Development of Bacterial Cellulose-Based Films Incorporated with Epigallocatechin-3-Gallate for Active Food Packaging

Abstract

Recently, renewable biopolymers have gained growing attention as an alternative to petroleum-based materials in the packaging industry due to their eco-friendliness, biodegradability, and biocompatibility. This study introduces an innovative method for producing active films, which uses natural bacterial cellulose (BC) films as the matrix and incorporates (−)-Epigallocatechin-3-gallate (EGCG) through an immersion process. The incorporation of EGCG improves the barrier performance against oxygen and UV of the BC-based active films while preserving their tensile strength without compromising their opacity. More importantly, the active films exhibited significant antibacterial effects, with the efficacy increasing with the concentration of EGCG. Specifically, the diameters of the inhibition zones enlarged progressively against both S. aureus (from 13.88 to 16.25 mm, p < 0.05) and E. coli (from 12.38 to 14.13 mm). Correspondingly, the antibacterial rate of the active films increased from 61.4% to 80.61% (p < 0.05) against S. aureus and from 57.38% to 60.38% against E. coli. Additionally, the BC-based active films developed in this work exhibit excellent biodegradability, being capable of achieving complete biodegradation within 21 days of soil burial. This breakthrough exhibits considerable potential of BC-based active films as eco-friendly packaging materials, showing exceptional promise for sustainable active food packaging applications.

1. Introduction

With the growing consumption of petroleum-based plastic packaging, the associated challenge of poor biodegradability has become a serious threat to both ecosystems and the food packaging industry [1,2]. In response to this challenge, researchers have focused on developing biopolymer-based films [3,4,5]. Biopolymers are favored for their abundant availability, eco-friendliness, non-toxicity, biodegradability and biocompatibility [3,6]. Moreover, they serve as excellent carriers for natural active substances. At present, numerous outcomes have been achieved in the development of biopolymer-based packaging films, including renewable and biodegradable natural biopolymers such as chitosan [7], plant cellulose [8], gelatin [9], and lipids [10].

Bacterial cellulose (BC), composed of cellulose, is a linear high-molecular-weight polysaccharide synthesized by the polymerization of D-glucopyranose units via β-1,4-glycosidic bonds [11,12]. Its repeating unit is cellobiose, consisting of two linked glucose molecules, with each glucose monomer containing three polar hydroxyl groups [12,13]. Natural BC gel film can be produced through microbial static fermentation [11,12,13] utilizing cost-effective and environmentally sustainable industrial by-products, such as sugarcane hydrolysate [14], orange peel hydrolysate [15], and cheese whey [16], thereby expanding its application potential. BC film exhibits distinct advantages. First, BC gel film is produced via bacterial fermentation with a purity of approximately 99%, significantly higher than that of plant cellulose (65%), substantially reducing purification costs. Second, the abundant hydroxyl groups in BC film promote the generation of extensive intramolecular and intermolecular hydrogen bonds, leading to a high crystallinity of 80%~90% and consequently excellent tensile strength [11,13]. Third, BC gel film forms an ultrafine nanoscale three-dimensional network structure driven by the extensive hydrogen bonds [13]. This unique porous structure can adsorb various substances through its hydroxyl groups, which is essential for its use as a film carrier.

Natural active substances are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA) [17] and have attracted considerable attention for their applications in food preservation [18,19]. (−)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic compound catechin in green tea, accounts for approximately 65% of the total catechin content [20,21]. As a typical natural bioactive substance, EGCG is characterized by its biocompatibility, odorlessness and low volatility [20,21], making it an ideal candidate for active food packaging films. Moreover, owing to its molecular structure rich in phenolic hydroxyl groups [21,22], EGCG exhibits broad-spectrum antimicrobial activity [23,24]. For example, it inhibits a variety of bacterial strains, including Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Helicobacter pylori, Mycoplasma pneumoniae, Salmonella spp. and Staphylococcus spp., with particular efficacy against S. aureus and E. coli. Its antibacterial mechanisms involve disrupting bacterial cellular structures, interfering with gene expression, and inhibiting intercellular communication [22,25,26].

The incorporation of bioactive substances, such as polyphenols [27], essential oils [3,4], and plant extracts [5], provides a potential strategy to endow films with antibacterial activity while also serves as an effective approach to modulate their physical properties, including water vapor permeability [4], tensile strength [5,27], elongation at break [3] and oxygen barrier property [5], UV barrier properties [27], and thermal stability [27]. These studies have laid the methodological foundation for the present work. Based on the excellent tensile strength of natural BC film, this study seeks to develop a novel bioactive BC-based film incorporating EGCG through an immersion method, and to evaluate its physical properties, microstructure, antimicrobial activity and biodegradability. This work will provide a novel sustainable method for the development of active packaging materials with highly efficient antibacterial activity, offering promising potential for food industry applications.

2. Materials and Methods

2.1. Materials

The natural BC gel films were provided by Guilin Qihong Technology Co., Ltd. (Guilin, China). Glycerol (Gly, Purity ≥ 99.5%), xanthan gum (XG) powder, sodium hydroxide (NaOH) powder, EGCG powder, tryptone powder, yeast extract powder, sodium chloride (NaCl) powder and agar powder were acquired from Sigma-Aldrich Co., Ltd. (Shanghai, China). The Gram-positive S. aureus (ATCC 25923) and Gram-negative E. coli (ATCC 25922) were obtained from the Institute of Microbiology, Chinese Academy of Sciences (IMCAS).

2.2. Methods

2.2.1. Purification Method of BC Film

Following the purification protocol described previously [28], BC films with a thickness of 3 mm were immersed in 0.1 M NaOH solution and bathed in water at 90 °C for 2 h to remove the bacterial cells and other impurities derived from culture medium. Subsequently, the films were washed repetitively using deionized water until the pH of the rinse water stabilized at approximately 7.0. At this stage, the BC films appeared as a milky white, semi-transparent hydrogel. Finally, they were stored in deionized water at 4 °C until further use.

2.2.2. Preparation of BC-Based Active Films

XG and Gly were incorporated to enhance the physical properties of BC film, including elongation and opacity, so as to better meet the basic requirements for food packaging application. Meanwhile, EGCG was introduced to impart antibacterial activity, thereby facilitating the development of active packaging films. Accordingly, the preparation method of the BC-based active films in this study is described as follows.

First, a mixed solution containing 1% XG and 2% Gly was mechanically stirred at 80 °C for 1 h to ensure full dissolution and homogenization, followed by cooling to room temperature. Next, EGCG was added to the solution at varying concentrations (0, 0.1%, 0.2%, and 0.3%), followed by magnetic stirring for 2 h to ensure homogeneity. Then, the BC gel films were fully immersed in the prepared mixtures containing varying EGCG concentrations, followed by magnetic stirring at room temperature for 24 h. Subsequently, the film surfaces were gently rinsed with deionized water, laid flat on a polytetrafluoroethylene (PTFE) plate, and air-dried at room temperature for 72 h. Additionally, the dried pure BC film was prepared by placing it directly on a PTFE plate and air-drying at room temperature for 72 h. Finally, all dried films were peeled off the PTFE plates and equilibrated at 25 °C and 50% relative humidity for 48 h. Five replicate samples were prepared for each formulation. The films, with their specific compositions detailed in Table 1, were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e). According to the protocol of Wang et al. [29], the aforementioned films were subsequently stored in a desiccator at room temperature for 2 days to equilibrate and remove residual moisture prior to analysis.

Table 1.

The specific compositions of films.

Film	Film Matrix	XG (%)	Gly (%)	EGCG (%)
S-a	BC	-	-	-
S-b	BC	1	2	-
S-c	BC	1	2	0.1
S-d	BC	1	2	0.2
S-e	BC	1	2	0.3

Since this study primarily focuses on investigating the effects of EGCG incorporation on the physical and antibacterial properties of BC-based active films (S-c, S-d and S-e), the BC/1% XG/2% Gly composite film (S-b) was designated as the control group, while the films S-c, S-d and S-e served as the experimental groups.

2.2.3. Film Thickness Measurement

Thickness is a critical factor affecting the performance of food packaging films, including mechanical, barrier and optical properties [30]. In this study, film thickness was measured at 15 randomly selected locations on each film using a Beslands spiral micrometer (Shenzhen, China) with an accuracy of 0.001 mm. The average values of these measurements were used as the film thickness to ensure consistency and reliability of the results.

2.2.4. Physical Performance

Mechanical Properties

Food packaging materials possess adequate mechanical strength to withstand the mechanical stress encountered during food transportation or storage [31], thereby extending the shelf life of packaged food. Mechanical properties mainly depend on the internal structure and intermolecular interaction forces within the film matrix and are typically evaluated by tensile strength (TS) and elongation at break (EB) [32]. TS is defined as the maximum tensile stress that a film can withstand before failure, while EB indicates its maximum extensibility prior to breaking. Therefore, the two parameters characterize the tensile resistance and flexibility of the film, respectively [33], which are critical properties determining its suitability for practical applications. In China’s food packaging industry, evaluations are commonly performed in accordance with the Chinese national standard GB/T 28118-2011 [34] Food Packaging Plastic and Aluminum Foil Composite Film. This standard specifies that food packaging films need to possess adequate tensile strength (≥35 MPa) to prevent fracture or tearing during practical application.

The standard test method of American Society for Testing and Materials (ASTM) D882-02 was slightly modified for this experiment [35]. Briefly, the TS and EB of the films were measured using a tensile testing machine (Instron, Norwood, MA, USA) equipped with a 2530-50N load cell. First, the films were cut into rectangular shapes with dimensions of 5 cm × 1 cm. Then, the films were clamped using self-tightening rollers with an initial gauge length of 20 mm, with a tensile load of 5 kN and a crosshead speed of 1 mm/min applied during testing. All final results are presented as mean values, and the calculation formulas for TS and EB are shown below [36]:

$$\mathrm{T}\mathrm{S}\left(\mathrm{M}\mathrm{P}\mathrm{a}\right)={\displaystyle \frac{\mathrm{F}\mathrm{m}\mathrm{a}\mathrm{x}}{\mathrm{A}}}$$ (1)

$$\mathrm{E}\mathrm{B}\left(\%\right)={\displaystyle \frac{\mathsf{\Delta }\mathrm{L}}{{\mathrm{L}}_0}}\times 100\%$$ (2)

In the formula, F_max is the maximum tensile load (N) sustained by the film prior to fracture; A denotes the cross-sectional area of the film (mm²); L₀ represents the initial gauge length of the film before stretching (20 mm); ΔL refers to the elongation of the film at the point of break (mm).

Barrier Properties

Water vapor permeability (WVP) and oxygen transmission rate (OTR) refer to the capacity of water vapor and oxygen, respectively, to permeate through a film from the external environment into the packaged food. Both properties serve as critical barrier indicators for biopolymer-based packaging materials [37] and crucial factors affecting food quality and safety. By inhibiting the transmission of small-molecule gases, these barrier properties help reduce moisture loss, limit microbial contamination, as well as slow the oxidation of proteins and lipids in food [38,39], thereby playing a vital role in extending food shelf life. In accordance with the Chinese national standard GB/T 28117-2011 [40] Multilayer Co-extruded Films and Bags for Food Packaging, the specified OTR value should be less than 20 cm³/(m²·day·atm).

WVP reflects the volume of water vapor permeating through a unit area of the film per unit time and per unit pressure difference, expressed in g·m/(m²·d·kPa). The WVP of the films were measured following a slightly modified ASTM E96/E96M-16 standard test method [41]. Briefly, 50 g of deionized water was added to 8 OZ Mason jars (Huntersville, NC, USA). Circular film samples with a diameter of 7 cm were placed over the mouths of the jar and sealed with silicone rubber gaskets and metal nuts. The sealed assemblies were then placed in a desiccator maintained at 23 °C and 50% relative humidity for 7 days, with daily weight recordings taken. The WVP values were calculated using the formula given below [36]:

$$\mathrm{W}\mathrm{V}\mathrm{P}={\displaystyle \frac{\mathsf{\Delta }\mathrm{w}\times \mathrm{d}}{\mathrm{A}\times \mathrm{t}\times {\mathrm{P}}_0\times ({\mathrm{R}}_1-{\mathrm{R}}_2)}}$$ (3)

In the formula, Δw represents the total mass change in the Mason jar (g); d denotes the average film thickness (m); A is the area of the jar opening (m²); t is the testing duration (days); P₀ is the saturated vapor pressure at 23 °C (2.81 kPa); R₁ refers to the relative humidity inside the Mason jar (100%); and R₂ indicates the relative humidity of the testing environment (50%).

The OTR of the film serves as an effective indicator for preventing lipid oxidation in food [38]. It represents the volume of oxygen permeating through a unit area of the film per unit time under a unit pressure difference, expressed in cm³/(m²·day·atm).

The OTR of the films were determined according to a slightly modified ASTM D-1434 standard test method [42]. Briefly, the sample films were cut into 6 cm × 6 cm squares and then tested for oxygen permeability using a GTR-701R oxygen transmission tester (Jinan, China) at 23 °C and 65% relative humidity. The OTR was calculated using the formula given below:

$$\mathrm{O}\mathrm{T}\mathrm{R}={\displaystyle \frac{\mathrm{V}}{\mathrm{A}\times \mathrm{t}\times \mathrm{P}}}$$ (4)

In the formula, V denotes the volume of oxygen permeating through the film (cm³), A refers to the exposed film area (m²), t is the test duration (days), and P represents the standard atmospheric pressure (taken as 0.1 MPa, i.e., 1 atm).

Optical Properties

Optical properties are also key attributes of food packaging films, primarily including ultraviolet (UV) blocking capacity, opacity, and color characteristics. UV radiation induces extensive photochemical reactions, generating free radicals that accelerate the oxidation of lipids and proteins [43]. These oxidative processes degrade the sensory quality of food (e.g., causing discoloration and off-odors) and shorten its shelf life. Consequently, food packaging films with UV-blocking capacity help effectively decelerate the oxidation of lipids and proteins [44]. The UV spectrum is categorized into three regions: UV-A (315~400 nm), UV-B (280~315 nm), and UV-C (200~280 nm), with transmittance in these regions reflecting the film’s UV-blocking efficiency. High-transparency films enable consumers to visually inspect the appearance, quality, and freshness of food, providing an appealing visual presentation [45]. Opacity is a commonly used parameter to evaluate film transparency, where a lower opacity value corresponds to higher transparency [45].

The UV-blocking efficiency and opacity of the films were assessed according to a slightly modified ASTM D1746-15 standard test method [45]. Film samples were cut into rectangular strips (10 mm × 25 mm) and tightly attached to the inner transparent wall of a quartz cuvette, with an empty cuvette used as the control. UV absorbance over the wavelength range of 200~400 nm was recorded using a Spectramax M3 UV-Vis spectrophotometer (San Jose, CA, USA) [36]. The UV-blocking performance was quantified by calculating the UV transmittance. Opacity was defined as the film absorbance at 600 nm divided by its thickness, with the corresponding calculation formula [44] presented below:

$$\mathrm{O}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}={\displaystyle \frac{\mathrm{A}{\mathrm{b}\mathrm{s}}_{600}}{\mathrm{x}}}$$ (5)

In the formula, Abs₆₀₀ is the film absorbance measured at 600 nm, and x denotes the film thickness in millimeters (mm).

The color of food packaging film is generally characterized by three parameters: L value (lightness, ranging from 0 for black to 100 for white), a value (green–red axis), and b value (blue–yellow axis) [36]. Ideally, the color of food packaging films should align with the inherent visual characteristics of the food product while being visually appealing to consumers. In this experiment, the color attributes of the films were evaluated using a CR-400 colorimeter (Tokyo, Japan) with a D65 light source and a 2° standard observer angle. The instrument was calibrated on a white standard background prior to measurements. For each film, ten random locations were measured. By comparing the L, a, and b values of the film samples with those of a standard white reference plate (L = 95.26, a = −0.89, b = 1.18) [36], the overall color difference (ΔE*) was calculated using Equation (7).

$$\mathsf{\Delta }\mathrm{E}\mathrm{*}=\sqrt{{\left(\mathrm{L}-\mathrm{L}\mathrm{*}\right)}^2+{\left(\mathrm{a}-\mathrm{a}\mathrm{*}\right)}^2+{\left(\mathrm{b}-\mathrm{b}\mathrm{*}\right)}^2}$$ (6)

In the formula, L*, a*, and b* represent the color coordinates of the films, while L, a, and b denote those of the standard white reference plate.

2.2.5. Microstructure Characterization

Field Emission Scanning Electron Microscope (FE-SEM)

The surface morphology of the films was observed using a SU8010 high-resolution FE-SEM (Beijing, China). Prior to imaging, the samples were sputter-coated with a thin gold layer to improve electrical conductivity [46]. Images were captured at an accelerating voltage of 3.0 kV under high vacuum conditions [47,48], at a working distance of 9.0 mm, with magnifications of 10,000× and 30,000×.

Fourier Transform Infrared Spectroscopy (FTIR)

The physicochemical interactions among film-forming components during soaking and drying were investigated using a Nicolet iS50 FTIR spectroscopy (Waltham, MA, USA) [49]. Such molecular interactions alter the local electron density and vibrational frequencies, which are reflected in the FTIR spectra through shifts and intensity variations in characteristic absorption peaks. All measurements were performed with air as background, over a wavenumber range of 800~4000 cm⁻¹ at a resolution of 2 cm⁻¹, with 64 scans accumulated per sample.

Thermogravimetric Analysis (TGA)

TGA was employed to evaluate the thermal properties of the sample films, including their weight-loss behavior and thermal degradation characteristics [50], as well as to assess the correlation between temperature and sample mass. During heating, any mass loss of the films due to sublimation, vaporization, decomposition, or removal of crystalline water in the film can be detected. In this experiment, approximately 3.0 mg of each film sample was weighed and placed into a crucible of a TGA/DSC 1100 SF thermogravimetric analyzer (Columbus, OH, USA). Under a nitrogen atmosphere with a flow rate of 35 mL/min, the temperature was raised at a rate of 10 °C/min while the mass change in the samples was recorded over the range of 30 to 500 °C. In the resulting TG curve, mass is plotted on the vertical axis (decreasing from top to bottom), while temperature is displayed on the horizontal axis (increasing from left to right).

2.2.6. Antibacterial Activity

EGCG exhibits wide -spectrum antibacterial activity against both S. aureus and E. coli, which are major food spoilage microorganisms [24]. Therefore, S. aureus (ATCC 25923) and E. coli (ATCC 25922) were selected as the test microorganisms in this study to evaluate the antibacterial properties of the prepared films [51,52].

Solid Culture Method

The antibacterial effects of the films against two major food spoilage bacteria, S. aureus and E. coli, were evaluated using the agar-based disk diffusion method [51,52]. Briefly, 20 mL of sterilized sterile solid LB medium (containing 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 20 g/L agar, pH 7.30) was transferred into 90 mm diameter Petri dishes to form a uniform layer. After solidification, 100 μL of diluted bacterial suspension with an optical density at 600 nm (OD₆₀₀) of 0.2 (approximately 10⁸ CFU/mL) was evenly spread onto the surface of the solid medium and allowed to stand for 20 min. Subsequently, sample films (5 mm in diameter) that had been UV-irradiated for 30 min were placed onto the solid medium surface. All media were incubated at 5 °C for 2 days. After incubation, the antibacterial activity of the films was assessed by measuring the diameter of the inhibition zone formed around the films, with each sample tested in triplicate.

Liquid Culture Method

The S. aureus suspension was diluted with sterile culture medium to approximately 10⁸ CFU/mL (corresponding to OD₆₀₀ = 0.2). Subsequently, a 200 µL of the diluted bacterial suspension and a sample film disk (5 mm in diameter) were added into each well of a sterilized 96-well plate, followed by incubation at 5 °C for 24 h. The same procedure was applied for E. coli. After incubation, the OD₆₀₀ value of the bacterial suspension in each well was measured using a Multiskan SkyHigh microplate reader (Waltham, MA, USA) to determine the antibacterial rate of the films, with the corresponding calculation formula presented below:

$$\mathrm{A}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)={\displaystyle \frac{\mathrm{C}-\mathrm{A}}{\mathrm{C}}}\times 100\%$$ (7)

In the formula, C and A represent the OD₆₀₀ values of the control and the experimental groups (active films), respectively.

2.2.7. Biodegradability

As increasing amounts of packaging waste are entering landfills, there is a growing expectation that their degradation time can be significantly shortened. This would help minimize the landfill space required and allow more packaging waste to be accommodated [53]. Biodegradability denotes to the ability of a material to be degraded by microorganisms in nature, such as bacteria, fungi, and algae. The degradation rate depends on various factors, including environmental conditions (e.g., temperature, humidity, and pH value), the chemical structure of the material, as well as the type and abundance of microorganisms [1,39]. To investigate the biodegradability of the films, soil burial degradation tests were carried out. Film samples with a mass of approximately 40 mg were buried in natural soil at a depth of 15 cm (at a temperature of 24~28 °C and a relative humidity of 40%~50%). The samples were collected every 7 days until the films were completely degraded. After collection, the films were cleaned of surface soil, and dried in an oven at 50 °C for 24 h before being weighed, with the corresponding calculation formula presented below:

$$\mathrm{B}\mathrm{i}\mathrm{o}\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} (\%)={\displaystyle \frac{{\mathrm{W}}_0-\mathrm{W}}{{\mathrm{W}}_0}}\times 100\%$$ (8)

In the formula, W₀ and W represent the mass of the film samples before and after soil burial, respectively.

2.3. Data Analysis

All experiments were conducted in triplicate, and the results are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using IBM SPSS Statistics v20. Statistical differences among treatments were assessed by one-way analysis of variance (ANOVA), combined with the Tukey test and Duncan’s multiple range test, with a significance level set at p < 0.05.

3. Results and Discussion

3.1. Physical Performance Analysis

3.1.1. Film Thickness and Mechanical Properties

The films’ thicknesses and mechanical properties are presented in Table 2 and Figure 1. Compared with the control film (S-b, thickness: 0.0236 mm; TS: 125.47 MPa), the incorporation of EGCG did not significantly alter either the thickness or the TS of the active films (p > 0.05). Specifically, the thickness of the active films ranged from 0.0236 to 0.0241 mm, while TS values varied between 124.34 and 126.42 MPa. This lack of significant change is likely due to the low concentration of EGCG used. Notably, the TS values of active films were significantly higher than the threshold specified in GB/T 28118-2011 (TS ≥ 35 MPa), indicating their excellent tensile strength performance. The EB values of the S-c film and the S-d film were not significantly different from that of the control film (p > 0.05). However, the EB value of the S-e film decreased by 12% relative to the control, indicating an increase in film brittleness. Previous studies [54] have suggested that the effect of EGCG on the mechanical properties of film is concentration-dependent. An appropriate concentration of tea polyphenol (TP) can enhance the mechanical properties of the film [55], whereas inappropriate amounts may induce molecular aggregation and consequently impair its mechanical performance [56]. For instance, Lei et al. [54] developed pectin-konjac glucomannan composite films introduced with TP. The study suggested that with TP concentration increased from 0 to 5%, the TS value of the composite films rose first and then fell, while the EB value decreased continuously. Siripatrawan et al. [55] reported that green tea extracts (GTE) significantly affected the mechanical properties of chitosan-based films. TS and EB showed no significant changes as the GTE concentration rose from 0 to 5%, but significantly enhanced when the GTE concentration increased from 5% to 20%. Peng et al. [56] reported that both the TS and EB values significantly decreased when tea extracts at concentrations of 0.5%, 1%, and 2% were incorporated into chitosan films. Therefore, the effect of EGCG, as one of the tea polyphenols, on the mechanical properties of films cannot be generalized, as it depends primarily on its concentration and the specific film matrix [54].

Table 2.

Film thickness and mechanical properties.

Film	Thickness (mm)	TS (MPa)	EB (%)
S-a	0.0228 ± 0.0011 a	76.63 ± 1.12 b	1.89 ± 0.08 c
S-b	0.0236 ± 0.0004 a	125.47 ± 5.02 a	16.58 ± 0.95 a
S-c	0.0236 ± 0.0005 a	124.34 ± 4.07 a	16.96 ± 0.97 a
S-d	0.0241 ± 0.0004 a	126.42 ± 3.48 a	17.11 ± 0.34 a
S-e	0.024 ± 0.0001 a	125.82 ± 3.06 a	14.57 ± 1.04 b

Note: Mean values with different letters indicate a significant difference (p < 0.05). The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Figure 1.

Mechanical properties of the BC-based films. (A) Tensile strength (TS, MPa) and elongation at break (EB, %) of the BC-based films; (B) Stress-strain curves of the BC-based films. Mean values with different letters indicate a significant difference (p < 0.05). Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

3.1.2. Barrier Properties

As shown in Figure 2, compared with pure BC film (S-a) with an OTR of 1.01 cm³/(m²·d·atm) and a WVP of 0.019 g·m/(m²·d·kpa), the control sample (S-b) exhibited a higher OTR of 3.24 cm³/(m²·d·atm) and WVP of 0.032 g·m/(m²·d·kpa). This increase is attributed to the incorporation of Gly, a hydroxyl-rich small-molecule plasticizer used to enhance the EB of the BC-based films [57]. Gly readily infiltrates the three-dimensional porous network of the BC gel film [58], replacing water molecules and forming hydrogen bonds with cellulose polymer chains. Unlike water, Gly does not evaporate during drying but remains within the BC matrix, hindering the assembly and crystallization of BC chains. This results in enlarged interchain distances and a less compact microstructure [59,60], thereby facilitating the permeation of oxygen and water vapor.

Figure 2.

Barrier properties of the BC-based films. Mean values with different letters indicate a significant difference (p < 0.05). Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Compared with the control film, the WVP and OP values of the active films with an EGCG concentration of 0.1% to 0.3% increase to 0.0356~0.0376 g·m/(m²·d·kpa) and decrease to 2.43~2.71 cm³/(m²·day·atm), respectively. The incorporation of EGCG led to a certain increase in the WVP of the active films, whereas their OTR values decreased significantly (p < 0.05). This phenomenon can be attributed to the abundance of hydrophilic groups (e.g., phenolic hydroxyls) in EGCG, which can form extensive hydrogen bonds with BC and XG. Meanwhile, the unbound hydroxyl groups may also attract water molecules, thereby promoting water vapor transmission [61]. Notably, the OTR values of active films were significantly lower than the threshold specified in GB/T 28117-2011 (OTR ≤ 20 cm³/(m²·day·atm)), indicating their excellent oxygen barrier property. Due to the extensive hydrogen bonding among BC, XG, and EGCG, the active films exhibit a denser microstructure than the control film, thereby impeding oxygen diffusion through the active films [62]. These morphological changes in the active films can be observed by FE-SEM established on a qualitative, trend-based level, as shown in Figure 3. It is widely recognized that the dense microstructure of the food packaging film can reduce oxygen permeability, thereby slowing the oxidation of lipids, proteins, and other food components, which ultimately contributes to extending the shelf life of food products [38,39].

Figure 3.

FE-SEM micrographs of film surface at magnifications of 10,000× (column 1) and 30,000× (column 2). Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

3.1.3. Optical Properties

As is well known, films with UV blocking properties can prevent photoinduced degradation of nutrients such as lipids and proteins in food, thereby more effectively inhibiting lipid oxidation and extending the shelf life of packaged food products [63,64]. Figure 4 and Table 3 present the transmittance and opacity of film samples over the wavelength range of 200~800 nm. It was observed that the incorporation of EGCG (0.1%~0.3%) significantly enhanced the UV blocking performance of the active films (p < 0.05). Compared with the control film, the UV transmittance of the active films decreased by 96.77%~99.56% (p < 0.05), 77.66%~93.3% (p < 0.05), and 14.91%~27.77% (p < 0.05) in the UV-C, UV-B, and UV-A regions, respectively. The reduction in UV transmittance was negatively correlated with EGCG concentration, further confirming the outstanding UV-shielding capacity of EGCG. Liu et al. [63] similarly reported that EGCG significantly improves the UV absorption capacity of gelatin-based films. This phenomenon is attributed to the strong UV absorption peak of EGCG at 280 nm from its aromatic benzene ring structure [65,66], which contributes to retarding lipid oxidation in food products and thereby extending their shelf life. Moreover, the opacity of the active films exhibited no significant difference from that of the control film, indicating that low concentrations of EGCG do not alter the opacity of the films. These results confirm that the incorporation of EGCG effectively improves the UV-blocking capacity of the active films while maintaining opacity comparable to the control.

Figure 4.

Transmittance (A) and opacity (B) of the BC-based films. Mean values with different letters indicate a significant difference (p < 0.05). Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Table 3.

UV transmittance and opacity of the BC-based films.

Film	Transmittance (%)			Opacity (%)
	UV-C	UV-B	UV-A
S-a	0.28 ± 0.02 b	0.87 ± 0.09 e	2.36 ± 0.19 d	23.19 ± 0.12 a
S-b	6.82 ± 0.26 a	17.46 ± 0.34 a	25.89 ± 0.3 a	12.42 ± 0.58 b
S-c	0.22 ± 0.02 b	3.9 ± 0.3 b	22.03 ± 0.77 b	11.31 ± 0.64 b
S-d	0.04 ± 0.01 c	1.79 ± 0.12 c	18.86 ± 0.87 c	11.37 ± 0.71 b
S-e	0.03 ± 0.00 c	1.17 ± 0.27 d	18.7 ± 0.4 c	12.01 ± 0.66 b

Note: Mean values with different letters indicate a significant difference (p < 0.05). The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

The color parameters of the films are summarized in Table 4. The control film exhibited L*, a*, b*, and ΔE* values of 93.13, −0.21, 3.17, and 1.66, respectively. The incorporation of EGCG significantly lowered the L* value and increased the a*, b*, and ΔE* values of the active films (p < 0.05). Notably, the rise in b* and ΔE* values reflect a yellowish tint in the active films, attributable to the inherent pale-yellow color of EGCG [67,68]. Similar color changes induced by EGCG have also been reported in other bio-based films. For instance, Krisana et al. [68] observed that adding EGCG significantly lowered the L* value and increased the a*, b*, and ΔE* values of gelatin-based films (p < 0.05). Sun et al. [69] similarly noted that the incorporation of EGCG raised the b* and ΔE* values in the konjac glucomannan/carboxymethyl chitosan composite film, accompanied by a decrease in L* value. Dou et al. [65] investigated the effect of tea polyphenols (0~2.5 wt%) on the color of gelatin/sodium alginate edible films. They observed that the L* value decreased from 94.06 to 92.61, whereas a* and b* values increased from 0.13 to 1.15 and from 2.90 to 5.16, respectively, leading to slight darkening and yellowing of the films.

Table 4.

Color parameters (L*, a*, b* and ΔE*) of BC-based films.

Film	Color Parameters				Picture
	L*	a*	b*	ΔE*
S-a	92.69 ± 0.71 a	0.09 ± 0.01 d	2.7 ± 0.08 c	1.51 ± 0.23 c
S-b	93.13 ± 0.05 a	0.21 ± 0.02 d	3.17 ± 0.05 c	1.66 ± 0.05 c
S-c	90.38 ± 0.51 b	0.34 ± 0.03 c	8.29 ± 0.71 b	7.27 ± 0.86 b
S-d	89.54 ± 0.06 c	0.63 ± 0.07 b	8.4 ± 0.03 b	7.77 ± 0.04 b
S-e	89.26 ± 0.3 c	1 ± 0.27 a	9.28 ± 0.41 a	8.75 ± 0.46 a

Note: Mean values with different letters indicate a significant difference (p < 0.05). The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Although the active films show mild yellowing in this study, the color remained soft and did not impair their transparency, a characteristic considered acceptable for practical film applications.

3.2. Microstructure Analysis

3.2.1. FE-SEM

The morphological characteristics of film are closely correlated with their barrier performance. The surface morphology of the films is shown in the FE-SEM images (Figure 3) at magnifications of 10,000× (column 1) and 30,000× (column 2). The porous structure of the control group (S-b) could be clearly observed at a magnification of 30,000× (Column 2). Compared with the control, the incorporation of EGCG resulted in a tighter arrangement of BC fibers, a reduction in pore size, and a decrease in porosity within the active films, thus accounting for the corresponding decrease in the OP values of the active films. These morphological changes observed by FE-SEM are closely related to the enhanced hydrogen bonding in the BC-based active film, which is attributed to the abundant hydroxyl groups from EGCG [21,22]. Therefore, the additional hydrogen bonding interactions are formed due to the incorporation of EGCG, which can be fully verified and interpreted by the FTIR characterization results (Figure 5).

Figure 5.

(A) FTIR spectra in the wavenumber range of 800~4000 cm⁻¹; (B) magnified FTIR spectra in the wavenumber range of 800~2000 cm⁻¹; (C) the hydroxyl peak area index (I_a(OH)) within the range of 3100~3700 cm⁻¹. Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e). Mean values with different letters indicate a significant difference (p < 0.05).

3.2.2. FTIR

As shown in Figure 5A, the FTIR spectra of the active films (S-c, S-d, S-e) closely resembled that of the control group (S-b), indicating that the incorporation of EGCG did not alter the inherent structure of the control film. However, the incorporation of EGCG promoted significant changes in the O-H and C-H characteristic peaks of the active films. Specifically, the hydroxyl peak area index (Iₐ(OH)) within the range of 3100~3700 cm⁻¹ increased significantly (p < 0.05), from 1.79 to 2.14 (Figure 5C), indicating a denser and stronger hydrogen bond interactions within the active films. This enhanced hydrogen bonding promoted strong physical cross-linking among EGCG, BC, and XG, thereby promoting the formation of a tighter arrangement of BC fibers, a reduction in pore size, and a decrease in porosity within the active films, which could only be observed under magnification of 30,000× in the FE-SEM images (Figure 4, column 2). Furthermore, the C-H stretching vibration of CH₂ groups shifted from 2892 cm⁻¹ in the control to 2888 cm⁻¹ in the active films. In conclusion, these spectral changes collectively suggest the presence of physical interactions among XG, BC, and EGCG.

3.2.3. TGA

Figure 6 and Table 5 illustrate the thermal degradation behavior of BC-based films across all three weight-loss stages. First, across the temperature range of 30~110 °C, the active films showed an initial weight loss of 1.61%~2.1%, which is attributed to the volatilization of absorbed free water [70]. The second stage of mass loss for the active films, ranging from 39.82% to 41.94%, occurred between 110 °C and 270 °C, followed by a third stage with a mass loss of 27.52%~32.26% from 270 °C to 440 °C.

Figure 6.

TGA (A) and DTG (B) curves of the BC-based films. Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Table 5.

Thermal analysis of the BC-based films.

Film	Phase 1		Phase 2		Phase 3		T1 (°C)	R (%)
	T (°C)	M (%)	T (°C)	M (%)	T (°C)	M (%)
S-a	30~273	1.64	273~446	84.75	-	-	355	15.25
S-b	30~110	2.74	110~260	36.37	260~440	35.96	188/326	24.93
S-c	30~112	2.1	112~270	39.82	270~440	32.26	331	25.82
S-d	30~121	1.61	121~263	41.94	263~440	31.58	336	24.87
S-e	30~100	1.78	100~270	41.24	270~440	27.52	338	29.46

Note: T represents temperature; M denotes mass loss; T1 is the temperature at maximum degradation rate; R refers to residual mass. The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Compared with the control group (326 °C), the active films exhibited a higher temperature at maximum degradation rate, which increased from 331 °C to 338 °C and showed a positive correlation with EGCG concentration. In addition, the residual mass of the active films ranged from 24.87% to 29.46%, whereas that of the control group was 24.93%. In summary, the altered thermal degradation behavior of the active films is attributed to the formation of a denser and stronger hydrogen bond interactions among EGCG, BC, and XG [62], as revealed by FTIR characterization. The formation of the denser and stronger hydrogen bond interactions is evidenced by a significant increase in the Iₐ(OH) and a shift in the C-H stretching vibration in the active films, as shown in Figure 5 B. Accordingly, these interactions restrict the segmental mobility of the polymer chains and delay the thermal cleavage of hydroxyl groups, glycosidic bonds, and the carbon skeleton structure [71,72,73], thereby enhancing the thermal stability of the active films.

3.3. Antibacterial Activity Analysis

The antibacterial activity of the active films against S. aureus and E. coli was assessed using an agar disk diffusion assay, as shown in Figure 7. Obviously, no inhibition zone was observed for the control group against either S. aureus or E. coli, confirming its lack of antibacterial activity. In contrast, the active films exhibited significant antibacterial effects, with the efficacy positively correlated with the concentration of EGCG. Specifically, the diameters of the inhibition zones enlarged progressively against both S. aureus (from 13.88 to 16.25 mm, p < 0.05; Figure 7A) and E. coli (from 12.38 to 14.13 mm; Figure 7B). Correspondingly, the antibacterial rate of the active films increased from 61.4% to 80.61% (p < 0.05) against S. aureus and from 57.38% to 60.38% against E. coli (Figure 8). These findings demonstrate that EGCG possesses significant antibacterial activity, which can be attributed to its multi-faceted mechanisms, including disrupting bacterial cellular structures, inhibiting fatty acid synthesis, and suppressing enzymatic activity [74].

Figure 7.

Example of inhibition zones of S. aureus (A) and E. coli (B). Note: The films were designated as follows: BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Figure 8.

Inhibition zone (A) and antibacterial rate (B) of active films. Mean values with different letters indicate a significant difference. Note: The films were designated as follows: BC/1% XG/2% Gly composite film (S-b), BC/1% XG/2% Gly/0.1% EGCG active film (S-c), BC/1% XG/2% Gly/0.2% EGCG active film (S-d), and BC/1% XG/2% Gly/0.3% EGCG active film (S-e).

Furthermore, the antibacterial efficacy of EGCG against S. aureus was superior to that against E. coli, indicating that S. aureus is more sensitive to EGCG than E. coli. This difference can be attributed to the thinner cell wall of S. aureus compared to the more complex outer membrane structure of Gram-negative E. coli [75,76]. Specifically, the sophisticated outer membrane of E. coli, composed of polysaccharides, proteins, and lipopolysaccharide, which provides a more effective barrier against the infiltration of active substances [77]. Jamróz et al. [77] observed that alginate/gelatin films containing green tea or black tea extracts exhibited stronger antibacterial activity against S. aureus than against E. coli. Similarly, starch films incorporated with tea polyphenols demonstrated superior efficacy against S. aureus [74]. These previous reports support the present finding that the antibacterial efficacy against S. aureus is greater than that against E. coli.

3.4. Biodegradability Analysis

Given the environmental impacts of packaging waste, the biodegradability of packaging materials has become a crucial factor in the development, evaluation, and practical application of sustainable packaging [78]. Soil burial is a widely employed disposal method [79], during which microorganisms attack and cleave cellulose chains via metabolic and enzymatic activities. These chains are gradually broken down into lower molecular weight components, leading to a reduction in mass [80].

In this study, the biodegradability of BC film (S-a), control group (S-b), and active film (S-d) was evaluated and compared. As shown in Figure 9, the pure BC film exhibited the slowest degradation rate, with mass losses of 11.03%, 45.19%, and 91.09% after 7, 14, and 21 days of soil burial, respectively. After 7 days of soil burial, the degradation rates of films S-b and S-d reached 51.75% and 43.41%, respectively, which were 3.69 and 2.94 times higher than that of film S-a. By the 14th day of soil burial, the degradation rates of films S-b and S-d had risen to 95.94% and 93.54%, corresponding to 0.93 and 1.07 times that of the pure BC film, respectively. Finally, films S-b and S-d achieved complete degradation after 21 days of soil burial, whereas the pure BC film exhibited partial degradation, with a mass loss rate of 91.09%. These findings indicate that the incorporation of XG and EGCG significantly enhances the biodegradability of the BC-based films. Throughout the soil burial period, all films gradually developed cracks, exhibited shrinkage, and underwent a color change from milky white to yellowish (Figure 9). The degradation process involves the synergistic effects of soil moisture and soil microorganisms. Water molecules disrupt hydrogen bonds between polymer chains, thereby facilitating microbial colonization and enzymatic breakdown, which collectively accelerate the overall degradation of the films [53]. Compared with other biopolymer-based films reported in the literature, the BC-based active films in this work demonstrate excellent biodegradability. For example, Wrońska et al. [81] reported that chitosan/metal oxide composite films required a minimum of six weeks to achieve complete degradation in soil. Wang et al. [82] noted that polylactic acid (PLA) materials took a longer period of approximately 70 days to fully decompose. Whereas, Pérez et al. [83] observed an even lengthier duration of 80 days for a polyhydroxyalkanoate (PHA) film derived from peanut oil. In a study assessing degradation rates, Riaz et al. [84] found that chitosan films containing chive root extract (CRE) lost 47.36% of their mass after three weeks, in contrast to only 26.98% for plain chitosan films.

Figure 9.

Biodegradability (A) and physical appearance (B) of BC-based films. Mean values with different letters indicate a significant difference. Note: The films were designated as follows: BC film (S-a), BC/1% XG/2% Gly composite film (S-b), and BC/1% XG/2% Gly/0.2% EGCG active film (S-d).

In summary, the findings of this study are consistent with the packaging design philosophy oriented toward green, healthy, and cost-effective solutions, thereby offering a promising strategy for alleviating the environmental burden associated with plastic packaging waste.

4. Conclusions

This study demonstrates that EGCG can be effectively incorporated into biodegradable BC-based films, enhancing their antibacterial activity, particularly against S. aureus and E. coli. The incorporation of EGCG preserved the tensile strength of the BC-based active films without compromising its opacity. Furthermore, it improved the barrier performance against oxygen and UV of the BC-based active films. SEM analysis revealed a tighter arrangement of BC fibers, a reduction in pore size, and a decrease in porosity within the active films. FTIR spectroscopy confirmed that the incorporation of EGCG enhanced the area of the hydroxyl absorption peak, and shifted the wavenumber of C–H stretching vibration band associated with CH₂ groups, which suggests the presence of physical interactions among XG, BC, and EGCG. More importantly, the active films exhibited significant antibacterial effects, with the efficacy positively correlated with the concentration of EGCG. Specifically, the diameters of the inhibition zones enlarged progressively against both S. aureus (from 13.88 to 16.25 mm, p < 0.05) and E. coli (from 12.38 to 14.13 mm). Correspondingly, the antibacterial rate of the active films increased from 61.4% to 80.61% (p < 0.05) against S. aureus and from 57.38% to 60.38% against E. coli. Additionally, the BC-based active films developed in this work exhibit excellent biodegradability, being capable of achieving complete biodegradation within 21 days of soil burial.

This work underscores the potential of BC-based active films for food packaging applications, laying a solid foundation for the development of novel food packaging systems with highly efficient antimicrobial properties. In future work, we expect to evaluate the BC-based active films on meat products, such as pork, beef, etc., to assess the effect on the shelf life of the food.

Author Contributions

Conceptualization, R.Z., C.G. and Z.L.; methodology, R.Z., C.G. and Z.L.; software, Q.L. and Z.L.; validation, R.Z. and C.G.; formal analysis, R.Z. and Q.L.; investigation, R.Z., C.G. and W.F.; resources, J.D. and Q.Z.; data curation, R.Z. and Q.L.; writing—original draft preparation R.Z. and Z.L.; supervision, X.C. and Q.Z.; project administration W.F. and X.C.; funding acquisition, Q.Z. and R.Z. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was supported by the Key Research and Development Projects of Shandong Province (Grant No. 2023CXGC010315); the Postdoctoral Fellowship Program of CPSF (Grant No. GZC20250291); the China Postdoctoral Science Foundation (Grant No. 2025M780451); and the Independent Innovation Research Program of China University of Petroleum (East China) (Grant No. 25CX06053A).