Innovative Preparation of Salted Duck Egg White Lysozyme Functional Film and Its Application in Fresh Storage of Small Nectarines

Abstract

Carboxymethyl chitosan (CMCS) is ideal for active packaging due to its non-toxicity and degradability, but its poor film-forming performance (strong hydrophilicity, weak mechanical properties, and low antibacterial activity) limits practical use. This study prepared a new edible antibacterial presFervation film (SDEWL-CMCS) by adding salted duck egg white lysozyme (SDEWL) to CMCS (as the film-forming substrate). It investigated how SDEWL concentration affects the composite film’s properties (thickness, water solubility, moisture/oil resistance, mechanical properties, and antibacterial activity) and tested the film’s preservation effect on small nectarines. The results showed the composite film had significantly improved packaging and antibacterial properties: compared to pure CMCS film, it had higher tensile strength, lower water solubility, better oil resistance and water vapor barrier performance, and stronger antibacterial activity against Escherichia coli and Staphylococcus aureus (larger inhibition zone diameters). The SDEWL-CMCS film effectively preserved small nectarines by inhibiting surface bacteria, regulating the preservation environment, and delaying fungal decay. This study confirms the film’s potential as a sustainable fruit packaging alternative, providing a theoretical basis for developing new fruit/vegetable preservation packaging and reducing the food industry’s reliance on non-degradable petroleum-based packaging.

1. Introduction

Food packaging plays an indispensable role in retaining food’s nutritional value and freshness, and in turn extending its shelf life, throughout the stages of food storage and sales. Due to the complexity and diversity of packaging materials, the safety and quality of packaged food face great challenges [1]. Petroleum-based plastic films, such as polystyrene, polyethylene terephthalate and polypropylene, have been widely used in food packaging [2,3]. These plastic films with excellent physical and chemical properties and favorable corrosion resistance can provide protection during the storage and transportation of food, and their costs are low. However, they cannot effectively maintain the safety and quality of food. Most of the plastic films are non-biodegradable, causing serious environmental and ecological problems [4]. In response to the harm of plastic film waste, it is significant to develop new biodegradable packaging resources with low toxicity. Many studies have found that edible thin films prepared with biopolymers are safe and environmentally friendly. These biopolymers include polysaccharides, lipids, and proteins, and the edible thin films made from them are also featured by low cost and high variability [5,6]. Edible films were once considered the most promising alternative to plastic films [7,8]. Adding active compounds to edible films can better maintain the safety and quality of food, and further extend their shelf life [9,10].

Carboxymethyl chitosan (CMCS) is a derivative of the carboxylation of chitosan and is characterized by excellent film-forming capacity, biocompatibility [11], and degradability [12], and favorable hygroscopic properties [13,14]. It has been widely used in clinical medicine [15], food packaging [16,17], and biological preservation [18,19]. Liu et al. [20] prepared CMCS peptide conjugate nanoemulsion using hemp seed, maize, and casein as raw materials. Subsequently, an active packaging film was prepared through the combination of camellia essential oil and the conjugate-stabilized nanoemulsion. Using this active packaging film for the preservation of blueberries can effectively maintain their texture and prolong their shelf life. Zhang et al. [21] developed a freshness-preserving agent through free radical grafting, which involved combining carboxymethyl chitosan (CMCS) with epigallocatechin gallate (EGCG), and subsequently applied this agent to the preservation of fruit juice. The results showed that ECGC-CMCS preservative could effectively increase the total phenol content, total soluble solids, and titratable acidity in fresh juice. The ECGC-CMCS composite could well maintain the antioxidant capacity and sensory color of fruit juice, and inhibit microbial growth during storage. Wei et al. [22] used CMCS, Pullulan and galangal essential oil (GEO) as raw materials and prepared CMCS/Pul-GEO blend active film by casting. The CMCS/Pul-GEO film showed good mechanical properties and thermal stability. It was used to preserve mangoes and effectively extended their storage period. On the other hand, these studies emphasized that the mechanical properties and antimicrobial activity of CMCS films are insufficient to meet food packaging standards, thereby restricting their practical application. To expand the application of CMCS in food packaging, the research on multi-functional CMCS film has become a research hotspot.

Salted duck egg white lysozyme (SDEWL) refers to a type of lysozyme extracted from salted duck egg white; it exhibits excellent antibacterial activity and shares similar structural characteristics and properties with egg lysozyme. [23]. SDEWL, as a natural bioactive antibacterial substance, can selectively dissolve the cell wall of microorganisms, causing their death and inactivation [24]. SDEWL is the preferred choice for food preservation because it has few side effects on other nutrients in food and can retain the original nutrition and flavor of food [25]. In addition, SDEWL is stable under a wide range of pH values and temperatures, which affords it unique advantages in food preservation and packaging. In this sense, the use of SDEWL to prepare new antibacterial packaging materials has a good prospect. In the present study, salted duck egg white lysozyme (SDEWL) was incorporated into carboxymethyl chitosan (CMCS) to enhance the comprehensive packaging properties and antibacterial performance of the material. The active SDEWL-CMCS composite films with different SDEWL concentrations were prepared, and their morphological, mechanical, optical, waterproof, and antibacterial properties were measured. Furthermore, the structure of the composite films was characterized using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and ultraviolet (UV) absorption spectroscopy to explore the interaction between the groups of each component of the SDEWL-CMCS composite films. The SDEWL-CMCS composite films were applied to the preservation of small nectarines, aiming to provide an effective reference for the development and utilization of new materials in fresh-keeping packaging for fruits and vegetables.

2. Materials and Methods

2.1. Materials and Reagents

Small nectarines were purchased from a local supermarket (Wuhu, China). In the initial experiments of this laboratory, we innovatively employed the isoelectric point precipitation method, ultrafiltration method and cation exchange method to separate and purify the salted duck egg white lysozyme (SDEWL) from salted duck egg white (SDEW). The extraction method refers to the previously published paper [23], with the purity of SDEWL being 90% and the specific enzyme activity being 18,300 U/mg. CMCS (degree of substitution ≥ 80%) was purchased from Shanghai Maclin Biochemical Technology Co., Ltd. (Shanghai, China). Escherichia coli (E. coli) (ATCC 25922) and Staphylococcus aureus (S. aureus) (ATCC 29213) were acquired from Shanghai Conservation Biotechnology Center. Other reagents (analytically pure) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of SDEWL-CMCS Composite Films

SDEWL-CMCS composite films were prepared using the solution casting method [26]. First, 2 g of CMCS was put into a beaker with 100 mL of distilled water. Then the beaker was placed in a 40 °C water bath, and the solution was stirred for 1 h to fully dissolve the CMCS. By this approach, a 2% (mass concentration) CMCS solution was obtained. Different concentrations of SDEWL—specifically 0.20%, 0.40%, 0.60%, 0.80%, and 1.00%—were then incorporated into the 2% CMCS solution while sustaining constant stirring. At the same time, glycerin (mass concentration of 1%) was added to the mixed solution as a coupling agent and stirred evenly. Then the bubbles were ultrasonically dissolved to obtain a film-forming solution. Subsequently, 50 mL of the film-forming solution was poured into a plastic sheet measuring 13 × 13 cm. The plastic plate was dried continuously for 24 h in a vacuum drying oven (30 ± 2 °C), and the film was obtained by solidifying the film-forming solution. According to the differences in SDEWL content, the films were labeled as 0.00% SDEWL-CMCS (or pure CMCS), 0.20% SDEWL-CMCS, 0.40% SDEWL-CMCS, 0.60% SDEWL-CMCS, 0.80% SDEWL-CMCS, and 1.00% SDEWL-CMCS.

2.3. Properties of SDEWL-CMCS Composite Films

2.3.1. Measurement of Thickness

The thickness of the films was measured by a spiral micrometer (Sanmu, Japan). (Accurate within 0.001 mm). Six measurement locations were randomly selected for the same film. The final result is the average of the six data points.

2.3.2. Water Solubility (WS)

The WS was determined using the method employed by John et al. [27], with a slight modification. The specific procedure is as follows. The films were cut into 20 × 20 mm sample strips with scissors. The film sample strips were continuously dried in an oven (105 °C) for 24 h to reach a constant weight, and then they were taken out for weighing. Next, the film sample strips were soaked in a beaker of 50 mL of distilled water for 24 h. The soaked film sample strips were continuously dried in the oven (105 °C) for 24 h until their weight reached a constant value. After the drying treatment, they were taken out for weighing. The WS was calculated by Equation (1):

$$solubility\left(\%\right)={\displaystyle \frac{m_0-m_1}{m_0}}\times 100\%$$

(1)

where m₀ represents the initial dry weight (g); m₁ represents the final dry weight (g).

2.3.3. Water Vapor Transmittance Rate (WVTR)

The WVTR was determined according to the method reported by Gregor and Friderik [28], with a slight modification. First, 5 g of anhydrous CaCl₂ (180 °C constant temperature drying for 3 h) was put into a weighing bottle, with the distance between anhydrous CaCl₂ and the bottle mouth controlled between 5 and 6 cm. Then the bottle was sealed by a composite film of even thickness and without cracks. Immediately after sealing, the bottle was weighed. Then the weighing bottle was placed into a dryer with deionized water at the bottom and was weighed every 24 h. The WVTR of the film can be calculated according to Equation (2):

$$WVTR={\displaystyle \frac{∆m\times d}{A\times ∆t\times ∆p}}$$

(2)

where WVTR represents the water vapor transmittance rate, g·m·(m²·s·Pa)⁻¹; ∆m denotes the change in the mass of the weighing bottle before and after the test, g; d is the thickness of the composite film, m; A symbolizes the area of the composite film, m²; ∆p represents the water vapor pressure difference on both sides of the composite film [29], and the value of ∆p in this experiment is 3.17 × 10³ Pa; ∆t stands for the measurement time, s.

2.3.4. Oil Resistance Performance

The measurement of OR performance was based on the method reported by Zhang et al. [13]. The experiment was conducted with a slight modification to the original protocol. Specifically, 1 mL of salad oil was first pipetted into a clean test tube; afterward, the opening of the test tube was sealed using a piece of the composite membrane prepared earlier. To monitor oil permeation, a pre-weighed piece of filter paper (used to absorb any oil that might pass through the membrane) was placed in close contact with the outer surface of the sealed composite membrane. The weight of this filter paper was measured at three-day intervals continuously, and the measurement was stopped once the filter paper’s weight remained stable (with a weight variation error controlled to be less than 0.001 g). The calculation of the OR performance is shown in Equation (3)

$$OR\left(\%\right)={\displaystyle \frac{∆m\times L}{S\times T}}\times 100\%$$

(3)

where OR represents the oil resistance coefficient, g·mm·(cm²·d)⁻¹; ∆m is the weight difference in filter paper before and after the test, g; L denotes the thickness of the film, mm; S represents the area of the membrane, cm²; T is the time for the oil to pass through the film, d.

2.3.5. Mechanical Properties

The tensile strength (TS) and elongation at break (EAB) of the films were tested using a texture analyzer (model TA-XT-PLUS, Microstable System, London, UK) equipped with a 50 kg load cell probe and rubber anti-slip tensile clamps [30]. All tests followed the ASTM D882-18 standard for thin plastic sheets. Sample preconditioning: Films were placed in a constant temperature-humidity chamber (23 ± 2 °C, 50 ± 5% RH) for 48 h to eliminate moisture absorption and residual stress. Defective samples (with bubbles, cracks, or uneven thickness) were discarded, and valid films were cut into 100 mm × 20 mm rectangular specimens using a precision cutter. Test parameter setting: The initial clamp spacing was calibrated to 5 cm (50 mm) with a vernier caliper, and the crosshead stretching rate was fixed at 60 mm/min; the clamp force was adjusted to 4 N to prevent sample slippage or edge damage. Each film sample was measured five times, and the mean value was calculated. The TS can be calculated by Equation (4):

$$TS={\displaystyle \frac{F\left(max\right)}{L\times W}}$$

(4)

where TS stands for tensile strength, MPa; F(max) represents maximum tensile force, N; L denotes film thickness, mm; W is film width, mm.

The EAB was calculated according to Equation (5):

$$EAB\left(\%\right)={\displaystyle \frac{L_1-L_0}{L_0}}\times 100\%$$

(5)

where L₀ represents the initial film length (mm), and L₁ is the length of the film when it breaks, mm.

2.3.6. Antibacterial Properties

The antibacterial experiment of SDEWL-CMCS composite films was based on the method proposed by Zhang et al. [31], with a slight modification. The inhibition zone size of SDEWL-CMCS composite films against E. coli and S. aureus was measured by the agar diffusion method. The specific process is as follows. With a hole punch, the film samples were cut into uniform round pieces (diameter = 6 mm) and placed on a pure working table. The film samples were sterilized by a UV lamp for 30 min. The bacterial strains (200 μL, 104–105 CFU/mL) were uniformly covered on the surface of the agar plates. At the same time, composite films with different SDEWL concentrations (0.00%, 0.20%, 0.40%, 0.60%, 0.80%, 1.00%) were placed on the surface of the agar plates, with three membranes of the same concentration on each agar plate. After the plates were incubated at 37 °C for 28 h, the diameter of the inhibition zone was determined, and the average diameter of the inhibition zone of the three membrane samples with the same concentration was calculated.

2.4. Characterization of SDEWL-CMCS Composite Films

2.4.1. UV Spectral Analysis

The optical properties of the composite films were measured by a UV-visible spectrophotometer (UV-2550, Shimadzu Co., Ltd., Tokyo, Japan). The films were cut into strips of 10 × 40 mm using a utility knife and were then placed in a quartz colorimetric dish for full-wavelength detection. The wavelength scanning range is 200–600 nm [32].

2.4.2. FTIR Spectrometer

An FTIR spectrometer (IRPrestige-21, Shimadzu Co., Ltd., Tokyo, Japan) was used to characterize the interactions between the components of the composite films. The well-dried films were chopped with scissors, mixed with KBr, and ground into powder. Then the powder was compressed into a small round sheet and placed in a sample tank. Specific test conditions were as follows: the scanning wavelength is 400–4000 cm⁻¹, the scanning rate is 4 cm⁻¹, and the scanning frequency is 32 [33].

2.4.3. XRD Analysis

The crystalline microstructure of composite films was characterized by an X-ray diffractometer (D8 ADVANCE, Bruck, Germany). Specific test conditions were as follows. In the experiment, 40 kV and 40 mA Cu Ka were used as the radiation source. The diffraction angle is 2θ, the scanning range is 5–60°, and the incremental step size is 0.02°/s [34].

2.4.4. SEM Analysis

The microstructure of the composite films was observed by SEM (S-4800, Hitachi, Tokyo, Japan). The films were cut to a size of 5 × 5 mm and fixed on the sample table with double-sided conductive tape. The samples were then sputtered with a layer of gold. At a voltage of 5 kV, the samples were observed by SEM and photographed [35].

2.5. Application of SDEWL-CMCS Composite Films

Since 0.80% SDEWL-CMCS film showed advantages in properties, it was selected for the preservation experiment. A total of 24 small nectarines with good appearance, no mechanical damage, or visible mold were selected to evaluate the quality parameters. The small nectarines were rinsed with distilled water for 5 min and dried at room temperature. The CMCS film and 0.80% SDEWL-CMCS film were closely attached to the wrapping paper to make a good air-tight packaging bag. A bag without film covers was taken as a blank sample. The three samples were labeled as the control sample, the CMCS sample, and 0.80% SDEWL-CMCS sample. Ambient temperature and humidity, the physical and chemical properties of small nectarines were evaluated at an interval of 1.5 days, and they were photographed every three days for basic condition evaluation, as shown in Figure 1.

Figure 1. Photographic images of small nectarines preserved in different ways in relation to the storage period.

2.5.1. Soluble Solid Content (SSC)

The determination of SSC was mainly based on the method reported by Li et al. [36]. The SSC in the pulp homogenate of small nectarines was determined using a digital Abbe refractometer. For sample preparation, the pulp of small nectarines was first crushed, then homogenized in a blender to obtain a juice-like homogenate, which was subsequently used for SSC measurement. Two to three drops of small nectarine juice were added to the detection prism, and the cover plate was closed. The ruler knob was adjusted so that the chiarosculator in the visual field was at the middle crossing point, and SSC in the sample solution was read directly.

2.5.2. Weight Loss Rate (WLR)

The fruit WLR was calculated by Equation (6):

$$WLR={\displaystyle \frac{m_1-m_2}{m_1}}\times 100\%$$

(6)

where m₁ is the mass of small nectarines before preservation, g; m₂ is the mass of small nectarines after storage for a certain time, g.

2.5.3. Color

The method reported by Zhang et al. [37] was adopted to determine the skin color of small nectarines. A colorimeter (CR-₁₀ PLUS, Konica Minolta, Japan) was employed to measure the L, a*, and b* on the surface of small nectarines. The formula is shown in Equation (7):

$$∆E=\sqrt{∆a^2+∆b^2+∆L^2}$$

(7)

where ∆L = L*standard − L*sample; ∆a = a* standard − a*sample; ∆b = b*standard − b*sample

2.5.4. Ascorbic Acid Content

The content of ascorbic acid was determined by referring to the method of Niu et al. [38]. First, 20 g of small nectarine pulp was cut by a knife and added into the grinding cup, and after 20 g of oxalic acid solution was added, they were ground into pulp solution for later use. Then, 10 g fruit pulp solution was transferred with a pipette gun into a 100 mL volumetric bottle and diluted with an oxalic acid solution to the scale line. After that, the sample solution was filtered. During the experimental titration, 10 mL of the sample solution was transferred into a 50 mL beaker by a pipette and then titrated with 2, 6-dichloroindophenol solution until it turned pink and maintained the pink color for 30 s. The dosage of 2, 6-dichloroindophenol solution was recorded, and the ascorbic acid content was calculated.

$$\omega ={\displaystyle \frac{c\times V1\times D\times V3}{m1\times V2\times 1000}}$$

(8)

ω—Content of ascorbic acid in the pulp of small nectarines, with the unit of mg/100g; c—Concentration of the 2,6-dichloroindophenol (2,6-Dichloroindophenol, DCPIP) standard solution, with the unit of mmol/L; V1—Volume of the 2,6-dichloroindophenol standard solution consumed when titrating 10 mL of the sample solution, with the unit of mL; D—Total dilution factor of the sample solution; V3—Molar volume correction factor of ascorbic acid, with a value of 0.176 L/mmol; m1— Mass of the initially weighed small nectarine pulp, with the unit of g; V2—Volume of the sample solution used for titration, with the unit of mL; 1000—Unit conversion factor.

2.6. Statistical Analysis

IBM SPSS 23.0 (IBM Corporation, Armonk, NY, USA) software was used for variance analysis, and Duncan’s multiple comparison tests were used for significance analysis (p < 0.05). Origin Pro 2021 (OriginLab Corporation, Northampton, MA, USA) was employed for data processing and chart creation. All data were labeled as mean ± standard deviation. All measurements were in triplicate unless otherwise stated.

3. Results and Discussion

3.1. Analysis of Basic Properties of SDEWL-CMCS Composite Films

Film thickness uniformity is closely related to the mechanical and isolation properties of the film, thus determining the quality of the film. [39]. As shown in Table 1, the thickness of the composite films changed after adding SDEWL. The thickness of pure CMCS films was the lowest, about 0.051 mm. The thickness of the composite films containing 0.20–0.40% SDEWL did not change markedly. There was no significant difference in the thickness between SDEWL-CMCS composite films and CMCS films (p > 0.05). When the concentration of SDEWL increased to 0.60%, the thickness of the composite films was elevated to 0.062 mm, which was significantly higher than that of pure CMCS films (p < 0.05). The reason for the increase may be that the interaction between SDEWL and CMCS expands the network structure of the molecular chain of the CMCS polymer [40].

Table 1. Thickness, water solubility, water vapor permeability, and oil resistance of the SDEWL-CMCS composite films.

Z	Thickness (mm)	Water Solubility (%)	Water Vapor Transmittance Rate (10−10 g·m−1·s−1·Pa−1)	Oil Resistance (%)
0.00%	0.051 ± 0.003 b	31.66 ± 0.37 a	3.25 ± 0.08 a	2.70 ± 0.02 a
0.20%	0.058 ± 0.006 ab	27.90 ± 0.34 b	2.71 ± 0.04 b	2.37 ± 0.01 b
0.40%	0.056 ± 0.005 ab	27.25 ± 0.28 b	2.48 ± 0.03 c	2.14 ± 0.06 c
0.60%	0.062 ± 0.005 a	24.55 ± 0.14 c	2.39 ± 0.02 c	1.89 ± 0.03 d
0.80%	0.060 ± 0.004 a	23.33 ± 0.25 d	2.19 ± 0.02 d	1.77 ± 0.02 e
1.00%	0.059 ± 0.004 a	21.84 ± 0.19 e	2.16 ± 0.03 d	1.62 ± 0.03 f

Note: The film’s properties were measured using percentages, respectively. Different lowercase letters in the same column express significant differences (p < 0.05).

The Water Solubility (WS) is a parameter for the evaluation of the water resistance of film materials, which is of great significance for maintaining the integrity of packaging in practical applications [41]. Table 1 lists the WS values of SDEWL-CMCS films. CMCS films had the highest WS value, indicating that it has the worst water solubility. The WS value of the SDEWL-CMCS composite films was relatively low. The reason may be that SDEWL destroyed the hydrogen bond formed by the cross-linking of the hydrophilic groups of CMCS with water molecules, reducing the original hydrophilicity of CMCS, thus decreasing the solubility of the film in water [42].

The water resistance of food packaging film was evaluated by measuring WVTR. Table 1 lists the WVTR for all the films. The results showed that the WVTR of SDEWL-CMCS composite films was lower than that of pure CMCS films. The added SDEWL may crosslink with CMCS through coordination bonds or other strong molecular forces to form a denser polymer network structure, thus reducing the penetration rate of water vapor to the film [43]. The SEM analysis results of SDEWL-CMCS composite films supported this view.

The Oil Resistance (OR) is an important index of fried food packaging. In this study, the oil permeability coefficient was used to characterize the OR of SDEWL-CMCS composite films. The results showed that the OR value of SDEWL-CMCS composite films was lower than that of pure CMCS films, suggesting better oil-resistance performance of composite films. The possible reason is that the crosslinked products filled the structural gaps in the molecular chains of CMCS, preventing oil molecules from penetrating the film [44]. It can be predicted that SDEWL-CMCS composite films have huge application potential.

3.2. Analysis of Mechanical Properties of SDEWL-CMCS Composite Films

The TS and EAB are important parameters for studying the mechanical properties of food packaging films. The TS and EAB values of SDEWL-CMCS composite films prepared in this study are shown in Figure 2. The addition of SDEWL changed the toughness and strength of CMCS films, and the mechanical properties of the composite films were significantly improved (p < 0.05). It can be seen from the histogram that as the concentration of added SDEWL increased from 0.20% to 1.00%, the TS value of composite films elevated significantly from 37.39 MPa to 46.18 MPa, and EAB decreased from 27.16% to 20.68%. The reason for the TS increase is that the plasticizing effect of glycerol improved the kinematic ability of the intrinsic molecular chains and the toughness and strength of the composite films. The decrease in EAB is because the enhancement of electrostatic cross-linking and hydrogen bond networks when carboxymethyl chitosan (CMCS) combines with lysozyme. On the one hand, the negatively charged CMCS (-COO⁻) forms ionic bonds with the positively charged lysozyme (pH < 11), replacing the van der Waals forces between flexible molecular chains, resulting in a decrease in the sliding property of the molecular chains and an increase in brittleness; on the other hand, the amino/hydroxyl groups of lysozyme and the carboxyl/hydroxyl groups of CMCS form dense hydrogen bonds, reducing the freedom of movement of the chain segments and causing the loss of extensibility [45].

Figure 2. Mechanical properties of the different SDEWL-CMCS composite thin films. Different letters in the columns indicate significant differences (p < 0.05).

3.3. Analysis of Antibacterial Properties of SDEWL-CMCS Composite Films

The inhibitory effect of SDEWL-CMCS composite films against E. coli and S. aureus was measured by the agar disk diffusion method (expressed by the diameter of the inhibition zone). The experimental results are shown in Table 2. Pure CMCS films (0.00% SDEWL) did not exhibit good antibacterial activity against E. coli (Figure 3A). With the addition of SDEWL, the antibacterial properties were enhanced remarkably. When the concentration of SDEWL increased from 0.20% to 1.00%, the diameters of the inhibition zone of SDEWL-CMCS composite films improved significantly from 10.37 ± 0.04 mm to 15.06 ± 0.16 mm (p <0.05). As a cationic antibacterial protein, SDEWL can perforate the negatively charged bacterial cell membranes to form ion channels [22]. This ability is conducive to enhancing the penetration of CMCS into cells to absorb ionic substances, thus disrupting the physiological activities of cells or blocking bacterial DNA transcription and replication [46]. In sum, the two substances play a synergistic role in the SDEWL-CMCS composite films to inhibit bacterial growth.

Table 2. Bacteriostatic diameters of different SDEWL-CMCS composite films.

Diameter of Inhibition Zones (mm)
	0.00% SDEWL-CMCS	0.20% SDEWL-CMCS	0.40% SDEWL-CMCS	0.60% SDEWL-CMCS	0.80% SDEWL-CMCS	1.00% SDEWL-CMCS
E. coli	0.00 e	10.37 ± 0.04 d	10.95 ± 0.12 c	11.15 ± 0.17 c	13.34 ± 0.21 b	15.06 ± 0.16 a
S. aureus	0.00 d	0.00 d	0.00 d	10.54 ± 0.02 c	12.22 ± 0.36 b	13.09 ± 0.25 a

^a Note: Different letters within a line represent significant differences (p < 0.05).

Figure 3. Diameters of the inhibition zone of films with different SDEWL contents (0.00%, 0.20%, 0.40%, 0.60%, 0.80%, 1.00%) against E. coli (A) and S. aureus (B).

Pure CMCS films and low-concentration SDEWL-CMCS composite films showed a certain degree of bacteriostatic effect against S. aureus (Figure 3B). Additionally, bacteriostatic zones (10.54 ± 0.02 mm in diameter) were found when the concentration of SDEWL was increased to 0.60%. With the concentration of SDEWL further increased to 0.80% and 1.00%, the diameter of the inhibition zone was significantly elevated to 12.22 ± 0.36 mm and 13.09 ± 0.25 mm, respectively (p <0.05). The difference in antibacterial activity of this strain with E. coli may be due to the various cell wall composition structures [47]. A thick layer of peptidoglycan is found on the surface of the cell wall of S. aureus, and lysozyme directly acts on the peptidoglycan to rupture the cell wall, thus inhibiting bacterial reproduction [48]. The results of this experiment are not consistent with those reported by Wu et al. [49]. The possible reason is that pure CMCS films and low-concentration SDEWL-CMCS composite films failed to show a significant bacteriostatic effect against S. aureus at this particular concentration (200 μL, 104–105 CFU/mL). With the increase in SDEWL concentration, the bacteriostatic effect of lysozyme on S. aureus was enhanced. The results of this experiment also indicate that SDEWL-CMCS composite films have a good antibacterial effect and are degradable and can be decomposed by microorganisms.

3.4. UV Spectral Analysis of SDEWL-CMCS Composite Films

Light exposure has many adverse effects on food quality and nutrition. For example, the oxidation of oil in food can lead to the deterioration of the oil, oxidation of natural pigments, changes in food color, and loss of vitamin B and vitamin C [50]. However, the UV barrier property of the antibacterial film has a significant protective effect on food quality. It can prevent the chain reaction of food deterioration caused by light, while maintaining the stability of the antibacterial activity. In this sense, it is very important to study the UV blocking performance of thin films during food storage. In this study, the barrier properties of different SDEWL-CMCS composite films were evaluated based on UV-vis absorption spectra, and the results are shown in Figure 4. The prepared composite films all had a large absorption band at 230 nm and a small absorption band at 330 nm and realized π-π* transition at 360 nm. In contrast, the pure CMCS films did not show any significant absorption peak in the UV-vis spectra. The addition of SDEWL significantly increased the absorption value at 200–600 nm. In addition, UV-vis transmission scanning was performed at 200–600 nm, and the experimental results are shown in Figure 3. SDEWL-CMCS composite films had good transparency and a remarkable UV protection effect. At 400 nm, the transmittance (T%) of pure CMCS films was 69.45%, with the addition of SDEWL, the transmittance of SDEWL-CMCS composite films decreased to 41.95%, and with the increase in SDEWL content, the transmittance of the composite films decreased to 29.92%. In addition, with the increase in wavelength, the transmittance was slightly decreased, suggesting that the composite films have a certain UV barrier effect.

Figure 4. UV-vis absorption spectra (A) and transmission spectra (B) of different SDEWL-CMCS composite thin films.

3.5. FTIR Analysis of SDEWL-CMCS Composite Films

FTIR spectra of SDEWL-CMCS composite films are shown in Figure 5. The CMCS spectra exhibited strong C−O symmetric stretching vibration (C−O−C) and C=O, O−H stretching vibration. The characteristic peaks were 1056 cm⁻¹, 1629 cm⁻¹, and 3394 cm⁻¹, respectively, indicating a large number of epoxy groups, carboxyl groups, and hydroxyl groups on the surface of the films [51]. The curves of SDEWL-CMCS composite films with proportions of 0.02%, 0.04%, 0.06%, 0.08%, and 1.00% show characteristic peaks at wavenumbers (for example, around 1066 cm⁻¹, 1076 cm⁻¹, 1014 cm⁻¹, 1026 cm⁻¹, 1022 cm⁻¹) that correspond to the vibrations of functional groups in CMCS (C−O, C=O, O−H). There are no newly emerged distinct peaks that would indicate the formation of new covalent bonds (such as C−C, C−N, or vibrations of other novel functional groups). Although there are slight shifts in peak positions (for instance, the peak at approximately 1056 cm⁻¹ in CMCS shifts to the range of ~1066–1022 cm⁻¹ in composite films) and changes in peak intensities, these phenomena are consistent with non-covalent interactions (such as hydrogen bonding and van der Waals forces) between SDEWL and CMCS. Such interactions can alter the local chemical environment of functional groups (resulting in peak shifts) and affect the light transmittance at specific wavelengths (leading to changes in intensity) without breaking or forming covalent bonds. During the film-forming process, the molecular structure of CMCS remains unchanged because there is no disappearance or significant modification of its characteristic peaks. These findings were consistent with the results reported by Hu et al. [52]. FTIR spectra showed that the molecular compatibility of SDEWL and CMCS was good.

Figure 5. The FTIR spectra of the SDEWL-CMCS composite thin films.

3.6. XRD Analysis of SDEWL-CMCS Composite Films

The structure of SDEWL-CMCS composite films was studied by XRD pattern (Figure 6). As can be seen from the figure, the XRD curves of SDEWL—CMCS composite films with different proportions (0.20%, 0.40%, 0.60%, 0.80%, 1.00%) and the pure CMCS film (0.00%) show a characteristic variation law of diffraction peaks in the diffraction angle (2θ) dimension. For the pure CMCS film (0.00%), its diffraction peak appears at 2θ = 23.2°, and this characteristic peak is a direct manifestation of the ordered arrangement of CMCS molecular chains to form a semicrystalline structure, reflecting that CMCS itself has a certain crystallinity. The diffraction peaks of each curve of the SDEWL-CMCS composite films are very close to the characteristic peak position of the pure CMCS film. From the perspective of peak morphology, although the peak intensity fluctuates as the proportion of SDEWL increases, the overall peak position does not shift significantly. This is because the concentration of SDEWL is low, and its molecules are mainly combined with CMCS molecules through non-covalent interactions (such as hydrogen bonds) in the composite film, which does not have a destructive effect on the ordered arrangement of CMCS molecular chains (that is, the core framework of the crystalline structure) [53]. Therefore, the composite film can still exhibit crystallization properties similar to those of the pure CMCS film and retain the key characteristics of the semicrystalline structure of CMCS. This is consistent with the results reported by Svetlana et al. [54], indicating that the incorporation of SDEWL into CMCS does not change the crystal structure of CMCS.

Figure 6. The XRD diagram of the SDEWL-CMCS composite thin films.

3.7. SEM Analysis of SDEWL-CMCS Composite Films

The surface morphology and structure of composite films were analyzed by SEM. Pure CMCS films and composite films (0.80% SDEWL) showed different surface morphologies. Figure 7A shows that pure CMCS films exhibited an irregular rough surface with discontinuous bumps, uneven pits, and micron-sized particles; Figure 7B demonstrates that the 0.80% SDEWL-CMCS composite films had relatively smooth and uniform surfaces; Figure 7C presents the rough granular cross-sectional structure of pure CMCS films; Figure 7D shows the sectional structure of 0.80% SDEWL-CMCS composite films, which is characterized by more uniform and denser fracture surfaces, and discernible textures. The two polymer materials, SDEWL and CMCS, were compatible. The addition of SDEWL changed the structure of the films and promoted the melting and plasticization of the films, which was consistent with the research result reported by Soubhagya et al. [55]. Due to the interaction between the particles of CMCS and SDEWL, an electrostatic bond was formed, thus improving the mechanical strength of the film and changing its surface morphology.

Figure 7. SEM spectra of the 0.80% SDEWL/ CMCS composite thin films and the pure CMCS thin films (A) 0.80% SDEWL-CMCS composite film surface appearance; (B) CMCS film surface appearance; (C) 0.80% SDEWL-CMCS composite film cross-sectional profile; (D) CMCS film cross-sectional profile).

3.8. Effects of SDEWL-CMCS Composite Films on the Preservation of Small Nectarines

3.8.1. Influence of SDEWL-CMCS Composite Membranes on the SSC of Small Nectarines

Soluble solid content (SSC) is an important index to evaluate fruit maturity, determine the ideal time for harvesting, and assess fruit quality during storage [56]. Figure 8 shows that the SSC of small nectarines in each experimental group had varying degrees of change. During the initial three days of storage, the SSC in the group without covering films was reduced because respiration consumed part of the soluble solids. The SSC in the group covered by 0.00% SDEWL-CMCS composite film increased first and then decreased. This trend may be related to post-ripening phenomena. The accelerated decomposition of polysaccharides leads to an increase in SSC. With the extension of time, respiration consumes more soluble solids, resulting in a decrease in SSC. The SSC of the group covered by 0.80% SDEWL-CMCS composite films decreased first and then increased, which is related to the ratio of oxygen to carbon dioxide in the bag [57]. After three days of storage, the SSC uniformly showed a gradually decreasing trend. On the 6th day of storage, the SSC of small nectarines preserved with 0.80% SDEWL-CMCS composite film was 8.54%, which was 7.94% higher than that of the group preserved with pure CMCS film and 7.56% higher than that of small nectarines preserved without films. In conclusion, 0.80% SDEWL-CMCS composite film is more effective in slowing down tissue metabolism, inhibiting the reduction in SSC, and maintaining the nutrients of the fruit [58].

Figure 8. Changes in soluble solids (A), weight loss (B), color (C), and ascorbic acid content (D) during storage of small nectarines after different treatments. Different letters indicate significant differences (p < 0.05).

3.8.2. Effects of SDEWL-CMCS Composite Film on the WLR of Small Nectarines

Fruit transpiration will cause water loss inside fruit tissues, resulting in wilting and shriveling [59]. In this sense, the rate of mass loss is a key factor affecting the structure of the fruit epidermis. As can be seen from Figure 8B, the WLR of small nectarines is positively correlated with storage time. With the extension of storage time, the WLR of all experimental groups showed an increasing trend. Significant differences were observed in the trends of WLR in different groups. Among them, small nectarines packaged with 0.80% SDEWL-CMCS composite film had a relatively slight change in WLR. The group without films showed the most remarkable change in WLR, followed by the group covered with pure CMCS films. These results indicate that the packaging treatment of fruit by SDEWL-CMCS composite films is more effective in maintaining the tissue structure of the fruit epidermis, reducing the respiratory intensity and WLR, slowing down aging, and thus extending the shelf life [60].

3.8.3. Effects of SDEWL-CMCS Composite Films on the Color of Small Nectarines

In the process of fruit storage, chlorophyll undergoes enzymatic reaction and degradation, resulting in changes in fruit color, which indirectly reflects the storage tolerance of fruit [61]. As can be seen from Figure 8C, the color of small nectarines in each experimental group was changed with the extension of storage time, and the overall color differences among the no film group, the CMCS film group, and the 0.80% SDEWL-CMCS composite film group were large. On the 6th day of storage, ∆E values of the three groups were 8.63, 11.26, and 15.31, respectively. During the following storage days, the color changes in the three groups have a certain difference. This indicates that SDEWL-CMCS composite films can effectively protect the cell film system of the epidermis, reduce the chlorophyll reaction and cause the color change in the pericarp, and delay the aging and metamorphism of the fruit, thus maintaining the quality of the fruit [18].

3.8.4. Effects of SDEWL-CMCS Composite Films on the Ascorbic Acid Content of Small Nectarines

The ascorbic acid content in fruit is an important index to measure the degree of fruit senescence. As can be seen from Figure 8D, the ascorbic acid content in all experimental groups generally presents a downward trend with the extension of storage time. The initial ascorbic acid content in small nectarines was more than 8 mg/100 g. After 6 days of storage, the ascorbic acid content of small nectarines covered without films, with pure CMCS films, and with 0.80% SDEWL-CMCS composite films was 3.6 mg/100 g, 4.8 mg/100 g and 5.7 mg/100 g, respectively. It can be concluded that the packaging treatment with SDEWL-CMCS composite films can well block the exchanges with the external environment, inhibit the oxidation of nutrients in fruits, and preserve ascorbic acid [62].

Notwithstanding these promising results, this work has several limitations that warrant acknowledgment. First, SDEWL is derived from egg white lysozyme, and its potential allergenicity in practical food contact scenarios remains unaddressed, which may restrict its application in allergen-sensitive markets. Second, the current antibacterial and preservative efficacy data are based on laboratory-scale experiments; large-scale production trials (e.g., continuous film casting) and long-term storage validation under real supply chain conditions (e.g., temperature fluctuations, transportation vibration) are still needed to confirm its industrial feasibility. Third, regulatory compliance—including adherence to food contact material standards (e.g., EU Regulation (EC) No. 10/2011 or FDA guidelines)—has not been evaluated, which is a critical prerequisite for commercialization. By addressing these issues in the future, SDEWL-CMCS composite films could be further developed into a competitive alternative to traditional synthetic preservative packaging, contributing to the advancement of sustainable and safe food preservation technologies.

4. Conclusions

In this study, CMCS-SDEWL composite films with different contents of SDEWL were prepared, and their mechanical properties, WS, water resistance, OR, and antibacterial properties were evaluated. The experimental results show that the above functional characteristics are concentration-dependent within the test range. With the increase in SDEWL concentration, the functional properties of the prepared active composite films were upgraded. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-Vis), and scanning electron microscopy (SEM) analyses were employed to investigate the intermolecular interactions in the CMCS-SDEWL composite films. These characterizations confirmed excellent compatibility between the two components, thereby verifying the successful preparation of the CMCS-SDEWL composite films. Notably, the CMCS-SDEWL composite films displayed superior antibacterial activity compared to pure CMCS films. Specifically, the composite film with 0.80% SDEWL showed potent antibacterial effects against Escherichia coli and Staphylococcus aureus, capable of inhibiting bacterial growth on surfaces. The 0.80% SDEWL-CMCS composite films showed strong antibacterial activity against E. coli and S. aureus. It can inhibit the growth of bacteria on the skin surface and delay the decay process caused by fungi. On this basis, they were used in the small nectarine storage experiment. The results show that 0.80% SDEWL-CMCS composite films with good adhesive properties can inhibit tissue metabolism and water loss, regulate the preservation environment, and extend the shelf life of small peaches. It is believed that after addressing these limitations in the future, the SDEWL-CMCS composite film is expected to become a strong alternative to traditional synthetic preservative packaging, promoting the development of sustainable and safe food preservation technologies.