Polyphenol-Driven Structural Alterations and Antibacterial Potency of the IgY-Catechin Complex

Polyphenol-Driven Structural Alterations and Antibacterial Potency of the IgY-Catechin Complex | 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 Polyphenol-Driven Structural Alterations and Antibacterial Potency of the IgY-Catechin Complex Lili Liu, Yanli Wang, Mengjun Zhang, Weiwei Cheng, Yue Ding, Jialiang He This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5017669/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract The binding of proteins with polyphenols can alter the properties of the proteins, thereby expanding their application scenarios. Yolk immunoglobulin (IgY), an easily obtainable protein from eggs, undergoes property changes upon binding with catechin, which is significant for broadening the application of IgY. This study investigates the binding modes, structural changes, stability, and antibacterial properties of the IgY-catechin complex using computational chemistry, spectroscopy, and antibacterial assays. Molecular dynamics simulations analysis revealed that catechin binds to residues PHE503, THR501, THR505, GLU511 of IgY respectively. Fourier transform infrared spectroscopy(FT-IR) and circular dichroism(CD) displayed an increase in hydrogen bond content within the complex, a 5.48% increase in α-helix content, and a decrease in random coil content. Scanning electron microscopy (SEM) showed that the complex had a smoother and more regular surface. The IgY-catechin complex exhibited improved acid-base stability but slightly reduced thermal stability compared to IgY. Fluorescence inverted microscopy and nucleic acid-protein leakage assays indicated that the complex disrupted the cell membranes of both Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ), leading to the leakage of intracellular genetic material. These findings confirmed that the interaction between catechin and IgY altered the protein structure of IgY, enhanced its bacteriostatic ability. This research provides a new approach to extending the application range of IgY and improving its comprehensive utilization. Yolk immunoglobulin catechin molecular dynamics simulation antibacterial activity mechanism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The majority of food spoilage is caused by microbial contamination, and common foodborne pathogens that mainly endanger human health are Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) (Mpatswenumugabo et al., 2023 ). The reduction in nutritional value of food due to foodborne pathogens is a pressing issue that needs to be addressed. While antibiotics can inhibit bacterial growth, their use can lead to the development of antibiotic-resistant bacteria. (Yip, A.Y.G. et al. 2023 ). Yolk immunoglobulin (IgY) has a high safety profile and exhibits a certain degree of stability (Li, et al. 2023) As a kind of new generation antibody, IgY has high application value and shows remarkable effect in human health care, disease diagnosis and prevention(Liu et al., 2023 ). Replacing antibiotics with IgY can alleviate the problem of antibiotic resistance and prevent bacterial diseases (Minquan Xia, et al. 2022 ). Heat resistance, acid and alkali resistance, and other physicochemical properties of IgY have a direct impact on its manufacture, storage, and application in a variety of sectors (Fang. et al. 2016). IgY has a certain degree of stability and its activity is less affected by temperature, pH, and enzymes within a certain concentration range (Bellingeri R V, et al. 2015 ). Under room temperature or refrigerated conditions, its activity remains stable after a longer period of storage (Nilsson E, et al. 2012 ). However, the activity of IgY decreased significantly, when the temperature exceeded 60°C, and was almost activity completely inactivated above 80°C. Current research has shown that the application of liposomes (Mahin Rigi, et al. 2022), chitosan-sodium alginate (Setiasih Siswati, et al. 2019), and other encapsulation treatments for IgY may alleviate its stability (Gandhi S, & Alshehri SM., 2021 ). The high production costs and operational complexities associated with these methods impede their scalability. Numerous studies have confirmed that natural polyphenols in plants possess antibacterial properties. For instance, research by Wang et al. (2023) has found that flavonoids from Sedum aizoon L. exhibit antifungal activity on grapes. Similarly, Maral et al. (2023) reported the use of curcumin as a substitute for synthetic antibacterial agents in the prevention and treatment of gray mold, demonstrating effective inhibition of bacterial growth. Catechin, which is polyphenol, possesses synergistic antibacterial and antioxidant properties(Tyagi, et al., 2021 ). It was used to prevent oxidative deterioration of meat products (Paturi G. et al., 2021 ). Many studies have shown that the interaction between proteins and polyphenols can significantly impact protein functionality (Jia, et al. 2016 ). Polyphenols have the capacity to interact with proteins, forming complexes that can be either reversible or irreversible, thus modifying the functional properties of protein (Jingxian Niu, et al., 2023). For example, Tan et al. (2022) studied the effects of phenolic compounds such as gallic acid (GA), chlorogenic acid (CA), epigallocatechin (EGC), epigallocatechin gallate (EGCG), and tannic acid (TA) on the modification of carp myofibrillar proteins. Their research indicated that these phenolic compounds alter both the secondary and tertiary structures of myofibrillar proteins. Additionally, the phenolic substances improve the thermal stability and gelation properties of the myofibrillar proteins. Xie et al. (2022) utilized multispectral methods to investigate the binding mechanisms of chlorogenic acid (CGA) and quercetin (QUE) with fish myofibrillar proteins. Circular dichroism analysis revealed that the addition of CGA does not affect the α-helix content of myofibrillar proteins, whereas QUE disrupts the β-turns and promotes the formation of α-helices in myofibrillar proteins. Liu et al. ( 2023 ) investigated the effect of the interaction between catechins and glycated porcine hemoglobin (G-PHb) on the antioxidant and structural properties of glycosylated hemoglobin. However, current research primarily focuses on the isolation, purification, and antibacterial properties of IgY. There is limited research on modifying IgY with catechins to alter its antibacterial performance, thermal stability, and pH stability, as well as on the structural characterization of catechin-IgY complex. This study prepared IgY-catechin complexes to investigate their structure and antibacterial properties. The structural changes and formation mechanism of the complex were investigated using molecular docking, molecular dynamics simulation, Fourier-transform infrared spectroscopy (FT-IR), and circular dichroism (CD). The antibacterial stability of the complex was assessed with IgY as a control. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the complex were determined. The inhibitory effect of the complex on bacterial growth was studied through growth curve, nucleic acid and protein leakage tests and fluorescence microscopy. This study investigates potential changes in IgY during its application in antibacterial and functional food development, providing a theoretical basis for these processes. The findings aim to lay a foundation for the subsequent development of IgY-related health foods. Materials and reagents Fresh eggs were obtained from Dazhang Supermarket, Luoyang, China. Propidium iodide (PI) dye was acquired from Shanghai McLean Biochemical Technology Co., and catechin was purchased from Hefei Bomei Biotechnology Co., Luria-Bertani(LB) solid medium was bought from Beijing Baoxing Biotechnology Co., E. coli and S. aureus were obtained from the microbiology lab of the Food and Bioengineering college, Henan University of Science and Technology. All reagents were analytically pure. All solutions and suspensions were prepared with deionized water. Experimental method Preparation of IgY and IgY-Catechin Complex Refer to the Lin et al. (2014) method to obtain IgY solution by ammonium sulfate precipitation. Afterwards, the solution was desalted at 4°C using a membrane with a molecular weight cutoff (MWCO) of 8000–14000 Da for 48 hours. IgY powder was obtained by vacuum freeze-drying in a vacuum freeze-dryer (Shanghai Danding International Trading Co., China). The 1:1 mass concentration of IgY and catechin was placed in a magnetic stirrer (Changzhou Ronghua Instrument Manufacture Co., China) and stirred for 6 h at 40°C to obtain the complex solution. And then the complex powder was then obtained by vacuum freezing. Molecular Docking of catechin and IgY The IgY structure (PDB ID: 2W59) and catechin molecules were sourced from the RCSB Protein Data Bank and PubChem database( https://pubchem.ncbi.nlm.nih.gov ), respectively. AutoDockTools-1.5.6 software was used for preprocessing and conducting molecular docking simulations of receptors and ligands. Semi-flexible docking involving 50 runs was performed. The model exhibiting the lowest binding energy was selected as the optimal configuration. Visualization of the simulation outcomes and illustration generation were performed using PyMOL software. Molecular dynamics simulation Molecular dynamics simulations were conducted using the downloaded PDB file and catechin molecule file from previous step. The protein and small molecule files were opened using Discovery Studio for protein preprocessing. Flexible peptide chains were removed, retaining only those with minimal thermal vibrations and more precise positioning. Water molecules were removed from the protein, and the protein was solvated and ionized to balance the solution using the CHARMM36 force field. The steepest descent algorithm was employed to resolve any initial bad contacts within the system, followed by the conjugate gradient method to ensure a low-energy starting point for subsequent dynamics. The simulation time was set to 20 ps for the molecular dynamics simulation. Visualization was performed using PyMOL and Discovery Studio. Molecular dynamics simulation analysis Root Mean Square Deviation (RMSD) was used to measure the overall change in a protein, calculating the average deviation of atomic positions at a given time point from a reference structure, which is typically the initial structure or a stable structure. Root Mean Square Fluctuation (RMSF) was used to measure the fluctuation of a particular atom (or residue) relative to its average position over the entire simulation, reflecting the local flexibility of each atom or residue. The formulas for calculating RMSD and RMSF are as follows: $$\:\text{RMSD=}\sqrt{\frac{\text{1}}{\text{N}}\sum\:_{\text{i=1}}^{\text{N}}{\left({\text{r}}_{\text{i}}\left(\text{t}\right)\text{-}{\text{r}}_{\text{i}}^{\text{ref}}\right)}^{\text{2}}}$$ $$\:\text{\:}{\text{r}}_{\text{i}}^{\text{ref}}$$ Here, N represents the number of atoms, and rii(t) is the position of the i-th atom at time t,while is the position of the atom in the reference structure. $$\:\text{RMSF=}\sqrt{\frac{\text{1}}{\text{T}}\sum\:_{\text{t=1}}^{\text{T}}{\left({\text{r}}_{\text{i}}\left(\text{t}\right)\text{-}\stackrel{-}{{\text{r}}_{\text{i}}}\right)}^{\text{2}}}$$ $$\:\stackrel{-}{{\text{r}}_{\text{i}}}$$ Where rii(t) is the position of the i-th atom at time, is the average position of this atom over the entire simulation, and T is the number of time steps. Scanning electron microscopy (SEM) To examine the effect of the dried protein samples, the structure morphologies of IgY and IgY-Catechin complex were determined by SEM (S–3000N, Hitachi Limited, Japan). Before imaging, the sample powder was evenly scattered on the conductive adhesive (Yang, Tu, Wang, Li, & Tian, 2017). The sample was sprayed with 5nm thick gold. After that, the morphology of the dried protein samples was observed using SEM. Collection of images magnified 500 times. Fourier transform infrared spectroscopy (FT-IR) The secondary structures of IgY and IgY-catechin complex were characterized with a FT-IR instrument (Nicoler-SX-170, Thermo Nicolet Corporation, USA). The samples were subjected to infrared spectroscopy using the potassium bromide compression method. The IgY, IgY-catechin complex and KBr were first dried for 12 h in advance. The dried particles were first ground into a very fine powder. IgY and IgY-catechin complex were mixed with potassium bromide powder at a mass ratio of 100:1, respectively. Then, the mixture was pressed into light-transmitting sheets (1–2 mm thick) in a sample tank at a pressure of 15–20 MPa. The scanning resolution was 4 cm − 1 and the scanning range was 4000 − 400 cm − 1 . The medium (KBr) with no protein was recorded as the background spectrum. OMNIC software was used to analyze the FT-IR spectral data. Circular dichroism spectrum Changes in protein secondary structure were analyzed using circular dichroism (Chirascan VX, Applied Photophysics, UK). Preparation of 0.25 mg/ml solution of IgY and IgY-catechin complex. The secondary structure of the protein was measured with 1 ml of the solution. The wavelength scanning range was 190–250 nm with a spectral resolution of 0.1 nm. The scanning speed was 100 nm/s and the response time was 0.5 s. The calibration was first scanned with air and deionized water and repeated twice. The samples were scanned three times and averaged. The measured spectra were smoothed twice to obtain a circular dichroism chromatogram. Finally, CDNN software was used for data analysis. Preparation of Bacterial suspensions Bacterial suspensions of E. coli and S. aureus were prepared according to the method of Ihuma, (2022). The concentration of the bacterial suspension was adjusted to 10 6 ~10 8 CFU/mL. Determination of Inhibitory activity The suspensions of E. coli and S. aureus were mixed with sterilised LB solid medium at a ratio of 1:100 and the homogeneous bacterial plates made with sterile smear rings. Determination of bacterial inhibitory activity was performed using the perforation method. The IgY and IgY-catechin complex were mixed with anhydrous ether (1:1 volume ratio) to form a mixture, respectively. Sterilized circular filter paper (diameter d = 0.5 cm) was then clamped with sterile tweezers, immersed in the mixture for 20 minutes, and subsequently air-dried. The filter paper sheets were pasted on each test bacterial plate, each plate was pasted with 3 pieces in a triangular shape. Anhydrous ether was used as a blank control. Determination of Inhibition rate Referring to the method of Srikacha, et al (2020) with slight improvement. Several tubes were sterilized after the addition of 4 mL of liquid medium to each tube. Subsequently, test tubes numbered 1 through 5 were sterilized and added with 100 µL of E. coli bacterial solution and 1 mL of a specified concentration of the sample solution, resulting in final concentrations of 0.025, 0.05, 0.1, 0.15, and 0.2 mg/mL, respectively. Tube number 6 was used as the positive control, receiving 1 mL of sterilized distilled water and 100 µL of E. coli bacterial solution, while the remaining tubes served as negative controls. The tubes were incubated in a constant temperature shaker (Shanghai Yiheng Scientific Instrument Co., China) at 37°C for 12 hours, followed by measurement of optical density (OD) at 600 nm. The inhibition rate was calculated using the following formula, Thermal and Acid-base Stability Tests The experimental protocol, based on Noriyuki et al. (2023) with slight modifications, included preparing samples in a 0.5 mg/mL solution. These samples were then treated at temperatures ranging from 40℃ to 90℃ for 20 minutes, and the optical density (OD) at 600 nm was measured to assess inhibition rates and investigate stability across different temperature conditions. Similarly, the bacteriostatic acid-base stability was determined using the same approach as described above. The sample solution's pH was varied to 5, 6, 7, 8, 9, and 10 to assess its bacteriostatic stability across different pH conditions. MIC and MBC of IgY-catechin Complex Referring to the method of Aigbogun et al.(2023) and improved it. Preparation of 9 tubes were numbered and were ready for use on an ultra-clean bench (Xinbeisi Biotechnology Co., China). Tubes 1–8 received 4 mL of sterilised LB liquid medium and 100 µL of bacterial suspension. Additionally, tubes 1–7 were supplemented with 1 mL of complex solutions at concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL, respectively. 1 mL of physiological saline was added to tube 8 in place of the complex solution, and only culture medium was added to tube 9. MIC values were obtained by observation after incubation was used in a thermostatic incubation shaker (Yiheng Scientific Instrument Co., China). The MBC was determined by the plate method, where 100 µL of the culture solution from each sample was streaked onto LB solid medium. The MBC value was obtained by watching the growth of colonies in the plate after incubation. Growth curve To test tubes containing LB liquid medium, 1 mL of a IgY-catechin complex solution was added to achieve final concentrations of MIC and 2 MIC. As a control, 1 mL of distilled water was added. Each tube then received 100 µL of bacterial culture grown to the logarithmic phase. The tubes were incubated at 37°C for 24 hours, with samples taken every 2 hours to measure OD 600nm . Measurement of Nucleic Acid Protein Leakage The method described by Chang (2021) was employed to assess the leakage of nucleic acid of E. coli and S. aureus . Bacterial suspensions at approximately 1×10 9 CFU/mL were treated with varying multiples of the MIC, ranging from 0 to 2 times MIC. The suspension was incubated at 37℃ for 12 h after preparation. Every 2 h, 200 µL of the culture solution was centrifuged at 10,000 r/min for 10 min and the supernatant was collected. The absorbance at 260 nm (OD 260 ) of the supernatants was then measured using a multimode plate reader (Tecan, Infinite M200 PRO, Männedorf, Switzerland). Observation of Fluorescence Inverted Microscopy Refer to the method of Han Yingjie, et al. (2021). The bacterial solution was washed, resuspended, diluted to the appropriate concentration and dispensed into test tubes. The samples were prepared to achieve final concentrations equivalent to the MIC and twice the MIC, then incubated at 37°C for 3 hours. After that, PI dye was added to it, and the reaction was carried out for 20 min under the dark condition. At the end of the reaction a small amount of liquid was pipetted onto a slide and allowed to dry, and observed by fluorescence inverted microscope(Olympus, Japanese). Statistical Analysis All experiments were performed thrice in parallel, and the results were presented as mean value ± SD. The statistical significance of the data was analyzed using SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). The analyses of variance were performed via the ANOVA procedure. The significance level of data was set at P < 0.05. The correlation analysis was carried out by using Origin 2021. Results and Discussion Results of Molecular Docking Simulation Molecular docking visualizes structural changes in protein molecules post-interaction with small molecules and elucidates the intermolecular forces and binding conditions involved in their interaction. The structure of molecular docking is shown in Fig. 1. From Fig. 1, it can be clearly seen that IgY showed insignificant changes in protein and catechin structure upon interaction with catechins. The final intermolecular energy was − 34.84 kJ/moL, Among these, the van der Waals forces(vdW), Hbond, desolv Energy made a large contribution. There were five hydrogen bonding site for the binding of catechins and IgY. Catechin binds to residues PHE503, THR501, THR505, GLU511 of IgY respectively, and was accompanied by six possible flips. This time the predicted free energy of binding was − 27.34 kJ/mol, indicated that the ligand-receptor interaction is stable. In conclusion, the molecular docking results showed stable ligand-receptor interactions, mainly driven by vdW, hydrogen bonding and desolvation energies. (a)2D Schematic Diagram of IgY and Catechin Molecular Docking. (b)3D Detailed View of IgY and Catechin Molecular Docking. (c)Comprehensive Structural Diagram of IgY and Catechin Molecular Docking Result of Molecular Dynamics Simulation Based on molecular docking analysis, it can be confirmed that there were interactions between IgY and catechin. Molecular dynamics simulations using Discovery Studio software were conducted to study the structure and binding modes of the IgY-catechin complex more precisely. First, the electrostatic potential of the protein was calculated and visualized using PyMOL. Figure 2 (a) and (b) showed the electrostatic potentials of IgY and the IgY-catechin complex, respectively. Catechin, consisting of two benzene rings and a dihydropyran ring with a hydroxyl group at carbon 3, carries a negative charge in aqueous solution(Chen, et, al. 2023). The charge and polarity of catechin make it prone to binding with positively charged regions on the protein(Gabriela, et, al. 2019). In the figures, the red areas represent positive charge regions, where catechin binds near the intersection of chains A and B of IgY with positively charged amino acid residues. According to Fig. 2 (c), during the binding process of IgY with catechin, amino acids PRO359, GLY393, THR409, SER425, GLY451, GLY481, ALA515, GLY519, ASP523, and ASN539 exhibit lower RMSF values, indicating their role in stabilizing the protein conformation. Amino acids with higher RMSF values were key binding sites for catechin. In the Fig. 2 (d) hydrogen bond heatmap, red indicates the presence of hydrogen bonds, representing interactions between amino acids. The color gradient from blue to red signifies an increase in the duration or number of hydrogen bonds. The data show that hydrogen bonds between ARG472 and THR505, and between LYS503 and GLU511, were the most numerous and long-lasting, consistent with the molecular docking results. Hydrogen bonds effectively maintain the stability of the complex, endowing it with certain functional properties(Li, et, al. 2022). The Fig. 3 (a) showed the temperature variation over time, where the temperature of system fluctuates between 296 K and 304 K, with an average temperature of 300 K. The temperature variation has a minimal impact on the total energy changes(Alexander, et, al. 2020). Figure 3 (b) displayed the total energy variation over time, showing energy oscillations centered around − 106,200 kcal/mol. The energy values demonstrated fluctuations during the simulation, suggesting the dynamic equilibrium of system. Combining Fig. 3 (b) and (c), the RMSD variation with conformation changes can be analyzed. The RMSD value increases gradually from 1.2 to 2.4 and then decreases, indicating significant conformational changes during the simulation. In the 0–30 frame stage, the total energy was relatively stable, with a low and stable RMSD, suggesting that the system was near equilibrium with minimal protein structural changes and higher system stability. During the 30–70 frame stage, energy fluctuations increased, indicating dynamic changes in the system. The RMSD increased, particularly after frame 40, showing substantial conformational changes of IgY. During this stage, synchronous increased in energy fluctuations and RMSD demonstrated that the protein was undergoing conformational adjustments, exploring new stable or transition states (Pearl, et,al.2019). In the final stage, energy fluctuations persist but showed signs of stabilization, indicating a transition to a new equilibrium state of complex. RMSD peaks at frame 80 and then decreases, but fluctuations remain. The energy of system and RMSD trends were moving towards stability, indicating that the IgY-catechin complex had reached a new equilibrium conformation, which enhanced overall system stability (Cheng, et, al. 2023). Significant conformational changes, shown by increasing RMSD, were accompanied by pronounced energy fluctuations, suggesting that energy variations drive these changes (Zhou, et, al. 2023). Following the RMSD peak, both energy fluctuations and RMSD gradually stabilize, indicating that the system has settled into a new stable conformation, with energy decreasing to a lower value. The RMSF plot illustrated that regions with residue numbers 1–50 and 350–450 exhibit high fluctuations with elevated RMSF values. High fluctuation regions may correspond to highly flexible loops of IgY, surface-exposed areas, or active sites interacting with other molecules (Zhou, et, al. 2023). Regions with residue numbers 100–300 show lower RMSF values and minimal fluctuations, indicating core structures or stable domains with higher conformational stability. Combining Fig. 3 (b) and (d), significant energy fluctuations correlate with substantial conformational changes or adjustments in high-RMSF regions, where large residue fluctuations significantly impact the system's energy state. When IgY and catechin have completed binding, the overall energy of the system tends to stabilize, and the complex forms a stable structure. Result of SEM Using SEM to observe the protein reveals the microscopic structural differences between IgY and the IgY-catechin complex. As shown in Fig. 4 (a), IgY displayed an approximately spherical morphology with a granular structure and a distinctly rough surface. Most of the particle surfaces were covered with smaller particles or block-like substances, indicating an aggregated form. Conversely, Fig. 4 (b) illustrated that the IgY-catechin complex adopted a sheet-like structure with a smooth surface and well-defined edges, lacking noticeable pore structures and exhibiting a well-organized layered structure. The sheet-like structure's surface was interspersed with small particles, possibly incompletely reacted polyphenols, protein residues, or minute composite particles (Ran et al., 2023). The formation of this sheet-like structure was likely attributed to the binding mechanism between the protein and polyphenols via hydrogen bonding (Zhang et al., 2021 ). Results of FT-IR Fourier infrared spectroscopy can offer structural information about proteins. FT-IR can obtain information about the secondary structure of proteins (e.g., Helix, β-Turn, etc.) by analyzing vibrational modes in the protein molecule, which can reveal the characteristics of the protein's structure. The changes in the protein conformation of IgY and IgY-catechin complex were further investigated by FT-IR, and the results were shown in Fig. 5 . Figure 5 (a) showed that relative to IgY, a discernible shift occurred in the peak positions of the amide I and amide II bands in the complex. The shifts moved from 1646.90 cm − 1 to 1631.14 cm − 1 and from 1529.49 cm − 1 to 1520.94 cm − 1 , respectively. This shift suggested a modification in IgY secondary structure upon the addition of catechins. This finding aligns with previous study of our team (Liu, et al. 2023 ). The absorption peaks within the 3200 cm − 1 to 3500 cm − 1 range correspond to characteristic O-H bonds. As depicted in Fig. 5 (a), the IgY-catechin complex exhibited broader peak shapes in this range compared to IgY. This suggested the formation of novel hydrogen bonds within the IgY-catechin complex, fostering increased association relative to IgY. The amide I band underwent deconvolution analysis. Gaussian fitting of peak areas was used to calculate the relative percentage content of various secondary structures, as outlined in Fig. 5 (b). Upon examination of Fig. 5 (b), it is evident that, compared to IgY, there were no notable alterations in the content of β-turns and β-sheets within the IgY-catechin complex. However, there was an increase in the content of α-helices, coupled with a decrease in random coils. α-helices, akin to β-sheets, represent predominant ordered structures in protein secondary configurations, sustained by intramolecular hydrogen bonds. The elevated α-helical content implies that the introduction of catechins fosters the development of fresh intramolecular hydrogen bonds within the protein. The rearrangement of hydrogen bonds may stem from the binding of catechin phenolic hydroxyl groups with carbonyl groups in IgY. This inference aligns with the preceding findings. Result of CD Circular dichroism (CD) was the most effective and commonly used method to study the conformational changes of proteins. Interaction between polyphenols and proteins occurs, which leads to inevitable modification of the spatial structure of proteins. Combined with FT-IR, it can reveal protein secondary structure changes more clearly. From the Fig. 6 , it is evident that the CD spectra of the IgY-catechin complex and IgY show significant differences, indicating that catechin has a notable impact on the structure of IgY. At 208 nm, the negative peak of the IgY-catechin complex was significantly enhanced, suggesting an increase in α-helix content. The stability of proteins is closely related to the α-helix content. An increase in the α-helix content of the IgY-catechin complex indicates an improvement in stability compared to IgY. This is also supported by previous stability tests. A decrease in random coils at 195 nm suggests that the IgY-catechin complex has formed more stable secondary structures, such as α-helices. Based on molecular docking and FTIR results, it can be inferred that the IgY-catechin complex possesses a more stable overall structure. Bacteriostatic Properties IgY has demonstrated the ability to cause cell aggregation and inhibit the growth of various pathogenic microorganisms by reducing the hydrophobicity and adhesion of bacterial cell membrane surfaces (Zhang Qian, et al., 2015). This antibacterial activity is particularly significant when inhibition occurs during the intestinal digestive process, facilitating the elimination of harmful bacteria. As a result, IgY has become a promising substitute for antibiotics. The antibacterial properties of both IgY and the IgY-catechin complex were examined, with results presented in Table 1 . Table 1 shows that at concentrations below 0.05 mg/mL, the IgY-catechin complex exhibited higher inhibition rates against E. coli than IgY alone. The complex showed the best inhibition effects at concentrations greater than 0.05 mg/mL, with an average increase of 15.74% against E. coli compared to IgY. Similarly, the inhibition rate of the IgY-catechin complex against S. aureus was significantly higher than that of IgY alone, with an increase of 135.8%. These results indicated that the IgY-catechin complex has superior antibacterial effects, suggesting that the binding of IgY with catechins enhances its antibacterial capability. Table 1 Bacteriostatic rate of IgY, and IgY-catechin complex Strains inhibition rate/% 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL 0.15 mg/mL 0.2 mg/mL E. coli IgY 22.08 ± 0.86 b 23.98 ± 0.44 b 29.88 ± 0.21 b 30.75 ± 0.11 b 32.11 ± 1.15 b IgY-catechin complex 22.33 ± 0.86 b 24.38 ± 0.82 b 34.14 ± 0.21 a 35.41 ± 0.62 a 37.83 ± 0.76 a S. aureus IgY 24.97 ± 1.27 c 24.85 ± 0.91 c 26.99 ± 1.13 c 41.85 ± 1.86 c 54.49 ± 1.53 c IgY-catechin complex 68.29 ± 0.30 a 72.16 ± 0.83 a 75.06 ± 0.49 a 79.15 ± 0.29 a 80.79 ± 1.12 a Columns values with different letters were significantly different( P < 0.05)。 Results of Stability of IgY-catechin Complex To exclude the effect of catechin on the antibacterial rate of the IgY-catechin complex, IgY and catechin were used as controls to analyze the heat stability of the IgY-catechin complex under different temperature treatments. The results were shown in Table 2 . The results showed that under identical conditions, the IgY-catechin complex exhibited the highest inhibition rates against E. coli . As the temperature increased, the inhibition rates of all three samples decreased, with a significant reduction for the IgY-catechin complex observed above 60°C. The thermal stability of the IgY-catechin complex was lower compared to IgY and catechin, yet it maintained good antibacterial activity at certain temperatures. For S. aureus , catechin exhibited the highest inhibition rate at each temperature, followed by the IgY-catechin complex, and then IgY. The IgY-catechin complex maintained better antibacterial activity against E. coli at temperatures where IgY alone was less effective, although its thermal stability declined significantly at temperatures above 60°C. At temperatures exceeding 70°C, the inhibition rates of both catechin and IgY against S. aureus and E. coli declined markedly. The observed changes in antibacterial activity may be attributed to the interaction between catechin and IgY, which alters the secondary structure of the protein macromolecules, exposing more active groups and enhancing antibacterial activity (Jie Liu et al., 2023 ). Although this structural change leads to reduced thermal stability and earlier loss of antibacterial activity compared to IgY, it does not negate the overall improvement in antibacterial performance of the IgY-catechin complex. Table 2 Bacteriostatic thermal stability of three substances Condition Inhibition rate/% E. coli S. aureus IgY catechin IgY-catechin complex IgY catechin IgY-catechin complex 30°C 22.02a 25.87a 31.23a 45.64a 89.36a 80.15a 40°C 22.50a 24.99a 31.70a 42.73a 92.30b 80.61b 50°C 21.77a 25.32a 31.61a 44.24c 91.01c 79.93c 60°C 20.21b 25.02a 30.59b 45.98a 90.44c 79.38d 70°C 18.10c 22.73b 24.67c 46.39b 89.21d 71.48e 80°C 11.11d 22.18b 21.69d 34.75d 78.92e 67.13f 90°C 8.20g 16.51c 17.67e 20.77e 62.64f 57.97g pH4 15.03a 81.39c 77.22b 18.04a 25.97b 28.00c pH5 31.89c 87.27c 80.95c 23.32b 27.25c 30.95c pH6 34.73c 89.43c 93.31c 25.47b 30.17b 35.61c pH7 37.26c 92.87c 93.67c 27.71b 29.55b 34.89c pH8 33.23c 92.69c 93.83c 22.85a 19.52b 35.01c pH9 24.12b 68.46b 71.77b 22.57a 15.64a 34.03c pH10 19.83a 58.42b 69.84b 12.17c 14.37a 27.18b Different letters denote significant differences (P < 0.05). Table 2 also presents the acid-base Stability of IgY and IgY-catechin complexes The IgY-catechin complex exhibited the highest inhibition rate against E. coli under identical conditions, with relatively small changes under different pH conditions. Compared to IgY and catechin alone, the IgY-catechin complex demonstrated improved inhibitory acid-base stability against E. coli . Table 2 shows that the inhibition rates of the complex and catechin against S. aureus were notably higher than that of IgY, following a similar trend as pH changed. The inhibition rate decreased significantly when pH exceeded 8. The superior acid-base stability of the IgY-catechin complex in inhibiting S. aureus could be attributed to the interaction between IgY and catechin, resulting in a complex that reduces the ionization degree of catechin’s hydroxyl groups (Liu. et al., 2023; He. et al., 2022). This interaction likely facilitates the binding of the complex to bacterial cell membranes and lipoproteins, synergistically enhancing its bacteriostatic activity and improving its acid-base stability. Results of MIC, MBC and Growth Curve MIC and MBC are crucial indicators for evaluating the antibacterial performance of a substance (P. Sruthi, et al., 2023). The MIC for the IgY-catechin complex against E. coli was determined to be 0.5 mg/mL. For S. aureus , the MIC was 0.125 mg/mL. After confirming the MIC values, MBC data were collected by identifying the lowest concentration of the IgY-catechin complex at which no bacterial growth was observed. The MBC of the complex was found to be 2 mg/mL for E. coli and 1 mg/mL for S. aureus . At lower concentrations, bacterial colonies were denser, while higher concentrations resulted in progressively fewer colonies. The inhibitory ability of the IgY-catechin complex on the bacterium was further determined by measuring the effect of the IgY-catechin complex on the growth curve of the bacterium. As can be seen from Fig. 7 (a). When the IgY-catechin complex were added at 0, MIC, and 2 MIC, the growth trends of E. coli were closer to each other in the 0–24 h time period. And all of them had obvious growth retardation, logarithmic, and stabilisation phases. However, the time for the growth of the bacteria to the retardation phase was prolonged compared to the non-addition of IgY-catechin complex. The absorbance was considerably lower than that of the untreated group at 2 MIC, which showed that the addition of the IgY-catechin complex exhibited an inhibitory effect on the growth of the bacterium. From Fig. 7 (b), S. aureus growth trends were similar and three phases of a normal growth curve existed when the complex was added at 0, MIC. The growth retardation period of the organisms was extended from 2 h to 10 h at MIC compared to 0. At 2 MIC, the absorbance was basically unchanged and the growth of the microbiological was severely inhibited. After the addition of the IgY-catechin complex, the lag phase of E. coli and S. aureus was significantly extended, the growth rate was notably reduced, and the overall bacterial count decreased, indicating that the IgY-catechin complex has significant potential in combating bacteria. Results of Nucleic Acid Protein Leakage Nucleic acid and intracellular protein leakage were measured to assess the impact of the complex on the membrane integrity and permeability of E. coli and S. aureus (Wang et al., 2018 ). The results of the nucleic acid and protein leakage assays are shown in Fig. 8 . From Fig. 8 (a), it was observed that the nucleic acid leakage in the control group was minimal and remained relatively stable over four hours. At MIC concentration, the nucleic acid leakage had significantly increased compared to the control group. As time progressed, the nucleic acid leakage gradually rose, indicating that the IgY-catechin complex at MIC concentration had caused some damage to the bacterial cells, leading to nucleic acid leakage. After 2 hours, the nucleic acid leakage tended to stabilize and did not rise significantly, suggesting that the IgY-catechin complex at MIC concentration had a certain bacteriostatic effect, capable of damaging the cell membrane of E. coli . When the concentration of the IgY-catechin complex reached 2MIC, the nucleic acid leakage was significantly higher than that of the control and MIC groups. The leakage markedly escalated within the first 1 to 2 hours and continued to rise from 2 to 4 hours. As can be seen in Fig. 8 (b), the leakage of nucleic acids rised with time and with the amount added under the effect of the IgY-catechin complex. The destructive effect was most obvious in the first 1 h, and the trend and mechanism of action were similar to the trend of E. coli . Form Fig. 8 (b) the leakage of intracellular proteins from S. aureus was similar to that of Escherichia coli. Under the MIC concentration of the complex, there is a significant increase in protein leakage within the first two hours. At a 2MIC concentration of the complex, the most pronounced leakage occurs within the first hour, after which the leakage does not show a significant increase. The complex also exhibits antibacterial activity against S. aureus . Catechin analogs have the ability to interact with components of cell membranes and alter their physical properties (Jun Sato, et al., 2022 ). This capability leads to leakage of cellular contents and interferes with normal cellular metabolism. The mechanism of bacteriostatic action of IgY was to inhibit the growth and reproduction of bacteria by causing cell aggregation (Mahenthiran, R., et al., 2021 ). The mechanism of inhibition of the IgY-catechin complex, similar to that of IgY and catechins, which was the inhibition of the growth of the organism through disruption of the cell membrane and the leakage of intracellular genetic material. The cell membrane plays a critical role in microorganisms like E. coli and S. aureus The cell membrane was the dividing line between the internal and external environments of the microbial cell (Dylan Gerard Ryan, et al., 2023), and had the role of screening and defense. The cell membrane of E. coli was highly resistant to osmotic pressure(Emma K. Eriksson, et al., 2019 ), enabling E. coli to maintain cellular stability in harsh environments. The biofilm of S. aureus is also a key structure in resisting antibiotics and other antimicrobial substances(Paul Payel, et al., 2023 ). If it is possible to disrupt a biofilm such as a cell membrane, it can easily result in the death of the microorganism. Results of Fluorescence Inverted Microscope PI dye can enter the bacterium when the cell membrane of the bacterium is disrupted or the bacterium had died(Chiaraviglio, et al., 2016 ). The PI dye chimerises with the DNA of the bacterium and then showed a red fluorescence under an inverted fluorescence microscope. When the cell membrane of the bacterium was intact, the PI dye was not able to pass through the cell membrane(Yawei Ning, et al., 2017 ). Therefore, the degree of growth inhibition of the bacterium was determined by observing the amount of red fluorescence in the image. (abc was plotted against Escherichia coli at a sample concentration of 0 MIC 2 MIC, def was plotted against Staphylococcus aureus at a sample concentration of 0, MIC, and 2 MIC, respectively.) The impact of the IgY-catechin complex on the cell membranes of E. coli and S. aureus was further assessed through observation using inverted fluorescence microscopy. As depicted in Fig. 10, negligible fluorescence was detected in the E. coli control group. Conversely, in the MIC-treated group, a noticeable increase in red fluorescence intensity was observed, with a substantial quantity of red fluorescence becoming apparent at a sample concentration of 2 MIC. Similarly, in the case of S. aureus , minimal fluorescence was observed in the control, while the intensity of red fluorescence progressively heightened with increasing sample concentration. These findings indicated that the IgY-catechin complex adversely affected the cell membrane of the bacteria, leading to a significant inhibition of E. coli and S. aureus growth, ultimately culminating in bacterial death. These observations were consistent with the results of previous studies on the influence of IgY-catechin complex on bacterial cell membrane rhabdomeres. CONCLUSION Polyphenols, when combined with proteins, alter the structure and properties of the proteins. Molecular dynamics simulations confirmed the interactions between IgY and catechin, highlighting key amino acids that stabilize the protein complex. The RMSF and RMSD analyses revealed significant conformational changes and energy fluctuations during the binding process, ultimately leading to a stable equilibrium state of the IgY-catechin complex. Catechin binds to residues PHE503, THR501, THR505, GLU511 of IgY respectively, and an increase in hydrogen bond content within the complex, a 5.48% increase in α-helix content, and a decrease in random coil content. Molecular docking techniques predicted the interaction sites and binding forms between catechins and proteins. FT-IR and CD studies of the complex's secondary structure revealed changes, including an increase in α-helix content and the formation of hydrogen bonds, consistent with software simulation results. Hydrogen bonds were identified as the primary binding force between catechins and IgY, indicating a more stable combination, preventing the complex's structure from being easily disrupted. Subsequent stability and antibacterial tests showed that the complex has lower thermal stability compared to IgY, and that pH has minimal impact on its antibacterial performance. This suggests that the complex has potential applications in antibacterial fields related to pH. The MIC of the complex against E. coli was 0.5 mg/mL and 2 mg/mL, while the MBC against S. aureus was 0.125 mg/mL and 1 mg/mL. After the addition of the IgY-catechin complex, the lag phase of E. coli and S. aureus was significantly extended, the growth rate was notably reduced, and the overall bacterial count decreased, indicating that the IgY-catechin complex has significant potential in combating bacteria. This provides strong evidence for its application as an antimicrobial agent. Nucleic acid-protein leakage analysis and fluorescence microscopy results indicated that the complex inhibits bacterial growth by disrupting the cell membrane, leading to the release of intracellular genetic material. It is plausible to envision the further expansion of its application in food processing as natural bacteriostatic agents, as well as in a broader spectrum of applications in future investigations. Declarations Ethics Approval and Consent to Participate Not applicable Competing Interests The authors declare no competing interests. Conflict of Interest The Authors declare that there is no conflict of interest. Funding Major Science and Technology Projects in Henan Province (No. 221100110500); Henan Province Science and Technology Research and Development (242102110092); National Key R&D Program of China (2022YFF1101600); Research Funding for Distinguished Professor at Henan University of Science and Technology (13510004); the Leading Talent Program for Science and Technology Innovation in Central China (234200510020) Author Contribution Lili Liu: Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing – original draft, Writing – review & editing. Yanli Wang: Data curation, Formal analysis, Writing – original draft, Conceptualization, Validation. Mengjun Zhang Writing – review & editing, prepared figures. Weiwei Cheng: prepared figures, Writing – review & editing. Ding: Writing – review & editing. Jialiang He: Writing – review & editing. All authors reviewed the manuscript. 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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-5017669","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348825887,"identity":"f2d9dcd2-eb2e-47a2-ae89-30e362108f90","order_by":0,"name":"Lili Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYLCCBwYMDGzsIFaFhBw/UVoSQFqYQawzFsaSDURpAREgLYxtFYkbCGkxOH728IuEgsOJfczMzx5+nSfBuIGB+eGjG/i0nMlLs0gwOJzYxsxmbiy7TYLZnIHN2DgHn5YDOWYGEC0MZtKS2yTYLBt42KTxajn/BqaF/Zu05BwJHoMDhLTcyDF+ANHCYyb5sUFCgqAWyRtvzICBnG4M1FImzXBMwkCymYBf+M7nGH/48Mdadn57+zbJHzV19f3szQ8f49OicICBTQJIOzYACWYekBAzHuUgIN/AwPwBSNuDOIw/CKgeBaNgFIyCkQkAImdJ58SuUVcAAAAASUVORK5CYII=","orcid":"","institution":"National Experimental Teaching Demonstration Center for Food Processing and Security, Henan University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Lili","middleName":"","lastName":"Liu","suffix":""},{"id":348825888,"identity":"e487f3a2-c8e5-4d26-b8e2-6753528e500a","order_by":1,"name":"Yanli Wang","email":"","orcid":"","institution":"National Experimental Teaching Demonstration Center for Food Processing and Security, Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Wang","suffix":""},{"id":348825889,"identity":"732460a0-3699-4d8f-b411-62e13ed4b771","order_by":2,"name":"Mengjun Zhang","email":"","orcid":"","institution":"National Experimental Teaching Demonstration Center for Food Processing and Security, Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Mengjun","middleName":"","lastName":"Zhang","suffix":""},{"id":348825890,"identity":"cf05e86b-2e39-4643-8f1c-11439f0bf682","order_by":3,"name":"Weiwei Cheng","email":"","orcid":"","institution":"National Experimental Teaching Demonstration Center for Food Processing and Security, Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Cheng","suffix":""},{"id":348825891,"identity":"1e04dbaf-7179-4f90-bf81-a88aecd74eb6","order_by":4,"name":"Yue Ding","email":"","orcid":"","institution":"National Experimental Teaching Demonstration Center for Food Processing and Security, Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Ding","suffix":""},{"id":348825892,"identity":"3578bb4e-d2f9-4d46-854a-f54086c1ebf7","order_by":5,"name":"Jialiang He","email":"","orcid":"","institution":"National Experimental Teaching Demonstration Center for Food Processing and Security, Henan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jialiang","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-09-02 10:51:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5017669/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5017669/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65916215,"identity":"6e01be6d-2c41-40c8-be08-bf64aa20e6dc","added_by":"auto","created_at":"2024-10-04 10:47:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":247459,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the interaction between IgY and catechins\u003c/p\u003e\n\u003cp\u003e(a)2D Schematic Diagram of IgY and Catechin Molecular Docking. (b)3D Detailed View of IgY and Catechin Molecular Docking. (c)Comprehensive Structural Diagram of IgY and Catechin Molecular Docking\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/605b606b10f85247d4dad903.jpg"},{"id":65917217,"identity":"21bbb77a-9709-44f8-b9de-a5c02f051051","added_by":"auto","created_at":"2024-10-04 10:55:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74710,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Electrostatic Potential Diagram of IgY and Catechin Before Molecular Docking. (b)Electrostatic Potential Diagram of IgY and Catechin After Molecular Docking. (c)RMSF Variation Diagram of the Main Chain and Side Chains. (d)Heat Map of Hydrogen Bonds.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/bdca34fadbdc3870bece2e23.jpg"},{"id":65916214,"identity":"35e24c63-149a-4b04-859b-816213f5fe7f","added_by":"auto","created_at":"2024-10-04 10:47:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42361,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The temperature variation of the system over time. (b)The total energy variation of the system over time. (c)The RMSD plot of the conformations. (d) The RMSF plot of the amino acids\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/206424826b7b4100c13d3c1f.jpg"},{"id":65916218,"identity":"ee7f9621-be55-41f5-b090-8f52c4b11086","added_by":"auto","created_at":"2024-10-04 10:47:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51167,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of IgY (a) and IgY-catechin complex (b) at 500× magnification.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/1f150d6c4e688415aa043eee.jpg"},{"id":65917218,"identity":"72fd1fbf-053c-40a6-b402-cb7496be291f","added_by":"auto","created_at":"2024-10-04 10:55:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26236,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fouriertransform infrared spectra of IgY and IgY-catechin complex. (b) The relative content of secondary structure of protein in amide I band of IgY and IgY-catechin complex\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/babed535a97783445f2c77f1.jpg"},{"id":65916216,"identity":"30225de6-5e57-4d34-9c94-2bb833394cda","added_by":"auto","created_at":"2024-10-04 10:47:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":556372,"visible":true,"origin":"","legend":"\u003cp\u003eCircular Dichroism Spectra of IgY and IgY-Catechin Complex (0.25 mg/mL) Scanned from 190-250 nm\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/f9743cf630ee9ddbba3caf61.jpg"},{"id":65917312,"identity":"cd538c6d-c120-4d48-af7c-03e1be96ea54","added_by":"auto","created_at":"2024-10-04 11:03:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1825074,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5017669/v1/3781373d-7772-432d-931a-195b5d02927c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polyphenol-Driven Structural Alterations and Antibacterial Potency of the IgY-Catechin Complex","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe majority of food spoilage is caused by microbial contamination, and common foodborne pathogens that mainly endanger human health are \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) (Mpatswenumugabo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The reduction in nutritional value of food due to foodborne pathogens is a pressing issue that needs to be addressed. While antibiotics can inhibit bacterial growth, their use can lead to the development of antibiotic-resistant bacteria. (Yip, A.Y.G. et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYolk immunoglobulin (IgY) has a high safety profile and exhibits a certain degree of stability (Li, et al. 2023) As a kind of new generation antibody, IgY has high application value and shows remarkable effect in human health care, disease diagnosis and prevention(Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Replacing antibiotics with IgY can alleviate the problem of antibiotic resistance and prevent bacterial diseases (Minquan Xia, et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Heat resistance, acid and alkali resistance, and other physicochemical properties of IgY have a direct impact on its manufacture, storage, and application in a variety of sectors (Fang. et al. 2016). IgY has a certain degree of stability and its activity is less affected by temperature, pH, and enzymes within a certain concentration range (Bellingeri R V, et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Under room temperature or refrigerated conditions, its activity remains stable after a longer period of storage (Nilsson E, et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, the activity of IgY decreased significantly, when the temperature exceeded 60\u0026deg;C, and was almost activity completely inactivated above 80\u0026deg;C. Current research has shown that the application of liposomes (Mahin Rigi, et al. 2022), chitosan-sodium alginate (Setiasih Siswati, et al. 2019), and other encapsulation treatments for IgY may alleviate its stability (Gandhi S, \u0026amp; Alshehri SM., 2021 ). The high production costs and operational complexities associated with these methods impede their scalability.\u003c/p\u003e \u003cp\u003eNumerous studies have confirmed that natural polyphenols in plants possess antibacterial properties. For instance, research by Wang et al. (2023) has found that flavonoids from Sedum aizoon L. exhibit antifungal activity on grapes. Similarly, Maral et al. (2023) reported the use of curcumin as a substitute for synthetic antibacterial agents in the prevention and treatment of gray mold, demonstrating effective inhibition of bacterial growth. Catechin, which is polyphenol, possesses synergistic antibacterial and antioxidant properties(Tyagi, et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e ). It was used to prevent oxidative deterioration of meat products (Paturi G. et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Many studies have shown that the interaction between proteins and polyphenols can significantly impact protein functionality (Jia, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Polyphenols have the capacity to interact with proteins, forming complexes that can be either reversible or irreversible, thus modifying the functional properties of protein (Jingxian Niu, et al., 2023). For example, Tan et al. (2022) studied the effects of phenolic compounds such as gallic acid (GA), chlorogenic acid (CA), epigallocatechin (EGC), epigallocatechin gallate (EGCG), and tannic acid (TA) on the modification of carp myofibrillar proteins. Their research indicated that these phenolic compounds alter both the secondary and tertiary structures of myofibrillar proteins. Additionally, the phenolic substances improve the thermal stability and gelation properties of the myofibrillar proteins. Xie et al. (2022) utilized multispectral methods to investigate the binding mechanisms of chlorogenic acid (CGA) and quercetin (QUE) with fish myofibrillar proteins. Circular dichroism analysis revealed that the addition of CGA does not affect the α-helix content of myofibrillar proteins, whereas QUE disrupts the β-turns and promotes the formation of α-helices in myofibrillar proteins. Liu et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) investigated the effect of the interaction between catechins and glycated porcine hemoglobin (G-PHb) on the antioxidant and structural properties of glycosylated hemoglobin. However, current research primarily focuses on the isolation, purification, and antibacterial properties of IgY. There is limited research on modifying IgY with catechins to alter its antibacterial performance, thermal stability, and pH stability, as well as on the structural characterization of catechin-IgY complex.\u003c/p\u003e \u003cp\u003eThis study prepared IgY-catechin complexes to investigate their structure and antibacterial properties. The structural changes and formation mechanism of the complex were investigated using molecular docking, molecular dynamics simulation, Fourier-transform infrared spectroscopy (FT-IR), and circular dichroism (CD). The antibacterial stability of the complex was assessed with IgY as a control. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the complex were determined. The inhibitory effect of the complex on bacterial growth was studied through growth curve, nucleic acid and protein leakage tests and fluorescence microscopy. This study investigates potential changes in IgY during its application in antibacterial and functional food development, providing a theoretical basis for these processes. The findings aim to lay a foundation for the subsequent development of IgY-related health foods.\u003c/p\u003e"},{"header":"Materials and reagents","content":"\u003cp\u003eFresh eggs were obtained from Dazhang Supermarket, Luoyang, China. Propidium iodide (PI) dye was acquired from Shanghai McLean Biochemical Technology Co., and catechin was purchased from Hefei Bomei Biotechnology Co., Luria-Bertani(LB) solid medium was bought from Beijing Baoxing Biotechnology Co., \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were obtained from the microbiology lab of the Food and Bioengineering college, Henan University of Science and Technology. All reagents were analytically pure. All solutions and suspensions were prepared with deionized water.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eExperimental method\u003c/h2\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003ePreparation of IgY and IgY-Catechin Complex\u003c/h2\u003e\n \u003cp\u003eRefer to the Lin et al. (2014) method to obtain IgY solution by ammonium sulfate precipitation. Afterwards, the solution was desalted at 4\u0026deg;C using a membrane with a molecular weight cutoff (MWCO) of 8000\u0026ndash;14000 Da for 48 hours. IgY powder was obtained by vacuum freeze-drying in a vacuum freeze-dryer (Shanghai Danding International Trading Co., China). The 1:1 mass concentration of IgY and catechin was placed in a magnetic stirrer (Changzhou Ronghua Instrument Manufacture Co., China) and stirred for 6 h at 40\u0026deg;C to obtain the complex solution. And then the complex powder was then obtained by vacuum freezing.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular Docking of catechin and IgY\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe IgY structure (PDB ID: 2W59) and catechin molecules were sourced from the RCSB Protein Data Bank and PubChem database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003c/span\u003e), respectively. AutoDockTools-1.5.6 software was used for preprocessing and conducting molecular docking simulations of receptors and ligands. Semi-flexible docking involving 50 runs was performed. The model exhibiting the lowest binding energy was selected as the optimal configuration. Visualization of the simulation outcomes and illustration generation were performed using PyMOL software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular dynamics simulation\u003c/h2\u003e\n \u003cp\u003eMolecular dynamics simulations were conducted using the downloaded PDB file and catechin molecule file from previous step. The protein and small molecule files were opened using Discovery Studio for protein preprocessing. Flexible peptide chains were removed, retaining only those with minimal thermal vibrations and more precise positioning. Water molecules were removed from the protein, and the protein was solvated and ionized to balance the solution using the CHARMM36 force field. The steepest descent algorithm was employed to resolve any initial bad contacts within the system, followed by the conjugate gradient method to ensure a low-energy starting point for subsequent dynamics. The simulation time was set to 20 ps for the molecular dynamics simulation. Visualization was performed using PyMOL and Discovery Studio.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular dynamics simulation analysis\u003c/h2\u003e\n \u003cp\u003eRoot Mean Square Deviation (RMSD) was used to measure the overall change in a protein, calculating the average deviation of atomic positions at a given time point from a reference structure, which is typically the initial structure or a stable structure. Root Mean Square Fluctuation (RMSF) was used to measure the fluctuation of a particular atom (or residue) relative to its average position over the entire simulation, reflecting the local flexibility of each atom or residue. The formulas for calculating RMSD and RMSF are as follows:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\text{RMSD=}\\sqrt{\\frac{\\text{1}}{\\text{N}}\\sum\\:_{\\text{i=1}}^{\\text{N}}{\\left({\\text{r}}_{\\text{i}}\\left(\\text{t}\\right)\\text{-}{\\text{r}}_{\\text{i}}^{\\text{ref}}\\right)}^{\\text{2}}}$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e$$\\:\\text{\\:}{\\text{r}}_{\\text{i}}^{\\text{ref}}$$\u003c/div\u003e\n \u003c/div\u003eHere, N represents the number of atoms, and rii(t) is the position of the i-th atom at time t,while is the position of the atom in the reference structure.\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e$$\\:\\text{RMSF=}\\sqrt{\\frac{\\text{1}}{\\text{T}}\\sum\\:_{\\text{t=1}}^{\\text{T}}{\\left({\\text{r}}_{\\text{i}}\\left(\\text{t}\\right)\\text{-}\\stackrel{-}{{\\text{r}}_{\\text{i}}}\\right)}^{\\text{2}}}$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Equd\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e$$\\:\\stackrel{-}{{\\text{r}}_{\\text{i}}}$$\u003c/div\u003e\n \u003c/div\u003eWhere rii(t) is the position of the i-th atom at time, is the average position of this atom over the entire simulation, and T is the number of time steps.\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eScanning electron microscopy (SEM)\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003cp\u003eTo examine the effect of the dried protein samples, the structure morphologies of IgY and IgY-Catechin complex were determined by SEM (S\u0026ndash;3000N, Hitachi Limited, Japan). Before imaging, the sample powder was evenly scattered on the conductive adhesive (Yang, Tu, Wang, Li, \u0026amp; Tian, 2017). The sample was sprayed with 5nm thick gold. After that, the morphology of the dried protein samples was observed using SEM. Collection of images magnified 500 times.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eFourier transform infrared spectroscopy (FT-IR)\u003c/h2\u003e\n \u003cp\u003eThe secondary structures of IgY and IgY-catechin complex were characterized with a FT-IR instrument (Nicoler-SX-170, Thermo Nicolet Corporation, USA). The samples were subjected to infrared spectroscopy using the potassium bromide compression method. The IgY, IgY-catechin complex and KBr were first dried for 12 h in advance. The dried particles were first ground into a very fine powder. IgY and IgY-catechin complex were mixed with potassium bromide powder at a mass ratio of 100:1, respectively. Then, the mixture was pressed into light-transmitting sheets (1\u0026ndash;2 mm thick) in a sample tank at a pressure of 15\u0026ndash;20 MPa. The scanning resolution was 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the scanning range was 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The medium (KBr) with no protein was recorded as the background spectrum. OMNIC software was used to analyze the FT-IR spectral data.\u003c/p\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003eCircular dichroism spectrum\u003c/h2\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003cp\u003eChanges in protein secondary structure were analyzed using circular dichroism (Chirascan VX, Applied Photophysics, UK). Preparation of 0.25 mg/ml solution of IgY and IgY-catechin complex. The secondary structure of the protein was measured with 1 ml of the solution. The wavelength scanning range was 190\u0026ndash;250 nm with a spectral resolution of 0.1 nm. The scanning speed was 100 nm/s and the response time was 0.5 s. The calibration was first scanned with air and deionized water and repeated twice. The samples were scanned three times and averaged. The measured spectra were smoothed twice to obtain a circular dichroism chromatogram. Finally, CDNN software was used for data analysis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of Bacterial suspensions\u003c/h2\u003e\n \u003cp\u003eBacterial suspensions of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were prepared according to the method of Ihuma, (2022). The concentration of the bacterial suspension was adjusted to 10\u003csup\u003e6\u003c/sup\u003e~10\u003csup\u003e8\u003c/sup\u003e CFU/mL.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of Inhibitory activity\u003c/h2\u003e\n \u003cp\u003eThe suspensions of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e were mixed with sterilised LB solid medium at a ratio of 1:100 and the homogeneous bacterial plates made with sterile smear rings. Determination of bacterial inhibitory activity was performed using the perforation method. The IgY and IgY-catechin complex were mixed with anhydrous ether (1:1 volume ratio) to form a mixture, respectively. Sterilized circular filter paper (diameter d\u0026thinsp;=\u0026thinsp;0.5 cm) was then clamped with sterile tweezers, immersed in the mixture for 20 minutes, and subsequently air-dried. The filter paper sheets were pasted on each test bacterial plate, each plate was pasted with 3 pieces in a triangular shape. Anhydrous ether was used as a blank control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eDetermination of Inhibition rate\u003c/h2\u003e\n \u003cp\u003eReferring to the method of Srikacha, et al (2020) with slight improvement. Several tubes were sterilized after the addition of 4 mL of liquid medium to each tube. Subsequently, test tubes numbered 1 through 5 were sterilized and added with 100 \u0026micro;L of \u003cem\u003eE. coli\u003c/em\u003e bacterial solution and 1 mL of a specified concentration of the sample solution, resulting in final concentrations of 0.025, 0.05, 0.1, 0.15, and 0.2 mg/mL, respectively. Tube number 6 was used as the positive control, receiving 1 mL of sterilized distilled water and 100 \u0026micro;L of \u003cem\u003eE. coli\u003c/em\u003e bacterial solution, while the remaining tubes served as negative controls. The tubes were incubated in a constant temperature shaker (Shanghai Yiheng Scientific Instrument Co., China) at 37\u0026deg;C for 12 hours, followed by measurement of optical density (OD) at 600 nm. The inhibition rate was calculated using the following formula,\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eThermal and Acid-base Stability Tests\u003c/h2\u003e\n \u003cp\u003eThe experimental protocol, based on Noriyuki et al. (2023) with slight modifications, included preparing samples in a 0.5 mg/mL solution. These samples were then treated at temperatures ranging from 40℃ to 90℃ for 20 minutes, and the optical density (OD) at 600 nm was measured to assess inhibition rates and investigate stability across different temperature conditions.\u003c/p\u003e\n \u003cp\u003eSimilarly, the bacteriostatic acid-base stability was determined using the same approach as described above. The sample solution\u0026apos;s pH was varied to 5, 6, 7, 8, 9, and 10 to assess its bacteriostatic stability across different pH conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eMIC and MBC of IgY-catechin Complex\u003c/h2\u003e\n \u003cp\u003eReferring to the method of Aigbogun et al.(2023) and improved it. Preparation of 9 tubes were numbered and were ready for use on an ultra-clean bench (Xinbeisi Biotechnology Co., China). Tubes 1\u0026ndash;8 received 4 mL of sterilised LB liquid medium and 100 \u0026micro;L of bacterial suspension. Additionally, tubes 1\u0026ndash;7 were supplemented with 1 mL of complex solutions at concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL, respectively. 1 mL of physiological saline was added to tube 8 in place of the complex solution, and only culture medium was added to tube 9. MIC values were obtained by observation after incubation was used in a thermostatic incubation shaker (Yiheng Scientific Instrument Co., China).\u003c/p\u003e\n \u003cp\u003eThe MBC was determined by the plate method, where 100 \u0026micro;L of the culture solution from each sample was streaked onto LB solid medium. The MBC value was obtained by watching the growth of colonies in the plate after incubation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eGrowth curve\u003c/h2\u003e\n \u003cp\u003eTo test tubes containing LB liquid medium, 1 mL of a IgY-catechin complex solution was added to achieve final concentrations of MIC and 2 MIC. As a control, 1 mL of distilled water was added. Each tube then received 100 \u0026micro;L of bacterial culture grown to the logarithmic phase. The tubes were incubated at 37\u0026deg;C for 24 hours, with samples taken every 2 hours to measure OD\u003csub\u003e600nm\u003c/sub\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eMeasurement of Nucleic Acid Protein Leakage\u003c/h2\u003e\n \u003cp\u003eThe method described by Chang (2021) was employed to assess the leakage of nucleic acid of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. Bacterial suspensions at approximately 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL were treated with varying multiples of the MIC, ranging from 0 to 2 times MIC. The suspension was incubated at 37℃ for 12 h after preparation. Every 2 h, 200 \u0026micro;L of the culture solution was centrifuged at 10,000 r/min for 10 min and the supernatant was collected. The absorbance at 260 nm (OD\u003csub\u003e260\u003c/sub\u003e) of the supernatants was then measured using a multimode plate reader (Tecan, Infinite M200 PRO, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eObservation of Fluorescence Inverted Microscopy\u003c/h2\u003e\n \u003cp\u003eRefer to the method of Han Yingjie, et al. (2021). The bacterial solution was washed, resuspended, diluted to the appropriate concentration and dispensed into test tubes. The samples were prepared to achieve final concentrations equivalent to the MIC and twice the MIC, then incubated at 37\u0026deg;C for 3 hours. After that, PI dye was added to it, and the reaction was carried out for 20 min under the dark condition. At the end of the reaction a small amount of liquid was pipetted onto a slide and allowed to dry, and observed by fluorescence inverted microscope(Olympus, Japanese).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003cp\u003eAll experiments were performed thrice in parallel, and the results were presented as mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical significance of the data was analyzed using SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). The analyses of variance were performed via the ANOVA procedure. The significance level of data was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The correlation analysis was carried out by using Origin 2021.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eResults of Molecular Docking Simulation\u003c/h2\u003e\n \u003cp\u003eMolecular docking visualizes structural changes in protein molecules post-interaction with small molecules and elucidates the intermolecular forces and binding conditions involved in their interaction. The structure of molecular docking is shown in Fig.\u0026nbsp;1. From Fig.\u0026nbsp;1, it can be clearly seen that IgY showed insignificant changes in protein and catechin structure upon interaction with catechins. The final intermolecular energy was \u0026minus;\u0026thinsp;34.84 kJ/moL, Among these, the van der Waals forces(vdW), Hbond, desolv Energy made a large contribution. There were five hydrogen bonding site for the binding of catechins and IgY. Catechin binds to residues PHE503, THR501, THR505, GLU511 of IgY respectively, and was accompanied by six possible flips. This time the predicted free energy of binding was \u0026minus;\u0026thinsp;27.34 kJ/mol, indicated that the ligand-receptor interaction is stable. In conclusion, the molecular docking results showed stable ligand-receptor interactions, mainly driven by vdW, hydrogen bonding and desolvation energies.\u003c/p\u003e\n \u003cp\u003e(a)2D Schematic Diagram of IgY and Catechin Molecular Docking. (b)3D Detailed View of IgY and Catechin Molecular Docking. (c)Comprehensive Structural Diagram of IgY and Catechin Molecular Docking\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003eResult of Molecular Dynamics Simulation\u003c/h2\u003e\n \u003cp\u003eBased on molecular docking analysis, it can be confirmed that there were interactions between IgY and catechin. Molecular dynamics simulations using Discovery Studio software were conducted to study the structure and binding modes of the IgY-catechin complex more precisely. First, the electrostatic potential of the protein was calculated and visualized using PyMOL. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (a) and (b) showed the electrostatic potentials of IgY and the IgY-catechin complex, respectively. Catechin, consisting of two benzene rings and a dihydropyran ring with a hydroxyl group at carbon 3, carries a negative charge in aqueous solution(Chen, et, al. 2023). The charge and polarity of catechin make it prone to binding with positively charged regions on the protein(Gabriela, et, al. 2019). In the figures, the red areas represent positive charge regions, where catechin binds near the intersection of chains A and B of IgY with positively charged amino acid residues.\u003c/p\u003e\n \u003cp\u003eAccording to Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (c), during the binding process of IgY with catechin, amino acids PRO359, GLY393, THR409, SER425, GLY451, GLY481, ALA515, GLY519, ASP523, and ASN539 exhibit lower RMSF values, indicating their role in stabilizing the protein conformation. Amino acids with higher RMSF values were key binding sites for catechin. In the Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (d) hydrogen bond heatmap, red indicates the presence of hydrogen bonds, representing interactions between amino acids. The color gradient from blue to red signifies an increase in the duration or number of hydrogen bonds. The data show that hydrogen bonds between ARG472 and THR505, and between LYS503 and GLU511, were the most numerous and long-lasting, consistent with the molecular docking results. Hydrogen bonds effectively maintain the stability of the complex, endowing it with certain functional properties(Li, et, al. 2022).\u003c/p\u003e\n \u003cp\u003eThe Fig.\u0026nbsp;3 (a) showed the temperature variation over time, where the temperature of system fluctuates between 296 K and 304 K, with an average temperature of 300 K. The temperature variation has a minimal impact on the total energy changes(Alexander, et, al. 2020). Figure\u0026nbsp;3 (b) displayed the total energy variation over time, showing energy oscillations centered around \u0026minus;\u0026thinsp;106,200 kcal/mol. The energy values demonstrated fluctuations during the simulation, suggesting the dynamic equilibrium of system.\u003c/p\u003e\n \u003cp\u003eCombining Fig.\u0026nbsp;3 (b) and (c), the RMSD variation with conformation changes can be analyzed. The RMSD value increases gradually from 1.2 to 2.4 and then decreases, indicating significant conformational changes during the simulation. In the 0\u0026ndash;30 frame stage, the total energy was relatively stable, with a low and stable RMSD, suggesting that the system was near equilibrium with minimal protein structural changes and higher system stability. During the 30\u0026ndash;70 frame stage, energy fluctuations increased, indicating dynamic changes in the system. The RMSD increased, particularly after frame 40, showing substantial conformational changes of IgY. During this stage, synchronous increased in energy fluctuations and RMSD demonstrated that the protein was undergoing conformational adjustments, exploring new stable or transition states (Pearl, et,al.2019). In the final stage, energy fluctuations persist but showed signs of stabilization, indicating a transition to a new equilibrium state of complex. RMSD peaks at frame 80 and then decreases, but fluctuations remain. The energy of system and RMSD trends were moving towards stability, indicating that the IgY-catechin complex had reached a new equilibrium conformation, which enhanced overall system stability (Cheng, et, al. 2023). Significant conformational changes, shown by increasing RMSD, were accompanied by pronounced energy fluctuations, suggesting that energy variations drive these changes (Zhou, et, al. 2023). Following the RMSD peak, both energy fluctuations and RMSD gradually stabilize, indicating that the system has settled into a new stable conformation, with energy decreasing to a lower value.\u003c/p\u003e\n \u003cp\u003eThe RMSF plot illustrated that regions with residue numbers 1\u0026ndash;50 and 350\u0026ndash;450 exhibit high fluctuations with elevated RMSF values. High fluctuation regions may correspond to highly flexible loops of IgY, surface-exposed areas, or active sites interacting with other molecules (Zhou, et, al. 2023). Regions with residue numbers 100\u0026ndash;300 show lower RMSF values and minimal fluctuations, indicating core structures or stable domains with higher conformational stability. Combining Fig.\u0026nbsp;3 (b) and (d), significant energy fluctuations correlate with substantial conformational changes or adjustments in high-RMSF regions, where large residue fluctuations significantly impact the system\u0026apos;s energy state. When IgY and catechin have completed binding, the overall energy of the system tends to stabilize, and the complex forms a stable structure.\u003c/p\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eResult of SEM\u003c/h2\u003e\n \u003cp\u003eUsing SEM to observe the protein reveals the microscopic structural differences between IgY and the IgY-catechin complex. As shown in Fig. 4 (a), IgY displayed an approximately spherical morphology with a granular structure and a distinctly rough surface. Most of the particle surfaces were covered with smaller particles or block-like substances, indicating an aggregated form. Conversely, Fig. 4 (b) illustrated that the IgY-catechin complex adopted a sheet-like structure with a smooth surface and well-defined edges, lacking noticeable pore structures and exhibiting a well-organized layered structure. The sheet-like structure\u0026apos;s surface was interspersed with small particles, possibly incompletely reacted polyphenols, protein residues, or minute composite particles (Ran et al., 2023). The formation of this sheet-like structure was likely attributed to the binding mechanism between the protein and polyphenols via hydrogen bonding (Zhang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003eResults of FT-IR\u003c/h2\u003e\n \u003cp\u003eFourier infrared spectroscopy can offer structural information about proteins. FT-IR can obtain information about the secondary structure of proteins (e.g., Helix, \u0026beta;-Turn, etc.) by analyzing vibrational modes in the protein molecule, which can reveal the characteristics of the protein\u0026apos;s structure. The changes in the protein conformation of IgY and IgY-catechin complex were further investigated by FT-IR, and the results were shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (a) showed that relative to IgY, a discernible shift occurred in the peak positions of the amide I and amide II bands in the complex. The shifts moved from 1646.90 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1631.14 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and from 1529.49 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1520.94 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. This shift suggested a modification in IgY secondary structure upon the addition of catechins. This finding aligns with previous study of our team (Liu, et al. \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The absorption peaks within the 3200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 3500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range correspond to characteristic O-H bonds. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (a), the IgY-catechin complex exhibited broader peak shapes in this range compared to IgY. This suggested the formation of novel hydrogen bonds within the IgY-catechin complex, fostering increased association relative to IgY. The amide I band underwent deconvolution analysis. Gaussian fitting of peak areas was used to calculate the relative percentage content of various secondary structures, as outlined in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (b). Upon examination of Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (b), it is evident that, compared to IgY, there were no notable alterations in the content of \u0026beta;-turns and \u0026beta;-sheets within the IgY-catechin complex. However, there was an increase in the content of \u0026alpha;-helices, coupled with a decrease in random coils. \u0026alpha;-helices, akin to \u0026beta;-sheets, represent predominant ordered structures in protein secondary configurations, sustained by intramolecular hydrogen bonds. The elevated \u0026alpha;-helical content implies that the introduction of catechins fosters the development of fresh intramolecular hydrogen bonds within the protein. The rearrangement of hydrogen bonds may stem from the binding of catechin phenolic hydroxyl groups with carbonyl groups in IgY. This inference aligns with the preceding findings.\u003c/p\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003eResult of CD\u003c/h2\u003e\n \u003cp\u003eCircular dichroism (CD) was the most effective and commonly used method to study the conformational changes of proteins. Interaction between polyphenols and proteins occurs, which leads to inevitable modification of the spatial structure of proteins. Combined with FT-IR, it can reveal protein secondary structure changes more clearly.\u003c/p\u003e\n \u003cp\u003eFrom the Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, it is evident that the CD spectra of the IgY-catechin complex and IgY show significant differences, indicating that catechin has a notable impact on the structure of IgY. At 208 nm, the negative peak of the IgY-catechin complex was significantly enhanced, suggesting an increase in \u0026alpha;-helix content. The stability of proteins is closely related to the \u0026alpha;-helix content. An increase in the \u0026alpha;-helix content of the IgY-catechin complex indicates an improvement in stability compared to IgY. This is also supported by previous stability tests. A decrease in random coils at 195 nm suggests that the IgY-catechin complex has formed more stable secondary structures, such as \u0026alpha;-helices. Based on molecular docking and FTIR results, it can be inferred that the IgY-catechin complex possesses a more stable overall structure.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n \u003ch2\u003eBacteriostatic Properties\u003c/h2\u003e\n \u003cp\u003eIgY has demonstrated the ability to cause cell aggregation and inhibit the growth of various pathogenic microorganisms by reducing the hydrophobicity and adhesion of bacterial cell membrane surfaces (Zhang Qian, et al., 2015). This antibacterial activity is particularly significant when inhibition occurs during the intestinal digestive process, facilitating the elimination of harmful bacteria. As a result, IgY has become a promising substitute for antibiotics. The antibacterial properties of both IgY and the IgY-catechin complex were examined, with results presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows that at concentrations below 0.05 mg/mL, the IgY-catechin complex exhibited higher inhibition rates against \u003cem\u003eE. coli\u003c/em\u003e than IgY alone. The complex showed the best inhibition effects at concentrations greater than 0.05 mg/mL, with an average increase of 15.74% against \u003cem\u003eE. coli\u003c/em\u003e compared to IgY. Similarly, the inhibition rate of the IgY-catechin complex against \u003cem\u003eS. aureus\u003c/em\u003e was significantly higher than that of IgY alone, with an increase of 135.8%. These results indicated that the IgY-catechin complex has superior antibacterial effects, suggesting that the binding of IgY with catechins enhances its antibacterial capability.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBacteriostatic rate of IgY, and IgY-catechin complex\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eStrains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003einhibition rate/%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.025 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2 mg/mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIgY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIgY-catechin complex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIgY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIgY-catechin complex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eColumns values with different letters were significantly different(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)。\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\n \u003ch2\u003eResults of Stability of IgY-catechin Complex\u003c/h2\u003e\n \u003cp\u003eTo exclude the effect of catechin on the antibacterial rate of the IgY-catechin complex, IgY and catechin were used as controls to analyze the heat stability of the IgY-catechin complex under different temperature treatments. The results were shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe results showed that under identical conditions, the IgY-catechin complex exhibited the highest inhibition rates against \u003cem\u003eE. coli\u003c/em\u003e. As the temperature increased, the inhibition rates of all three samples decreased, with a significant reduction for the IgY-catechin complex observed above 60\u0026deg;C. The thermal stability of the IgY-catechin complex was lower compared to IgY and catechin, yet it maintained good antibacterial activity at certain temperatures. For \u003cem\u003eS. aureus\u003c/em\u003e, catechin exhibited the highest inhibition rate at each temperature, followed by the IgY-catechin complex, and then IgY. The IgY-catechin complex maintained better antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e at temperatures where IgY alone was less effective, although its thermal stability declined significantly at temperatures above 60\u0026deg;C. At temperatures exceeding 70\u0026deg;C, the inhibition rates of both catechin and IgY against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e declined markedly. The observed changes in antibacterial activity may be attributed to the interaction between catechin and IgY, which alters the secondary structure of the protein macromolecules, exposing more active groups and enhancing antibacterial activity (Jie Liu et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although this structural change leads to reduced thermal stability and earlier loss of antibacterial activity compared to IgY, it does not negate the overall improvement in antibacterial performance of the IgY-catechin complex.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBacteriostatic thermal stability of three substances\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eCondition\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eInhibition rate/%\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgY\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ecatechin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgY-catechin complex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgY\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ecatechin\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIgY-catechin complex\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.87a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.23a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.64a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.36a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.15a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.50a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.99a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.70a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.73a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.30b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.61b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.77a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.32a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.61a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.24c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91.01c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.93c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.21b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.02a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.59b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.98a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.44c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.38d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e70\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.10c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.73b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.67c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.39b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.21d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.48e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.11d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.18b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.69d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.75d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.92e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.13f\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.20g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.51c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.67e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.77e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.64f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.97g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.03a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.39c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.22b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.04a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.97b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.00c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.89c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.27c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.95c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.32b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.25c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.95c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.73c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.43c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.31c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.47b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.17b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.61c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.26c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.87c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.67c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.71b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.55b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.89c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.23c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.69c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.83c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.85a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.52b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.01c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.12b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.46b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.77b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.57a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.64a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.03c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epH10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.83a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.42b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e69.84b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.17c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.37a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.18b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eDifferent letters denote significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e also presents the acid-base Stability of IgY and IgY-catechin complexes The IgY-catechin complex exhibited the highest inhibition rate against \u003cem\u003eE. coli\u003c/em\u003e under identical conditions, with relatively small changes under different pH conditions. Compared to IgY and catechin alone, the IgY-catechin complex demonstrated improved inhibitory acid-base stability against \u003cem\u003eE. coli\u003c/em\u003e. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the inhibition rates of the complex and catechin against \u003cem\u003eS. aureus\u003c/em\u003e were notably higher than that of IgY, following a similar trend as pH changed. The inhibition rate decreased significantly when pH exceeded 8. The superior acid-base stability of the IgY-catechin complex in inhibiting \u003cem\u003eS. aureus\u003c/em\u003e could be attributed to the interaction between IgY and catechin, resulting in a complex that reduces the ionization degree of catechin\u0026rsquo;s hydroxyl groups (Liu. et al., 2023; He. et al., 2022). This interaction likely facilitates the binding of the complex to bacterial cell membranes and lipoproteins, synergistically enhancing its bacteriostatic activity and improving its acid-base stability.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003eResults of MIC, MBC and Growth Curve\u003c/h2\u003e\n \u003cp\u003eMIC and MBC are crucial indicators for evaluating the antibacterial performance of a substance (P. Sruthi, et al., 2023). The MIC for the IgY-catechin complex against \u003cem\u003eE. coli\u003c/em\u003e was determined to be 0.5 mg/mL. For \u003cem\u003eS. aureus\u003c/em\u003e, the MIC was 0.125 mg/mL.\u003c/p\u003e\n \u003cp\u003eAfter confirming the MIC values, MBC data were collected by identifying the lowest concentration of the IgY-catechin complex at which no bacterial growth was observed. The MBC of the complex was found to be 2 mg/mL for \u003cem\u003eE. coli\u003c/em\u003e and 1 mg/mL for \u003cem\u003eS. aureus\u003c/em\u003e. At lower concentrations, bacterial colonies were denser, while higher concentrations resulted in progressively fewer colonies.\u003c/p\u003e\n \u003cp\u003eThe inhibitory ability of the IgY-catechin complex on the bacterium was further determined by measuring the effect of the IgY-catechin complex on the growth curve of the bacterium. As can be seen from Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e (a). When the IgY-catechin complex were added at 0, MIC, and 2 MIC, the growth trends of \u003cem\u003eE. coli\u003c/em\u003e were closer to each other in the 0\u0026ndash;24 h time period. And all of them had obvious growth retardation, logarithmic, and stabilisation phases. However, the time for the growth of the bacteria to the retardation phase was prolonged compared to the non-addition of IgY-catechin complex. The absorbance was considerably lower than that of the untreated group at 2 MIC, which showed that the addition of the IgY-catechin complex exhibited an inhibitory effect on the growth of the bacterium. From Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e (b), \u003cem\u003eS. aureus\u003c/em\u003e growth trends were similar and three phases of a normal growth curve existed when the complex was added at 0, MIC. The growth retardation period of the organisms was extended from 2 h to 10 h at MIC compared to 0. At 2 MIC, the absorbance was basically unchanged and the growth of the microbiological was severely inhibited. After the addition of the IgY-catechin complex, the lag phase of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e was significantly extended, the growth rate was notably reduced, and the overall bacterial count decreased, indicating that the IgY-catechin complex has significant potential in combating bacteria.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003eResults of Nucleic Acid Protein Leakage\u003c/h2\u003e\n \u003cp\u003eNucleic acid and intracellular protein leakage were measured to assess the impact of the complex on the membrane integrity and permeability of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e (Wang et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). The results of the nucleic acid and protein leakage assays are shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eFrom Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e (a), it was observed that the nucleic acid leakage in the control group was minimal and remained relatively stable over four hours. At MIC concentration, the nucleic acid leakage had significantly increased compared to the control group. As time progressed, the nucleic acid leakage gradually rose, indicating that the IgY-catechin complex at MIC concentration had caused some damage to the bacterial cells, leading to nucleic acid leakage. After 2 hours, the nucleic acid leakage tended to stabilize and did not rise significantly, suggesting that the IgY-catechin complex at MIC concentration had a certain bacteriostatic effect, capable of damaging the cell membrane of \u003cem\u003eE. coli\u003c/em\u003e. When the concentration of the IgY-catechin complex reached 2MIC, the nucleic acid leakage was significantly higher than that of the control and MIC groups. The leakage markedly escalated within the first 1 to 2 hours and continued to rise from 2 to 4 hours.\u003c/p\u003e\n \u003cp\u003eAs can be seen in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e (b), the leakage of nucleic acids rised with time and with the amount added under the effect of the IgY-catechin complex. The destructive effect was most obvious in the first 1 h, and the trend and mechanism of action were similar to the trend of \u003cem\u003eE. coli\u003c/em\u003e. Form Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e (b) the leakage of intracellular proteins from \u003cem\u003eS. aureus\u003c/em\u003e was similar to that of Escherichia coli. Under the MIC concentration of the complex, there is a significant increase in protein leakage within the first two hours. At a 2MIC concentration of the complex, the most pronounced leakage occurs within the first hour, after which the leakage does not show a significant increase. The complex also exhibits antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eCatechin analogs\u003c/em\u003e have the ability to interact with components of cell membranes and alter their physical properties (Jun Sato, et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). This capability leads to leakage of cellular contents and interferes with normal cellular metabolism. The mechanism of bacteriostatic action of IgY was to inhibit the growth and reproduction of bacteria by causing cell aggregation (Mahenthiran, R., et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The mechanism of inhibition of the IgY-catechin complex, similar to that of IgY and catechins, which was the inhibition of the growth of the organism through disruption of the cell membrane and the leakage of intracellular genetic material. The cell membrane plays a critical role in microorganisms like \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e The cell membrane was the dividing line between the internal and external environments of the microbial cell (Dylan Gerard Ryan, et al., 2023), and had the role of screening and defense. The cell membrane of \u003cem\u003eE. coli\u003c/em\u003e was highly resistant to osmotic pressure(Emma K. Eriksson, et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), enabling \u003cem\u003eE. coli\u003c/em\u003e to maintain cellular stability in harsh environments. The biofilm of \u003cem\u003eS. aureus\u003c/em\u003e is also a key structure in resisting antibiotics and other antimicrobial substances(Paul Payel, et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). If it is possible to disrupt a biofilm such as a cell membrane, it can easily result in the death of the microorganism.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eResults of Fluorescence Inverted Microscope\u003c/h3\u003e\n\u003cp\u003ePI dye can enter the bacterium when the cell membrane of the bacterium is disrupted or the bacterium had died(Chiaraviglio, et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The \u003cem\u003ePI dye\u003c/em\u003e chimerises with the DNA of the bacterium and then showed a red fluorescence under an inverted fluorescence microscope. When the cell membrane of the bacterium was intact, the PI dye was not able to pass through the cell membrane(Yawei Ning, et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, the degree of growth inhibition of the bacterium was determined by observing the amount of red fluorescence in the image.\u003c/p\u003e\n\u003cp\u003e(abc was plotted against Escherichia coli at a sample concentration of 0 MIC 2 MIC, def was plotted against Staphylococcus aureus at a sample concentration of 0, MIC, and 2 MIC, respectively.)\u003c/p\u003e\n\u003cp\u003eThe impact of the IgY-catechin complex on the cell membranes of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e was further assessed through observation using inverted fluorescence microscopy. As depicted in Fig.\u0026nbsp;10, negligible fluorescence was detected in the \u003cem\u003eE. coli\u003c/em\u003e control group. Conversely, in the MIC-treated group, a noticeable increase in red fluorescence intensity was observed, with a substantial quantity of red fluorescence becoming apparent at a sample concentration of 2 MIC. Similarly, in the case of \u003cem\u003eS. aureus\u003c/em\u003e, minimal fluorescence was observed in the control, while the intensity of red fluorescence progressively heightened with increasing sample concentration. These findings indicated that the IgY-catechin complex adversely affected the cell membrane of the bacteria, leading to a significant inhibition of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e growth, ultimately culminating in bacterial death. These observations were consistent with the results of previous studies on the influence of IgY-catechin complex on bacterial cell membrane rhabdomeres.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003ePolyphenols, when combined with proteins, alter the structure and properties of the proteins. Molecular dynamics simulations confirmed the interactions between IgY and catechin, highlighting key amino acids that stabilize the protein complex. The RMSF and RMSD analyses revealed significant conformational changes and energy fluctuations during the binding process, ultimately leading to a stable equilibrium state of the IgY-catechin complex. Catechin binds to residues PHE503, THR501, THR505, GLU511 of IgY respectively, and an increase in hydrogen bond content within the complex, a 5.48% increase in α-helix content, and a decrease in random coil content. Molecular docking techniques predicted the interaction sites and binding forms between catechins and proteins. FT-IR and CD studies of the complex's secondary structure revealed changes, including an increase in α-helix content and the formation of hydrogen bonds, consistent with software simulation results. Hydrogen bonds were identified as the primary binding force between catechins and IgY, indicating a more stable combination, preventing the complex's structure from being easily disrupted. Subsequent stability and antibacterial tests showed that the complex has lower thermal stability compared to IgY, and that pH has minimal impact on its antibacterial performance. This suggests that the complex has potential applications in antibacterial fields related to pH. The MIC of the complex against \u003cem\u003eE. coli\u003c/em\u003e was 0.5 mg/mL and 2 mg/mL, while the MBC against \u003cem\u003eS. aureus\u003c/em\u003e was 0.125 mg/mL and 1 mg/mL. After the addition of the IgY-catechin complex, the lag phase of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e was significantly extended, the growth rate was notably reduced, and the overall bacterial count decreased, indicating that the IgY-catechin complex has significant potential in combating bacteria. This provides strong evidence for its application as an antimicrobial agent. Nucleic acid-protein leakage analysis and fluorescence microscopy results indicated that the complex inhibits bacterial growth by disrupting the cell membrane, leading to the release of intracellular genetic material. It is plausible to envision the further expansion of its application in food processing as natural bacteriostatic agents, as well as in a broader spectrum of applications in future investigations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of Interest\u003c/strong\u003e \u003cp\u003eThe Authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eMajor Science and Technology Projects in Henan Province (No. 221100110500); Henan Province Science and Technology Research and Development (242102110092); National Key R\u0026amp;D Program of China (2022YFF1101600); Research Funding for Distinguished Professor at Henan University of Science and Technology (13510004); the Leading Talent Program for Science and Technology Innovation in Central China (234200510020)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLili Liu: Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Yanli Wang: Data curation, Formal analysis, Writing \u0026ndash; original draft, Conceptualization, Validation. Mengjun Zhang Writing \u0026ndash; review \u0026amp; editing, prepared figures. Weiwei Cheng: prepared figures, Writing \u0026ndash; review \u0026amp; editing. Ding: Writing \u0026ndash; review \u0026amp; editing. Jialiang He: Writing \u0026ndash; review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAigbogun, I. E., Bakare, A. O., Evbuomwan, S. A., Babatunde, O. S., Harrison, I., Christiana, A., \u0026amp; Anita, D. O. (2023). 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Changes in structural and functional properties of globulin\u0026ndash;polyphenol complexes in mung beans: Exploration under different interaction ratios and heat treatment conditions. \u003cem\u003eInternational Journal of Food Science and Technology\u003c/em\u003e, \u003cem\u003e57\u003c/em\u003e, 1920\u0026ndash;1935. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ijfs.15180\u003c/span\u003e\u003cspan address=\"10.1111/ijfs.15180\" 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":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Yolk immunoglobulin, catechin, molecular dynamics simulation, antibacterial activity, mechanism","lastPublishedDoi":"10.21203/rs.3.rs-5017669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5017669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe binding of proteins with polyphenols can alter the properties of the proteins, thereby expanding their application scenarios. Yolk immunoglobulin (IgY), an easily obtainable protein from eggs, undergoes property changes upon binding with catechin, which is significant for broadening the application of IgY. This study investigates the binding modes, structural changes, stability, and antibacterial properties of the IgY-catechin complex using computational chemistry, spectroscopy, and antibacterial assays. Molecular dynamics simulations analysis revealed that catechin binds to residues PHE503, THR501, THR505, GLU511 of IgY respectively. Fourier transform infrared spectroscopy(FT-IR) and circular dichroism(CD) displayed an increase in hydrogen bond content within the complex, a 5.48% increase in α-helix content, and a decrease in random coil content. Scanning electron microscopy (SEM) showed that the complex had a smoother and more regular surface. The IgY-catechin complex exhibited improved acid-base stability but slightly reduced thermal stability compared to IgY. Fluorescence inverted microscopy and nucleic acid-protein leakage assays indicated that the complex disrupted the cell membranes of both \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e), leading to the leakage of intracellular genetic material. These findings confirmed that the interaction between catechin and IgY altered the protein structure of IgY, enhanced its bacteriostatic ability. This research provides a new approach to extending the application range of IgY and improving its comprehensive utilization.\u003c/p\u003e","manuscriptTitle":"Polyphenol-Driven Structural Alterations and Antibacterial Potency of the IgY-Catechin Complex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 10:47:37","doi":"10.21203/rs.3.rs-5017669/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-09-03T08:47:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-02T22:51:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Food and Bioprocess Technology","date":"2024-09-02T10:47:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"food-and-bioprocess-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Food and Bioprocess Technology](https://www.springer.com/journal/11947)","snPcode":"11947","submissionUrl":"https://submission.nature.com/new-submission/11947/3","title":"Food and Bioprocess Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2582eb3a-8182-40ec-a26d-e885326beede","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-04T10:47:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-04 10:47:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5017669","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5017669","identity":"rs-5017669","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}