thinsp;=\u0026thinsp;O and C-N groups of OVA subunit [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Acid conditions increased the surface hydrophobicity of OVA\u003c/h2\u003e \u003cp\u003eSurface hydrophobicity is an important indicator for evaluating changes in protein conformation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Figure\u0026nbsp;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 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at pH 3.0 than at pH\u0026thinsp;=\u0026thinsp;7.4; however, the surface hydrophobicity of the OVA-T2A-9.0 was not significantly different from that of the OVA-T2A-7.4 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This finding confirmed the results of FT-IR.\u003c/p\u003e \u003cp\u003eIn 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 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6 OVA-T2A obtained maximum free sulfhydryl group at pH\u0026thinsp;=\u0026thinsp;3.0\u003c/h2\u003e \u003cp\u003eThe content of free -SH group represents alterations in the cleavage and accumulation of intramolecular and intermolecular disulfide bonds in the protein molecule [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. As indicated in Fig.\u0026nbsp;4B, the content of free -SH groups was in the order of pH 3.0 group\u0026thinsp;\u0026gt;\u0026thinsp;pH 7.4 group\u0026thinsp;\u0026ge;\u0026thinsp;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 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe free sulfhydryl group content of OVA and OVA-polyphenols followed the order of OVA-T2A\u0026thinsp;\u0026gt;\u0026thinsp;OVA-DA\u0026thinsp;=\u0026thinsp;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 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Additionally, cinnamic acid-based polyphenols exhibited higher antioxidant activity than benzoic acid-based polyphenols [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], 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 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Acid treatment and polyphenols led to larger particle size\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;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 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. On the other hand, the interactions between OVA and polyphenols are considered to be the main contributing factor [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. This result corroborates with previous findings of Chang, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and von Staszewski, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. These results confirm the conjecture proposed in the FA and FS results.\u003c/p\u003e \u003cp\u003eIt 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 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. T2A has a longer C-chain and C\u0026thinsp;=\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Acidic and neutral environments weakened the electrostatic repulsion of aggregates\u003c/h2\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. 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.\u0026nbsp;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, \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.9 OVA-T2A aggregates had the lowest intrinsic fluorescence intensity at pH\u0026thinsp;=\u0026thinsp;3.0\u003c/h2\u003e \u003cp\u003eTo investigate the structural changes of proteins after the addition of polyphenols or acid-base treatment, IFS spectroscopy was applied in the present study [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The introduction of polyphenols led to a significant reduction in the IFS of OVA (Fig.\u0026nbsp;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.\u003c/p\u003e \u003cp\u003eBoth acidic and alkaline treatments caused a decrease in fluorescence intensity of OVA and OVA-polyphenols, and they can be ranked as follows: pH\u0026thinsp;=\u0026thinsp;3.00 group\u0026thinsp;\u0026gt;\u0026thinsp;pH\u0026thinsp;=\u0026thinsp;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 [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.10 T2A strongly quenched the fluorescence intensity of OVA\u003c/h2\u003e \u003cp\u003e3-D fluorescence spectroscopy is an essential method for evaluating conformational changes in proteins [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Peak A (λex\u0026thinsp;=\u0026thinsp;275 nm) is the characteristic band of OVA, reflecting changes of protein tertiary structure, while peak B (λex\u0026thinsp;=\u0026thinsp;230 nm) is the characteristic band of the polypeptide backbone structure (C\u0026thinsp;=\u0026thinsp;O) of OVA, which reveals changes of secondary structure of protein [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The 3-D fluorescence spectra of aggregates under different pH treatments is given in Fig.\u0026nbsp;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.\u003c/p\u003e \u003cp\u003eFurthermore, the fluorescence intensity of peak A of OVA followed the following order: pH 3.0 groups\u0026thinsp;\u0026gt;\u0026thinsp;pH 7.4 groups\u0026thinsp;\u0026gt;\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.11 Mechanism of foam formation\u003c/h2\u003e \u003cp\u003eThe mechanism diagrams of foam formation of OVA-DA and OVA-T2A at pH 3.0, 7.4 and 9.0 were presented in Fig.\u0026nbsp;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.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eDA and T2A, one of the benzoic acid-based and cinnamic acid-based polyphenols, respectively, can induce protein conformational changes in OVA at different pH conditions, thus influencing the FA and FS of the formed OVA-polyphenol aggregates. Both the application of polyphenols and the acid treatment improved the FA and FS of OVA, which was due to the strong hydrophobic interactions between aggregates leading to greater protein conformational disorder and conversion or breakage of disulfide bonds, further creating an interfacial film with high cohesiveness and elasticity, ultimately contributing to the formation of stable and small density foams. The results of particle size and zeta-potential indicated that acidic treatment led to neutralization of protein surface charges, further inducing swelling and self-aggregation and promoting protein adsorption and rearrangement at the air-liquid interface. Notably, T2A, compared to DA, and acidic environment, compared to neutral and alkaline conditions, induced more alterations in disulfide bonds, protein conformation and non-covalent interaction forces, which in turn increased the positive effect on foaming properties of OVA. The IFS and 3-D fluorescence spectra experiments also confirmed that OVA-T2A aggregates at pH 3.0 had large altered non-covalent interaction forces and protein secondary and tertiary structures, which explained their high foaming properties and allowed them to serve as promising functional ingredients in food systems. This study may provide guidance on the use of polyphenols to regulate the foaming properties of proteins in the baking industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eAll authors have no conflict of interest.\u003c/p\u003e \u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eHedi Wen: Investigation; Methodology; Validation. Zhenzhen Ning: Methodology; Formal analysis; Data curation; Validation. Deju Zhang: Data curation; Formal analysis; Writing-original draft. Jingbo Liu: Investigation; Supervision; Project administration; Resources; Writing-review \u0026amp; editing. Zihao Li:Data curation; Formal analysis. Yan Zhang: Formal analysis. Ting Zhang: Writing-review \u0026amp; editing. Ting Yu: Conceptualization; Validation; Writing-review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by National Key R\u0026amp;D Program of China (2022YFD2101000).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGharbi N, Labbafi M (2019) Influence of treatment-induced modification of egg white proteins on foaming properties. Food Hydrocolloids 90:72\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodhyd.2018.11.060\u003c/span\u003e\u003cspan address=\"10.1016/j.foodhyd.2018.11.060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen HD, Zhang DJ, Ning ZZ, Li ZH, Zhang Y, Liu JB, Zhang T (2023) How do the hydroxyl group number and position of polyphenols affect the foaming properties of ovalbumin? 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Int J Biol Macromol 228:333\u0026ndash;345. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ijbiomac.2022.12.218\u003c/span\u003e\u003cspan address=\"10.1016/j.ijbiomac.2022.12.218\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"Jilin University","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polyphenols, pH, Ovalbumin, Protein-polyphenol interactions, Foaming properties","lastPublishedDoi":"10.21203/rs.3.rs-4011113/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4011113/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo broaden the application of ovalbumin (OVA) in food formulations, it is meaningful to improve its foaming characteristics. This study aimed to investigate the effect of benzoic acid-based (3,4-dihydroxybenzoic acid, DA) and cinnamic acid-based polyphenols (trans-2-hydroxycinnamic acid, T2A) on the foaming properties of OVA at acidic (pH 3.0), neutral (pH 7.4) and alkaline (pH 9.0) pH conditions. Both the addition of polyphenols and acid treatment enhanced the foaming properties of OVA. Surface hydrophobicity, circular dichroism, free sulfhydryl groups, and Fourier transform infrared spectroscopy results indicated that after acidic workup, the presence of stronger hydrophobic interactions in OVA-polyphenol aggregates induced more disordered protein conformation and conversion or breakage of disulfide bonds. Particle size and zeta potential indicated that acidic treatment neutralized protein surface charges, further inducing self-aggregation and swelling of OVA, ultimately enhancing foaming properties. Comparatively, T2A exhibited better foam-inducing capacity due to its stronger interaction with OVA, leading to the unfolding of the OVA structure and the exposure of more hydrophobic groups. The intrinsic and 3-D fluorescence spectra experiments also confirmed that OVA-T2A aggregates at pH 3.0 had greater altered non-covalent interaction forces and protein secondary and tertiary structures compared to other complexes. This study provides a theoretical basis for designing protein formulations with excellent foaming properties.\u003c/p\u003e","manuscriptTitle":"Effect of benzoic acid-based and cinnamic acid-based polyphenols on foaming properties of ovalbumin at acidic, neutral and alkaline pH conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 04:40:45","doi":"10.21203/rs.3.rs-4011113/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"436b1235-a78d-4b70-9bf8-c37ceb867ab8","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29108296,"name":"Food Science \u0026 Technology"}],"tags":[],"updatedAt":"2024-03-15T22:02:19+00:00","versionOfRecord":{"articleIdentity":"rs-4011113","link":"https://doi.org/10.1016/j.foodhyd.2024.109998","journal":{"identity":"food-hydrocolloids","isVorOnly":true,"title":"Food Hydrocolloids"},"publishedOn":"2024-03-01 22:02:19","publishedOnDateReadable":"March 1st, 2024"},"versionCreatedAt":"2024-03-05 04:40:45","video":"","vorDoi":"10.1016/j.foodhyd.2024.109998","vorDoiUrl":"https://doi.org/10.1016/j.foodhyd.2024.109998","workflowStages":[]},"version":"v1","identity":"rs-4011113","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4011113","identity":"rs-4011113","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}