Fabrication and antibacterial properties of self-assembled lysozyme nanofilms induced by reduced glutathione

Abstract

This study developed a self-assembled lysozyme nanofilm at the air-water interface using reduced glutathione (GSH) as an inducer, demonstrating broad-spectrum antibacterial activity and tunable functionality. Through thiol-disulfide exchange, GSH reduces the disulfide bonds in lysozyme, leading to protein unfolding and conformational transition from α-helix to β-sheet-rich structures. The assembly process, along with film morphology and surface properties, can be effectively modulated by varying lysozyme concentration and environmental pH, enabling the rational design of surface charge and hydrophobicity to optimize antibacterial performance. The resulting nanofilm demonstrates significant antibacterial efficacy against both Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria), attributed to a synergistic mechanism involving inherent enzymatic activity and membrane disruption mediated by exposed hydrophobic domains and positive surface charges. These findings provide a feasible strategy for designing eco-friendly protein-based antibacterial materials with potential applications in food packaging, biomedical coatings, and functional surfaces.

Graphical abstract

Highlights

• •Green self-assembly of lysozyme nanofilms using biocompatible GSH.
• •Tunable morphology, structure, and function via controlled pH and concentration.
• •Broad-spectrum efficacy against both Gram-positive and Gram-negative bacteria.
• •Enhanced efficacy via membrane disruption by hydrophobic and cationic surfaces.

1. Introduction

Protein-based packaging materials have attracted considerable interest due to their renewable origin, biodegradability, and food-grade status (Zhen et al., 2022). Certain proteins, such as lysozyme, exhibit inherent antimicrobial activity, providing a basis for developing natural antibacterial packaging (Wu et al., 2019). However, advancing these materials toward practical application faces notable challenges. Conventional processing often relies on synthetic chemicals or harsh conditions, which conflicts with the green and sustainable principles. Moreover, achieving robust and precisely tunable antibacterial performance in protein-based materials remains difficult, as their functional properties are inherently linked to complex structural states that are hard to control (Fang et al., 2025). Therefore, there is an urgent need for green fabrication strategies that can avoid hazardous reagents while enabling the precise regulation of protein assembly into structures with predictable and enhanced functions, particularly antibacterial efficacy.

Protein self-assembly, especially into amyloid-like structures, offers a promising route to meet this need. Protein structures are diverse, ranging from native and unfolded states to partially folded intermediates (Cao and Mezzenga, 2019; Dobson, 2004; Han et al., 2023). Through intermolecular interactions, unfolded or partially folded proteins can self-assemble into various aggregates, among which amyloid-like assemblies have attracted significant attention (Su et al., 2026). These assemblies are characterized by a cross-β-sheet-rich core structure, akin to that found in amyloid fibrils, but they can adopt diverse morphologies beyond the classical linear fibrils, including oligomers, particles, and films (Ren et al., 2024). Although protein amyloid fibrillation was initially regarded as a pathological phenomenon due to its association with neurodegenerative diseases, it is now widely recognized that amyloid and amyloid-like assemblies are a fundamental and evolutionarily conserved protein fold that serves diverse functional roles in nature (Su et al., 2026). This functional amyloid paradigm has inspired the engineering of amyloid-based materials with exceptional mechanical stability, biocompatibility, and tunable functionality for beneficial applications. The common β-sheet-rich architecture endows these amyloid-like materials with high mechanical strength and stability. Notably, when such assembly occurs at the air-water interface, the well-ordered, two-dimensional layered films are formed (Su et al., 2026). This interface-mediated, non-fibrillar assembly pathway provides a unique opportunity to fabricate protein-based films with precisely tunable nanostructures and surface properties.

Lysozyme is a natural antibacterial protein widely present in biological fluids (e.g., egg white, saliva, and tears) and is extensively utilized in the food, pharmaceutical, and cosmetic industries (Chen et al., 2022). It specifically hydrolyzes the β-(1-4) glycosidic bond in peptidoglycan, thereby exhibiting significant antibacterial activity against Gram-positive bacteria (Wu et al., 2019). Lysozyme consists of 129 amino acids with a molecular weight of 14.3 kDa, stabilized by four disulfide bonds that confer robust conformational stability (Huang et al., 2025). This inherent rigidity allows lysozyme to maintain its antibacterial activity even after adsorbing onto charged surfaces, making it a promising candidate for constructing antibacterial nanocomposites (Bouaziz et al., 2017).

Studies have demonstrated that lysozyme can undergo conformational transitions under harsh conditions (e.g., high temperature, extreme pH) or in the presence of strong chemical reducing agents such as tris(2-carboxyethyl) phosphine (TCEP) and dithiothreitol (DTT), promoting the formation of β-sheet-rich aggregates or amyloid fibrils (Chen et al., 2022; Guo et al., 2025; Ren et al., 2024). However, these strategies depend on synthetic reagents that raise safety and environmental concerns, limiting their suitability for food and biomedical applications.

To address these limitations, we turn to a green and biocompatible alternative, reduced glutathione (GSH). Our group previously observed that GSH can induce the self-assembly of lysozyme into macroscopically protein films at the air-water interface (Guo et al., 2023). This presents a promising pathway for creating protein-based materials under mild conditions. However, the detailed mechanism by which GSH influences the conformational changes and interfacial assembly behavior of lysozyme, as well as its subsequent effect on antibacterial activity, remains unclear. Therefore, this study aims to systematically investigate the GSH-induced conformational evolution of lysozyme and the regulatory effect on the surface properties and antibacterial performance of the self-assembled films. We focus on establishing clear relationships between processing parameters, film properties, and antibacterial function. The findings are expected to facilitate the development of a green, efficient and controllable protein-based antibacterial packaging material, offering a novel strategy for the high-value utilization of natural antibacterial agents and advanced food active packaging.

2. Materials and methods

2.1. Materials

Lysozyme (from egg white, 20000 U/mg) and GSH were supplied by Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The fluorescent probes N-(1-pyrenyl) maleimide (NPM) and 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer and Congo red were obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). Phosphate buffered saline (PBS) and glutaraldehyde fixative solution (2.5%) were obtained from Beijing Lanjieke Technology Co., Ltd. (Beijing, China). Luria-Bertani (LB) nutrient agar and LB broth medium were sourced from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, China). The Calcein-AM/PI Cell Viability and Cytotoxicity Assay Kit was acquired from Beyotime Biotechnology (Shanghai, China). All other reagents, such as sodium hydroxide (NaOH), were procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were of analytical grade and used without further purification.

2.2. Preparation of lysozyme nanofilms

Lysozyme and GSH were separately dissolved in 10 mM HEPES buffer (pH 6.0) to prepare stock solutions with lysozyme concentrations of 1.75, 3.5, 7, and 14 mg/mL, and a fixed GSH concentration of 7 mg/mL. The pH of the GSH solutions was adjusted to 6, 8, 10, and 12 using 4 M NaOH. Subsequently, lysozyme solutions at different concentrations were mixed with GSH solutions at various pH values in equal volume ratios under gentle stirring, and the mixtures were allowed to react under quiescent conditions.

The nanofilms were prepared according to the previously reported method (Zhu et al., 2025). Briefly, the pH-adjusted GSH solution and lysozyme solution were combined in equal volumes and mixed thoroughly. A certain volume of the mixture was pipetted onto a clean glass coverslip and incubated at 30 °C in a humid environment for 3 h. During incubation, phase separation occurred, leading to the formation of a colorless, transparent, two-dimensional protein film at the air-water interface. The resulting film was carefully transferred onto the surface of deionized water, where it floated and could be subsequently collected and deposited onto another clean coverslip. Finally, the film was air-dried for further use.

2.3. The behavior and structural changes of lysozyme in solution

2.3.1. The kinetics of thiol exposure of lysozyme induced by GSH

The exposure of thiol groups in lysozyme molecules was monitored using NPM as a fluorescent probe (Guo et al., 2025). Briefly, 1.4 mL of lysozyme solution was mixed with 200 μL of NPM solution (15 mM) and incubated in the dark for 10 min. Then, an equal volume of GSH solution was added, and the mixture was immediately transferred to a cuvette after thorough mixing. Time-dependent changes in fluorescence intensity were monitored using a fluorescence spectrophotometer (F-7000, Hitachi) with the excitation wavelength set at 330 nm and the emission wavelength at 380 nm. Both excitation and emission slit widths were set to 10 nm. The control groups included native lysozyme solution and GSH solution, each supplemented with NPM alone.

2.3.2. Analysis of hydrophobic group exposure

ANS was employed as a fluorescent probe to detect the exposure of hydrophobic regions during protein unfolding (Seth et al., 2023). Briefly, 1.4 mL of lysozyme solution was mixed with 200 μL of ANS solution (300 μM) and incubated in the dark for 10 min. An equal volume of GSH solution was then added to the mixture. After thorough mixing, the solution was immediately transferred to a cuvette for fluorescence measurement. Fluorescence emission spectra were acquired using a fluorescence spectrophotometer with the excitation wavelength set at 355 nm and the emission wavelength at 480 nm. Both the excitation and emission slit widths were set to 5 nm.

2.3.3. Measurement of particle size and zeta potential

The surface charge and hydrodynamic diameter of colloidal particles were determined using a particle size and zeta potential analyzer (Zetasizer Nano-ZS, Malvern). The lysozyme solution was mixed with GSH solution at a 1:1 vol ratio and allowed to stand for 30 min prior to measurement. All operations were performed under a constant temperature of 25 °C.

2.3.4. Changes in the secondary structure of lysozyme in the bulk phase

The changes in the secondary structure of the lysozyme were analyzed using far-ultraviolet circular dichroism (CD) spectroscopy as described previously (Ren et al., 2024). Equal volumes of lysozyme solution and GSH solution were mixed and allowed to react for 3 h. The resulting mixture was dialyzed (molecular weight cutoff of 1000 Da) against deionized water to remove GSH. The dialyzed sample was diluted to 0.1 mg/mL and loaded into a quartz cuvette with a 0.1 cm optical path length. The CD spectra were acquired on a J-1500 spectropolarimeter (Jasco) over a wavelength range of 190-240 nm with a bandwidth of 1 nm. The native lysozyme solution was used as the control group.

Fourier transform infrared (FT-IR) spectroscopy (Nicolet iS10, Thermo Fisher) was employed to analyze the changes in the secondary structure of lysozyme. The dialyzed sample was freeze-dried to obtain a powder, and the secondary structure of the unfolded lysozyme in the bulk phase was characterized using an attenuated total reflectance (ATR) FT-IR spectrometer over the wavenumber range of 1000-4000 cm⁻¹. The amide Ⅰ band of lysozyme was deconvoluted and subjected to peak fitting using PeakFit software. Native lysozyme was used as a control to evaluate structural alterations.

2.4. Characterization of the structure and physicochemical properties of lysozyme nanofilms

2.4.1. Analysis of the secondary structure of lysozyme films

Lysozyme solution was mixed with an equal volume of GSH solution and reacted for 3 h to induce the formation of a nanofilm. The resulting film was transferred onto a quartz substrate, which was then placed into a CD sample cell with an optical path length of 0.1 cm for measurement. The CD spectra were acquired with the following settings: wavelength range of 190-240 nm and a bandwidth of 1 nm.

The FT-IR spectrum of the lysozyme film was measured using an ATR-FTIR spectrometer in the wavenumber range of 1000-4000 cm⁻¹. The amide Ⅰ band of lysozyme was deconvoluted and subjected to peak fitting using PeakFit software.

2.4.2. Analysis of the amino acid composition in lysozyme films

The lysozyme films were prepared according to the aforementioned method. Approximately 50 layers of the films were stacked onto a glass coverslip to enhance the Raman signal, and spectra were collected over 400-4000 cm⁻¹. Peak fitting of the Raman spectra was performed using PeakFit software to analyze changes in the amino acid composition of the protein.

2.4.3. Analysis of the morphology and surface roughness of lysozyme films

The lysozyme films were prepared and transferred onto a silicon wafer substrate. Topographic imaging was performed using an atomic force microscope (AFM, Dimension Icon, Bruker) operating in tapping mode (Zhu et al., 2025). The scanning parameters were set as follows: the resonance frequency was 100-300 kHz, and the scan rate was 100 Hz. The obtained images were processed using NanoScope Analysis software, and the surface roughness was calculated and expressed as the root mean square (RMS) value.

2.4.4. Determination of the surface hydrophobicity of lysozyme films

The lysozyme films were prepared and transferred onto coverslips for contact angle measurement. A 3 μL droplet of ultrapure water was dispensed onto the film surface at a rate of 5 μL/s using a microsyringe. The droplet profile was fitted using the Laplace-Young equation to calculate the contact angle values.

2.5. Characterization of the antibacterial properties of lysozyme nanofilms

2.5.1. Determination of bacterial inhibition rate

The antibacterial activity of the lysozyme films was evaluated using a colony counting method with S. aureus and E. coli as model strains (Na et al., 2023). First, the revived strains were streaked onto LB agar plates and incubated at 37 °C for 16 h. Single colonies were then inoculated into LB broth and cultured in a shaking incubator at 37 °C until the logarithmic growth phase was reached. The bacterial suspension was adjusted to a concentration of 10⁸ CFU/mL using PBS, and further diluted to 10⁵ CFU/mL with LB broth for subsequent use. The glass coverslips coated with the lysozyme films were sterilized under ultraviolet light for 20 min.

The sterilized samples were placed in a six-well plate, and 20 μL of the diluted bacterial suspension was added to the surface of each sample. To ensure full contact, the inoculum was covered with another sterile coverslip. After incubation at 37 °C for 2 h, the samples were taken out and gently washed with 2 mL of PBS buffer. The bacterial suspension was collected and subjected to 10-fold serial dilution. A volume of 50 μL of the diluted suspension was spread onto LB agar plates and incubated at 37 °C for 16 h for colony counting. The sterile coverslips served as the negative control. The bacterial inhibition rate was calculated using the following formula:

$$R=\left(1-\frac{N_1}{N}\right)\times 100\%$$

where R is the bacterial inhibition rate (%), N is the average number of colonies in the control group, N₁ is the average number of colonies in the sample group.

2.5.2. Observation of bacterial morphology

The lysozyme films were prepared and transferred onto cell climbing slides according to the aforementioned method, followed by ultraviolet sterilization for 20 min. Overnight-cultured S. aureus and E. coli were diluted to 5 × 10⁵ CFU/mL. The slides were placed in a 48-well plate, and 500 μL of bacterial suspension was added to each well. After incubation at 37 °C for 24 h, the samples were collected and fixed with 2.5% glutaraldehyde solution at 4 °C for 4 h, followed by dehydration through an ethanol gradient series (30%, 50%, 70%, 90%, and 100%). The sterile cell climbing slides served as the control group. The samples were dried, sputter-coated with gold, and observed under a field emission scanning electron microscope (SEM, SU8010, Hitachi) to examine morphological changes of the bacteria.

2.5.3. Bacterial live/dead staining

The lysozyme films were prepared and sterilized as described above. The bacterial suspension was diluted to 5 × 10⁵ CFU/mL and co-cultured with the samples for 24 h. After incubation, the samples were gently washed twice with PBS. Then, 200 μL of Live/Dead BacLight bacterial viability staining solution (containing Calcein-AM and PI) was added. The reaction was carried out at 37 °C in the dark for 30 min. After staining, the samples were rinsed again with PBS to remove background fluorescence. Sterile cell climbing slides were used as the control group, and the fluorescent staining of bacteria was observed under a laser scanning confocal microscope (CLSM, Leica SP8) to evaluate membrane integrity and bactericidal efficacy.

2.6. Statistical analysis

All experiments were performed in three independent replicates. Statistical significance was determined by one-way analysis of variance (ANOVA) and two-way ANOVA. The results are expressed as mean ± standard deviation, and all statistical analyses were conducted using Origin 2021 and GraphPad Prism 9.5 software.

3. Results and discussion

3.1. The behavior and structural changes of lysozyme in solution

The thiol-disulfide exchange reaction between GSH and native lysozyme was initiated by mixing their buffer solutions. This reaction cleaved one disulfide bond in the protein, releasing free thiol groups that were subsequently detected using NPM fluorescence labeling. NPM reacts rapidly and specifically with free thiol groups to form highly fluorescent derivatives, resulting in a significant increase in fluorescence intensity at 380 nm (Xu et al., 2020). As shown in Fig. S1, in the presence of both GSH and lysozyme, the fluorescence intensity increased markedly over time, indicating disulfide bond cleavage and thiol groups exposure. In contrast, control groups containing only native lysozyme or only GSH exhibited no significant fluorescence change after incubation with NPM. This confirms that the fluorescence enhancement specifically resulted from the reductive cleavage of disulfide bonds in lysozyme by GSH, rather than from intrinsic fluorescence or non-specific interactions. These results demonstrate that GSH effectively induces disulfide bond reduction in lysozyme, promoting protein unfolding and exposure of reactive thiol groups.

ANS, as a fluorescent probe highly sensitive to hydrophobic microenvironments, was used to detect the exposure of hydrophobic regions during protein unfolding (Liu et al., 2025). Notably, even native lysozyme contains hydrophobic residues within its folded structure, which can partially bind to ANS and yield a measurable baseline fluorescence signal. In contrast, the small hydrophilic peptide GSH provides minimal ANS binding sites, resulting in a very low fluorescence background. As shown in Fig. 1A–D, the fluorescence intensity at 475 nm of the lysozyme-GSH mixture increased rapidly over 120 min. By contrast, control systems containing only lysozyme or only GSH with ANS showed no significant fluorescence change. This indicates that GSH effectively induces partial unfolding of lysozyme, destabilizing its native conformation and exposing originally buried hydrophobic residues to the solvent, thereby providing numerous specific binding sites for ANS. The fluorescence enhancement was concentration-dependent, with higher lysozyme concentrations yielding stronger signals (Fig. 1E and S2A), suggesting that more protein units are available for self-assembly, which exposes more hydrophobic regions and intensifies the ANS fluorescence. Furthermore, environmental pH significantly influenced this process (Fig. 1F and S2B). The fluorescence increased was more markedly under higher pH conditions, indicating that an alkaline environment facilitates lysozyme unfolding and the exposure of its hydrophobic regions.

Fig. 1.

ANS fluorescence spectra of (A) native lysozyme, (B) reduced glutathione (GSH), and (C) lysozyme treated with GSH. (D) Time-dependent changes in ANS fluorescence intensity of native lysozyme, GSH, and GSH-treated lysozyme. ANS fluorescence characterization of (E) lysozyme at different concentrations after mixing with GSH, and (F) GSH-treated lysozyme at different pH values.

Hydrodynamic particle size and zeta potential were measured for the lysozyme-GSH system to characterize its self-assembly behavior in solution. As shown in Fig. 2A–B, the hydrodynamic diameter of the assemblies increased significantly with lysozyme concentration. This is because a higher protein concentration leads to more unfolded molecules, exposing more hydrophobic regions. The resulting enhanced hydrophobic interactions promoted intermolecular aggregation, ultimately forming larger assemblies. Meanwhile, changes in surface charge were reflected in the zeta potential (Fig. 2C). At a low lysozyme concentration (1.75 mg/mL), the zeta potential was negative, likely due to the adsorption of anions from the HEPES buffer onto the protein surface, which partially masked its positive charges. As the lysozyme concentration increased, the zeta potential exhibited an upward trend, driven primarily by the inherent positive charge of lysozyme. The assembly behavior also varied markedly with pH (Fig. 2D–F). At pH 10 and 12, near the isoelectric point (pI) of lysozyme, reduced protonation and ionization decreased electrostatic repulsion, facilitating the formation of larger aggregates (Wang et al., 2010). Notably, a negative zeta potential was observed at pH 12, suggesting that the alkaline environment induced deprotonation of basic residues in lysozyme, thereby reducing its net positive charge and resulting in an overall negative surface charge. In summary, the self-assembly of the lysozyme-GSH system is governed by both hydrophobic interactions and electrostatic forces, where hydrophobic interactions promote the formation and growth of aggregates, while the electrostatic environment significantly influences their size and stability.

Fig. 2.

(A) Hydrodynamic diameter, (B) size distribution, and (C) zeta potential of lysozyme at different concentrations after mixing with GSH. (D) Hydrodynamic diameter, (E) size distribution, and (F) zeta potential of lysozyme after mixing with GSH at different pH values. Different letters indicate statistically significant differences among samples (p < 0.05).

The CD spectroscopy was used to analyze changes in the secondary structure of lysozyme. As shown in Fig. 3A, native lysozyme exhibited characteristic signals of α-helix structure, with distinct negative peaks near 208 nm and 222 nm (Wu et al., 2019). However, upon addition of GSH, the conformation of lysozyme underwent significant changes. Specifically, the characteristic signal of the α-helix structure markedly decreased, while a negative peak emerged near 216 nm and a positive peak appeared around 198 nm, which were both typical peaks of the β-sheet structure (Guo et al., 2025; Tu et al., 2021). These results indicated that lysozyme underwent a conformational transition from a predominantly α-helix structure to a β-sheet-rich configuration in the presence of GSH. This structural rearrangement is closely associated with the self-assembly behavior of the protein at the air-water interface and represents a critical step in the formation of nanofilms. The increase in β-sheet content, which is generally associated with ordered stacking and fibrillar aggregation, further confirms that structural reorganization occurs at the interface, thereby promoting film formation.

Fig. 3.

(A) Far-UV CD spectra, (B) FT-IR spectra, (C) Deconvoluted amide I band in FT-IR spectra, and (D) Secondary structure content analysis of native lysozyme, GSH-treated lysozyme, and lysozyme films.

3.2. The structure and physicochemical properties of lysozyme nanofilms

The secondary structure of the self-assembled lysozyme films was characterized by CD and FT-IR spectroscopy. The CD spectra revealed a significant decrease in α-helix content and a pronounced increase in β-sheet-related signals in the films compared to native lysozyme, indicating a conformational transition from α-helix to β-sheet (Fig. 3A).

FT-IR spectroscopy further corroborated this structural reorganization (Fig. 3B). An absorption band observed at 1624 cm⁻¹ in the amide I region arises from C=O stretching vibrations of the peptide backbone and is highly sensitive to changes in secondary structure (Zeng et al., 2024). Additionally, a strong absorption band near 1516 cm⁻¹ was attributed to the amide II band, primarily arising from N-H bending and C-N stretching vibrations (Asgharzadeh et al., 2025). To resolve overlapping vibrational modes, the amide I band was deconvoluted and fitted with Gaussian curves (Fig. 3C–D). The fitting results clearly indicated a significant increase in β-sheet content accompanied by a corresponding decrease in α-helix, consistent with the conclusions drawn from CD spectroscopy. Quantitative analysis revealed that the β-sheet content increased from 22% to 41% during the self-assembly of native lysozyme into the film, confirming that the formation of β-sheet-rich structures at the expense of α-helix reduction is a key molecular event in the film formation process.

To ascertain whether these β-sheet-rich structures possess amyloid-like characteristics, Congo red staining was performed. The resulting films exhibited the characteristic apple-green birefringence under polarized light (Fig. S3), a diagnostic feature of amyloid-like assemblies with cross-β-sheet architecture (Chibh et al., 2024). This result, combined with the spectroscopic evidence above, confirms the amyloid-like nature of the films.

Raman spectroscopy further revealed significant changes in amino acid microenvironments and chemical bonds during the transition of lysozyme from its native state to self-assembled nanofilms (Fig. 4). Compared with native lysozyme, the characteristic peak near 500 cm⁻¹ attributed to disulfide bond (S-S) stretching vibrations was significantly weakened in the film samples. This confirms that GSH successfully cleaved the intramolecular disulfide bonds and induced protein unfolding (Na et al., 2023). The enhanced Raman peaks at 760 cm⁻¹, 830 cm⁻¹, and 1003 cm⁻¹ were assigned to tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues, respectively (Fig. 4A–B). These changes indicates that these originally buried hydrophobic and reactive residues became progressively exposed on the molecular surface during unfolding and reassembly (Ren et al., 2024). The spectral bands at 1340 cm⁻¹ and 1360 cm⁻¹ originate from Fermi resonance within the indole ring of Trp. Notably, the intensity ratio I₁₃₄₀/I₁₃₆₀ is closely related to the hydrophobicity of the microenvironment surrounding the Trp residues (Chen et al., 2023). In the lysozyme nanofilms, this ratio increased with higher lysozyme concentration and pH (Fig. 4C–D), suggesting a change in the local environment around the indole rings and further confirming the increased exposure of Trp residues during film formation.

Fig. 4.

Raman spectra of (A) native lysozyme and lysozyme films formed at different lysozyme concentrations, and (B) native lysozyme and lysozyme films formed at different pH values. I₁₃₄₀/I₁₃₆₀ intensity ratio of (C) native lysozyme and lysozyme films formed at different lysozyme concentrations, and (D) native lysozyme and lysozyme films formed at different pH values. Different letters indicate statistically significant differences among samples (p < 0.05).

The formation of lysozyme nanofilms is based on interfacial aggregation and ordered accumulation of protein particles, following an assembly pathway of rapid nucleation and progressive fusion that ultimately yields a continuous, dense film (Tao et al., 2023). The effects of different preparation conditions on the morphology and surface roughness of the films were investigated through AFM analysis. First, the influence of lysozyme concentration was investigated. AFM images (Fig. 5A–B) indicated that lysozyme rapidly self-assemble at the air-water interface into a continuous coating composed of tightly packed oligomers. As the concentration increased from 1.75 to 14 mg/mL, the RMS roughness increased from 4.0 nm to 22.2 nm, demonstrating that higher protein concentrations promote rapid formation and growth of oligomers in solution. The resulting larger aggregates, upon interfacial accumulation and fusion, produce films with more pronounced surface undulations and higher roughness. The effect of pH was also studied (Fig. 5C–D). As the pH increased from 6 to 12, the film roughness decreased significantly, with RMS values decreasing from 12.8 nm to 2.2 nm. This can be attributed to the reduced electrostatic repulsion under higher pH conditions. The decrease in net surface charge not only attenuates intermolecular electrostatic repulsion but also enhances the relative contribution of hydrophobic interactions, as charge screening allows hydrophobic domains to associate more readily. These conditions promote the formation of uniform and densely packed oligomeric structures (Guo et al., 2023), and improve the surface activity and interfacial spreading of the protein (Norde, 2008). Consequently, the oligomers exhibit improved fusion capability when aligned at the interface, leading to a highly ordered packing arrangement and ultimately resulting in a smooth, continuous nanofilm.

Fig. 5.

(A) AFM topographic images of lysozyme films formed at different lysozyme concentrations. (B) Effect of lysozyme concentration on the surface roughness of the lysozyme films. (C) AFM topographic images of lysozyme films formed at different pH values. (D) Effect of pH on the surface roughness of the lysozyme films. Different letters indicate statistically significant differences among samples (p < 0.05). Scale bar = 1 μm.

The surface wettability of lysozyme nanofilms was characterized by contact angle measurements to evaluate the influence of preparation conditions. As shown in Fig. 6A–B, the film exhibited a contact angle of approximately 73°, indicating moderate hydrophilicity (Xu et al., 2020). Furthermore, the contact angle of the films was not significantly altered across different lysozyme concentrations, suggesting that although concentration affects oligomer packing density and film roughness, the consistent chemical composition and intrinsic wettability of the oligomers lead to stable macroscopic wetting behavior. In contrast, environmental pH had a pronounced effect on the surface wettability of the films (Fig. 6C–D). At pH 6 and 8, the films exhibited strongly hydrophilic, with contact angles below 30°. As the pH approached the pI of lysozyme, the contact angle significantly increased to 68° and 72°. This trend is attributed to pH-dependent changes in protein surface charge. Under pH conditions far from the pI, lysozyme carries a high net charge, resulting in strong hydrophilicity. In contrast, near the pI, the reduced net charge promotes exposure of hydrophobic residues, thereby enhancing the overall hydrophobicity of the film.

Fig. 6.

Contact angles of (A-B) lysozyme films formed at different lysozyme concentrations, and (C-D) lysozyme films formed at different pH values. Different letters indicate statistically significant differences among samples (p < 0.05).

3.3. The antibacterial properties of lysozyme nanofilms

The antibacterial properties of lysozyme-based films prepared under different conditions were evaluated using S. aureus (ATCC 6538, Gram-positive bacteria) and E. coli (ATCC 25922, Gram-negative bacteria) as model strains. The antibacterial activity was first assessed by the colony counting assay (Fig. 7). The results showed a pronounced concentration-dependent effect against S. aureus. With increasing lysozyme concentration, the inhibition rate rose significantly, reaching 76.67% against S. aureus and 69.27% for E. coli at the highest concentration (Fig. 7B). This difference aligns with the innate antibacterial mechanism of native lysozyme, which specifically hydrolyzes β-1,4-glycosidic bonds in the peptidoglycan layer of Gram-positive bacteria. In contrast, Gram-negative bacteria exhibit stronger resistance to native lysozyme due to the presence of an outer membrane (De France, Kummer, Ren, Campioni and Nyström, 2020; Wu et al., 2019). Notably, the self-assembled films still exhibited considerable antibacterial activity against E. coli, suggesting a mechanism beyond enzymatic hydrolysis alone. Based on the experimental results showing that the potential increases with the concentration (Fig. 2C), it can be inferred that the enriched positive charges promote electrostatic adhesion to the negatively charged bacterial membranes, leading to membrane destabilization and contributing to the observed bactericidal effect against E.coli.

Fig. 7.

(A) Colony counts and (B) corresponding bactericidal rates of S. aureus and E. coli after 2 h of contact with lysozyme films formed at different lysozyme concentrations. (C) Colony counts and (D) corresponding bactericidal rates of S. aureus and E. coli after 2 h of contact with lysozyme films formed at different pH values. ∗∗ and ∗∗∗∗ indicate p < 0.01 and 0.0001, respectively.

Furthermore, the antibacterial activity of the films exhibited a trend of initial decrease followed by increase as the environment pH rose (Fig. 7C–D). At low pH, the film surface is enriched with positive charges, facilitating antibacterial action primarily through electrostatic adsorption and physical disruption of the bacterial membrane (Abouhmad et al., 2017). As pH increases, although the net charge decreases, more hydrophobic groups become exposed. This progressive hydrophobic exposure is evidenced by Raman spectroscopy, which showed enhanced signals for aromatic residues in films formed at higher pH (Fig. 4), and is further corroborated by the macroscopic increase in water contact angle (Fig. 6C and D), indicating an overall rise in surface hydrophobicity. The enhanced hydrophobicity improves the capacity for insertion into and disruption of the microbial cell membrane (Sato et al., 2022), leading to the renewed increase in antibacterial efficacy at higher pH. These findings align with previous reports that lysozyme gains broad-spectrum antibacterial activity after forming amyloid aggregates (Wei et al., 2021). We hypothesize that the enhanced activity stems from structural reorganization during unfolding and film formation. Partial denaturation exposes additional hydrophobic regions and increases the density of surface positive charges. These two factors may act synergistically to disrupt bacterial cell membrane integrity. Hydrophobic microdomains can insert into the phospholipid bilayer, while positively charged residues engage in electrostatic interactions with negatively charged membrane components. Ultimately, this combined action disrupts the outer membrane structure and induces leakage of cellular contents.

SEM imaging confirmed the significant antibacterial activity of lysozyme nanofilms against both S. aureus and E. coli (Fig. 8). Compared with the control group, the films not only effectively reduced bacterial adhesion but also induced noticeable morphological alterations in the bacteria, including shrinkage, collapse, and rupture, indicating disruption of structural integrity and ultimately leading to bacterial death. This antibacterial effect can be attributed to the synergistic action of two key physicochemical properties of the lysozyme nanofilms: electrostatic adsorption mediated by positively charged surfaces, and membrane penetration capability facilitated by exposed hydrophobic amino acid residues (Na et al., 2023). The positively charged regions on the film surface attract the negatively charged bacterial membranes, while the hydrophobic domains insert into the phospholipid bilayer. Together, these actions disrupted microbial membrane integrity, resulting in the leakage of intracellular contents.

Fig. 8.

SEM images of S. aureus and E. coli after 24 h of incubation on lysozyme films formed at (A) different lysozyme concentrations, and (B) different pH values. Red arrows indicate severely deformed and ruptured bacterial cells. Scale bar = 3 μm.

CLSM observations provided further evidence (Fig. 9). In the control group, strong green fluorescence (live bacteria) was observed on the sample surface, whereas red fluorescence (dead bacteria) was nearly absent. In contrast, lysozyme film-treated groups showed a significant increase in red fluorescence intensity with rising lysozyme concentration, demonstrating elevated bacterial mortality. Notably, films prepared under two extreme pH conditions (pH 6 and pH 12) exhibited the strongest bactericidal effects. At pH 6, the film surface possessed the highest positive potential, maximizing electrostatic attraction to bacterial membranes. At pH 12, the film showed the strongest hydrophobicity, enhancing its membrane-disrupting capability. Under both conditions, the strongest red fluorescence signals were observed in CLSM images, further confirming the critical synergistic role of surface charge and hydrophobicity in the antibacterial mechanism.

Fig. 9.

CLSM images of S. aureus and E. coli after 24 h of incubation on lysozyme films formed at (A) different lysozyme concentrations, and (B) different pH values. Green and red fluorescence indicate live and dead bacteria, respectively. Scale bar = 20 μm.

Based on the comprehensive experimental evidence, which includes changes in surface charge (Fig. 2C and F), increased surface hydrophobicity (Fig. 4, Fig. 6), and direct observation of membrane damage via SEM and live/dead staining (Fig. 8, Fig. 9), we propose a coherent antibacterial mechanism. The conformational transition of lysozyme induced by GSH leads to the exposure of hydrophobic domains and the redistribution of cationic residues on the nanofilm surface. These modified surface properties act synergistically, such that the positively charged regions facilitate electrostatic adhesion to negatively charged bacterial membranes, while the hydrophobic microdomains promote insertion into the phospholipid bilayer, together resulting in membrane disruption and eventual cell death.

4. Conclusion

In this study, an antibacterial protein nanofilm was successfully constructed by GSH-induced self-assembly of lysozyme at the air-water interface. Film formation was initiated by the reductive cleavage of intramolecular disulfide bonds in lysozyme by GSH, which triggered a conformational transition from a native α-helix-rich structure to a β-sheet-rich assembly. This assembly then organized into an ordered film at the interface through hydrophobic interactions and electrostatic attraction. The results demonstrated that the preparation conditions (e.g., lysozyme concentration and environmental pH) significantly affected the physicochemical properties and antibacterial behavior of the films. Higher lysozyme concentration promoted oligomer formation and interfacial stacking, leading to increased film roughness. In contrast, pH conditions modulated the surface charge of the protein and the exposure of hydrophobic residues, thereby affecting the hydrophobicity, surface potential, and ultimate antibacterial performance of the film. The antibacterial experiments confirmed that the films possessed significant inhibitory ability against both S. aureus (Gram-positive bacteria) and E. coli (Gram-negative bacteria). The antibacterial mechanism not only dependent on the inherent enzymatic activity of lysozyme but also originated from the synergistic disruptive effect of enriched positive charges and hydrophobic microdomains on the bacterial cell membrane. These findings offer a feasible strategy for designing eco-friendly protein-based antibacterial materials with potential applications in food packaging, biomedical coatings, and functional surfaces. Future studies could employ molecular dynamics simulations to further elucidate the atomistic interactions between the nanofilm surface and bacterial membrane components.

CRediT authorship contribution statement

Can Wu: Investigation, Data curation, Formal analysis, Resources, Writing-original draft; Qiuyue Hou: Investigation, Methodology, Formal analysis, Software, Writing-original draft; Hongyu Liang: Investigation, Software; Xuefeng Zhang: Formal analysis, Validation; Bing Cui, Yuying Hu & Xin Shi: Investigation, Formal analysis; Bin Zhou: Conceptualization, Project administration, Resources, Supervision, Writing-review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Dr. Xing Chen

Appendix A. Supplementary data

The following is the Supplementary data to this article: