Development of PLA-PVA/LZ cellulose pads with antimicrobial activity and their application in instant cooked rice preservation

Abstract

This study developed an antibacterial pad by immobilizing lysozyme (LZ) within porous cellulose through polyvinyl alcohol (PVA) cross-linking, reinforced with polylactic acid (PLA) barrier layers. The PLA-PVA/LZ pad demonstrated significantly enhanced structural properties, including a 65.52% increase in TS, a 78.88% rise in WCA, and an 87.62% reduction in water absorption. At an optimal loading of 4% LZ, the pads exhibited potent antibacterial efficacy, inhibiting Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) growth by 93.93% and 86.73%, respectively. Mechanistic investigations revealed that the pad disrupted bacterial cell morphology, compromised membrane integrity, suppressed ATPase activity, and elevated AKP levels. When applied to cooked rice stored at 25 °C, the pad effectively maintained microbial counts below the detection limit for 48 h, extending shelf life by at least 12 h according to Chinese national standards.

Highlights

• •PLA enhances hydrophobicity PVA immobilizes lysozyme boosting antibacterial stability.
• •Multilayer cellulose PVA lysozyme PLA integration enables functional synergy.
• •Development of antimicrobial pad for preserving cooked rice at room temperature.
• •The antimicrobial mechanism of the pad against bacteria was investigated.

1. Introduction

Rice is a fundamental staple food in Asia and Southeast Asia, serving as a major source of nutrition for the human population (Verma & Srivastav, 2020). In recent years, instant cooked rice has grown increasingly popular among consumers owing to its convenience and ease of preparation(Li et al., 2022). It effectively preserves a texture and flavor similar to those of freshly cooked rice.

Driven by increasing market demand, the global instant cooked rice industry has experienced rapid growth (Li et al., 2022). Statistics show that annual demand for instant cooked rice in China has approached 40 billion units (Meng et al., 2019). Nonetheless, packaged instant cooked rice generally has a shelf life less than 2 days. Throughout transportation and distribution, external factors, including temperature variation, light exposure, enzymatic reactions, and microbial contamination, can adversely affect product quality, leading to a marked reduction in shelf life. Such constraints hinder the long-term expansion and advancement of the instant cooked rice industry. Refrigeration is commonly used to delay spoilage and extend the shelf life of cooked rice. However, the high starch content in cooked rice promotes starch retrogradation under cold storage conditions, resulting in textural hardening and a decline in flavor quality (Ma & Sun, 2009).

Antimicrobial pads, as an emerging active packaging material, provide new ideas to solve this problem. Among them, lysozyme (LZ), a natural enzyme with well-documented antibacterial properties, is particularly notable for its ability to inhibit the growth of pathogenic bacteria such as Staphylococcus aureus (S. aureus) (Kim et al., 2020), making it a promising active component in the development of antibacterial pads. However, the practical application of free LZ is limited by its poor stability and susceptibility to inactivation under the complex conditions of food systems(Zhang, Li, et al., 2024), and its performance needs to be improved by immobilization techniques.

Cellulose, as a biodegradable bio-based packaging material, exhibits high mechanical strength and features abundant porous structures(Khin et al., 2025; Mehmood et al., 2025), provides an ideal immobilization matrix by offering favorable loading space for LZ (Fu et al., 2025). However, conventional physical adsorption methods often lead to LZ desorption during storage due to weak interactions between cellulose and LZ, thereby reducing antimicrobial efficacy. Chemical immobilization, although effective, involves complex procedures and presents environmental risks, which limit its large-scale application (Bayazidi et al., 2018; Buruaga-Ramiro et al., 2020).

Polyvinyl alcohol (PVA) presents a viable approach to addressing this paradox. Wang G.Z et al. (Wang et al., 2008) found that PVA can be used as an additive to protect protein stability. Simona Morariu et al. (Morariu et al., 2023) further demonstrated that the interactions between PVA and LZ do not affect the higher-order structure of LZ. This finding provides valuable insights for the immobilization of LZ within cellulose matrices. However, the composite system of PVA and cellulose is excessively hydrophilic, which may result in the inactivation of antimicrobial agents and promote microbial growth (Rui et al., 2024; Zhang, Yu, et al., 2024). Therefore, hydrophobic modification is necessary.

In this study, a multilayered antibacterial pad was developed using LZ as the antibacterial agent, cellulose pads as the support substrate. PVA was utilized to immobilize LZ, while Polylactic acid (PLA) was applied as a water barrier layer, coated on both sides of the pad using a simple physical coating technique. The hydrophobicity, microstructure, and mechanical properties of the prepared antimicrobial pad were characterized. This study also investigated the antibacterial mechanism of the antibacterial pad using dominant spoilage bacteria in rice as the model organisms, and explored its potential for practical application in cooked rice preservation.

2. Materials and methods

2.1. Materials

LZ (25,000 U/mg) was purchased from Nanning Pangbo Biological Engineering Co., Ltd. (Nanning, China). PLA (molecular weight of 80 kDa) was provided by Shanghai MackLin Bio-chemical Technology Co., Ltd. (Shanghai, China). PVA (fineness: 1788) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). CH₂Cl₂ was purchased by China Pharmaceutical Group Co., Ltd. (Wuhan, China). Cellulose pads were provided by Shanghai Tongbei Absorbent Material Qidong Co., Ltd. (Shanghai, China). Staphylococcus aureus (S. aureus, CICC (B) 44,102) and Escherichia coli (E. coli, CICC (B) 26,003) were obtained from China Medical Bacterial Strain Preservation and Management Center (Beijing China). Ultra-micro Na⁺, K⁺-ATPase detection kit and AKP detection kit were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit bought from Biosharp (Beijing, China). All other chemicals were of analytical grade, and deionized water was utilized in experiments.

2.2. Preparation of PLA-PVA /LZ pads

As illustrated in Fig. 1A, the antibacterial pad consists of three layers: the cellulose pad as the substrate material, the PVA/LZ layer as the antibacterial layer, and the PLA as the hydrophobic layer. The preparation of the PVA /LZ and PLA solutions was carried out according to the following method.

Fig. 1.

(A) Schematic illustration of the preparation of PLA-PVA/LZ-based antibacterial pads using layer-by-layer assembly technique. (B) Application schematic of antibacterial pads in instant cooked rice.

First, 2 g of PVA and 4 g of LZ were added to 100 mL of distilled water and stirred at 40 °C on a magnetic stirrer set to 800 rpm for 30 min. After the PVA and LZ were fully dissolved, the solution was cooled to room temperature and degassed in an ultrasonic bath for 5 min to obtain the PVA/LZ solution.

Next, 2 g of PLA was dissolved in 100 mL of dichloromethane and stirred at 40 °C on a magnetic stirrer set to 800 rpm for 30 min. The solution was then degassed in an ultrasonic bath for 5 min to obtain the PLA solution.

A precisely measured 5 mL of the PVA/LZ solution was applied to the surface of a cellulose pad measuring 70 mm × 120 mm, which was placed in a mold of the same dimensions to ensure uniform boundary control. The solution was evenly spread using a sterile L-shaped spreader in a standardized pattern (10 horizontal and 10 vertical strokes) under consistent gentle pressure to achieve a homogeneous coating. The coated pad was then dried using a blow dryer (CMJ04LXW, Xiaomi Corporation Co., Ltd., China) with an air velocity of 20 m/s and a temperature of 40 °C for 8 min. This process was repeated three times to achieve a total coating of 15 mL of the PVA/LZ solution, resulting in the formation of a uniform PVA/LZ antibacterial layer. Subsequently, both sides of the PVA/LZ-coated pad were coated with 5 mL of the PLA solution per side, following the same coating and drying procedure, to form PLA layers on both surfaces.

Additionally, 2 g of PVA was dissolved in 100 mL of distilled water to obtain a PVA solution. Following the previously described steps, 15 mL of the PVA solution was applied to the surface of the cellulose pad in three coats. After the surface had dried, a PLA solution (5 mL) was applied to both sides of the pad, and the coating was dried using a blow dryer, resulting in the experimental control group PLA-PVA, Simultaneously, 4 g of LZ was dissolved in 100 mL of distilled water to prepare the LZ solution. Following the same procedure, 15 mL of the solution was applied to the cellulose pad to fabricate another set of experimental control LZ pads. The untreated original cellulose pad was used as a blank control.

Finally, all coated pads were placed in a vacuum drying oven (OV4–65, Lab Companion Co., Ltd., China) and dried for 12 h at 45 °C to remove the organic solvent.

In addition, Fig. 1B illustrates the application of the developed antimicrobial pads in the storage of instant cooked rice.

2.3. Physicochemical characterization of the PLA-PVA /LZ pads

2.3.1. WCA

The sample pad is designed for the preservation of instant cooked rice, where it is expected to effectively inhibit microbial growth while preventing the absorption of moisture from the instant cooked rice itself. Excessive hydrophilicity of the pad would cause active water uptake from the rice, which could lead to moisture loss and hardening of the rice grains, thereby compromising the overall eating quality. The prepared PLA–PVA/LZ pads were randomly cut into square specimens (20 mm × 20 mm). Prior to testing, all test samples were conditioned at 25 °C and 50% relative humidity for at least 24 h to reach equilibrium. The equilibrated samples were then fixed on glass slides, and the WCA was measured using a contact angle analyzer (JY-PHA, Chengde Youte Testing Instrument Manufacturing Co., Ltd., China). A 5 μL droplet of ultrapure water was carefully placed on the sample surface using a micro-syringe, and the droplet image was immediately captured by a high-resolution digital camera. For each group, at least five different samples were tested, and three random positions were measured on each sample. The average contact angle across the film surface was then calculated and reported.

2.3.2. Water uptake

The water uptake capacity of the prepared pads was evaluated following the method described by Sanyang et al. (Sanyang et al., 2016). In brief, the pads were cut into 50 mm × 50 mm pieces and dried in an oven (GZX-9070MBE, Shanghai Jiecheng Experimental Instrument Co., Ltd., China) at 105 °C for 24 h. After drying, the mass of the sample pad was weighed and recorded as M₁. The sample pad was then immersed in 50 mL of distilled water, and soaked at 25 °C for 12 h. After soaking, the pad was gently blotted with filter paper to remove surface water, and its mass was accurately measured and recorded as M₂. The water uptake rate of the sample pad was calculated according to Eq. (1).

$$\text{Water uptake rate}\left(\%\right)=M_2-M_1/M_1\times 100\%$$ (1)

where M₁ (g) is the mass of the dry sample pad, M₂ (g) is the mass of the sample pad after soaking.

2.3.3. Mechanical properties

Following the method of L. Zhang et al.(Zhang et al., 2023) with modifications, the TS and EAB of the sample pads were measured using a texture analyzer (TA.XTC-18, Shanghai Baosheng Industrial Development Co., Ltd., China). Before testing, the sample pads were equilibrated for 48 h at 60% relative humidity. The equilibrated pads were then cut into strips (20 mm × 60 mm) and fixed on a TA-DGA probe for tensile testing. The target displacement was set to 50 mm, the trigger point load to 0.1 N, and the test speed to 0.5 mm/s. Each sample underwent 8 repetitions. The TS and EAB were calculated using the Eqs. (2), (3).

$$\mathrm{TS}\left(\mathrm{MPa}\right)=F/A$$ (2)

$$\mathrm{EAB}\left(\%\right)=L_1-L_0/L_0\times 100\%$$ (3)

where F (N) is the applied force, A (m²) is the cross-sectional area of the sample, L₀ (mm) is the initial length of the sample, and L₁ (mm) is the length of the sample after stretching.

2.4. Morphology

The surface morphologies of the sample pads were examined using a SEM (S-4800, HITACHI, Japan) at an accelerating voltage of 10.0 kV. Prior to SEM observation, the samples were fixed onto short aluminum stubs using double-sided adhesive tape and coated with a thin layer of gold via sputtering to enhance conductivity.

2.5. Antibacterial activity

The antimicrobial activity testing of antimicrobial pads was evaluated using the oscillation method specified in the Chinese national standard GB/T 20944.3–2008, and on this basis, combined with the research methodology of Suryaprabha et al. (Suryaprabha et al., 2023) with minor modifications. The sample pads with different treatments were cut into 20 mm × 40 mm strips and sterilized under UV light for 30 min to ensure sterility. Subsequently, the prepared sample pads were immersed into 5 mL of bacterial suspension at a concentration of 1 × 10⁶ CFU/mL. The cellulose pads without antibacterial treatment were used as experimental controls, and bacterial solutions without the pads served as blank controls. The bacterial solutions were then incubated in a shaker (ZHTY-50 N, Shanghai Yisai Scientific Instrument Co., Ltd. China) at 37 °C with agitation at 160 rpm for 24 h. After incubation, the absorbance of the bacterial solution at 600 nm was measured using a spectrophotometer (T6 New Century, Beijing Persee General Instrument Co., Ltd., China) to evaluate the antibacterial effect. The antibacterial efficiency was calculated using the Eq. (4).

$$\text{Antibacterial efficiency}\left(\%\right)=A_0-A/A_0\times 100\%$$ (4)

where A₀ is the absorbance of the bacterial suspension in the blank control, and A is the absorbance in the experimental group.

2.6. Antibacterial mechanism

2.6.1. Micromorphological characterization of bacteria

Antibacterial pads were cut into 20 mm × 40 mm strips and sterilized under UV light for 30 min. The sterilized pads were immersed in 5 mL of bacterial suspension (1 × 10⁶ CFU/mL) and incubated at 37 °C with shaking at 160 rpm for 24 h. Following incubation, the bacterial pellets were collected by centrifugation at 3000 g for 10 min, and subsequently washed thrice with phosphate-buffered saline (PBS, pH 7.4). For fixation, the samples were treated with 2.5% glutaraldehyde solution at 4 °C for 6 h. Subsequently, the specimens underwent sequential ethanol dehydration steps (30%, 50%, 70%, 80%, 90%, 95%, and 100% v/v) with 15 min immersion at each concentration. Finally, resuspended bacterial cells were then deposited onto silicon wafers (5 mm × 5 mm) using 3 μL aliquots, air-dried at ambient temperature. After complete air-drying, the specimens were sputter-coated with gold prior to morphological observation under a SEM.

2.6.2. Fluorescence microscopy

The antibacterial pads were cut into 20 mm × 40 mm strips, sterilized by UV for 30 min, and then immersed in 5 mL of bacterial suspension at a concentration of 1 × 10⁶ CFU/mL. The suspension was incubated at 37 °C and 160 rpm for 24 h. After incubation, the bacterial pellet was collected by centrifugation at 3000 g for 10 min, and washed three times with PBS.

To assess bacterial viability, a dual fluorescent staining protocol was performed using a Live/Dead Bacterial Viability Kit according to the manufacturer's instructions. Briefly, the bacterial pellet was stained with Calcein-AM (4 μmol/L) and propidium iodide (PI, 6 μmol/L) provided in the kit. After incubation, the bacterial pellet was collected by centrifugation at 3000 g for 6 min, and the supernatant was discarded. The bacterial pellet was resuspended in 100 μL of PBS. Then, 10 μL of the stained bacterial pellet was placed on a microscope slide for observation under a fluorescence microscope (IX53, Olympus Corporation Co., Ltd. Japan).

2.6.3. Measurement of permeability and integrity of bacterial cell membrane

Following the method described by Yi Hao et al. (Hao et al., 2023) with slight modifications, different batches of the test pads were cut into 20 mm × 40 mm strips, sterilized under UV light for 30 min, and then immersed in 5 mL of bacterial suspension at a concentration of 1 × 10⁶ CFU/mL. The suspension was incubated at 37 °C and 160 rpm for 24 h. After incubation, the bacterial suspension was centrifuged at 3000 g for 6 min, and the supernatant was carefully removed.

The protein content in the supernatant was measured using the Coomassie Brilliant Blue method (Liu et al., 2023). A 5 mL solution of Coomassie Brilliant Blue G-250 was prepared, and 1 mL of the supernatant was subsequently added to it. After standing for 3 min, the absorbance at 595 nm was measured using a microplate reader (Enspire, PerkinElmer Co., Ltd. America). Similarly, following the same procedure, the supernatant was diluted and the absorbance at 260 nm was measured using a UV spectrophotometer to detect nucleic acids.

2.6.4. AKP and ATPase activities

The activities of bacterial AKP and ATPase were determined by modifying the method described by Ning et al. (Ning et al., 2019). A 5 mL aliquot of activated bacterial culture (1 × 10⁶ CFU/mL) was centrifuged at 3000 g for 10 min to collect pellets, which were resuspended in 1 mL of sterile physiological saline. Cell lysis was achieved (200 W, 3 s ultrasonic pulses with 10 s intervals, repeated 30 times) using an ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd. China). The lysate was then centrifuged at 8000 g for 10 min at 4 °C, and the supernatant was stored on ice for subsequent analysis. The activity of AKP and ATPase was determined by referring to the instructions of Ultra-micro Na+, K + -ATPase detection kit and AKP detection kit.

2.7. Application of the PLA-PVA /LZ pads to instant cooked rice

The polished rice (Indica) was purchased from a local supermarket for the preparation of instant cooked rice. The polished raw rice was rinsed twice with purified water. Then, the cleaned rice was placed into a rice cooker with purified water at a volume ratio of 1:1.3 and cooked for 20 min to obtain the cooked rice samples. An antibacterial pad, measuring 70 mm × 120 mm, was laid flat on the bottom of a 350 mL disposable meal box and sterilized by UV irradiation for 30 min. The prepared rice was evenly portioned into the boxes at 150 g per serving. Finally, the cooked rice samples were stored at 25 °C and 50% relative humidity.

The antibacterial activity of the pads was assessed by monitoring microbial growth of the cooked rice samples during storage. The total colony count of the rice samples was determined according to the GB4789.2–2022 method. Measurements were taken every 12 h.

After 60 h of storage, a sensory evaluation was conducted to assess changes in appearance, odor, and texture of the rice. The evaluation involved 10 trained judges (5 females and 5 males) aged between 20 and 30 years. Each sample was assigned a comprehensive score. The evaluation criteria were adapted from GB/T15682–2008 with modifications. The sensory evaluation standards for the rice samples are presented in Supplementary Table S1.

2.8. Data analysis

All experiments were performed in at least triplicate. Data were expressed as mean ± standard deviation, and one-way analysis of variance (ANOVA) was conducted using SPSS statistical program (SPSS 27, IBM) to determine differences between groups. A P value of <0.05 was considered statistically significant.

3. Results and discussion

3.1. Characterization of PLA-PVA /LZ pads

3.1.1. Water resistance

The water resistance of the sample pads was evaluated through WCA measurements and water absorption tests. As shown in Fig. 2A, the static WCA of the cellulose pad substrate could not be determined, as water droplets placed on the surface were rapidly and completely absorbed within 10 s. Compared to the unmodified cellulose substrate, immobilizing LZ on the cellulose substrate using PVA (PVA/LZ) resulted in a 25.1% increase in the WCA of the pad, likely due to the synergistic hydrophobic interactions between the hydrophobic modification of LZ and the spatial site-barrier effect of PVA. Upon further coating the PVA/LZ with PLA on both sides, the WCA increased substantially to 78.88%, indicating that the PLA coating significantly enhanced the water resistance of the pad. Furthermore, as illustrated in Fig. 2B, the cellulose substrate exhibited the highest water absorption rate at 700.26%. The water absorption rate of PVA/LZ pads showed a reduction, while the water absorption rate of PLA-PVA/LZ pads, decreased markedly to 86.67%. These findings further substantiate that the application of a PLA coating effectively improves the pad's resistance to water absorption.

Fig. 2.

(A) WCA, (B) water absorption, and (C) mechanical properties values of the antibacterial pad. (D) The SEM images of the surface of the cellulose pad, PVA/LZ pad, PLA-PVA/LZ pad, and (E) digital photos of the local parts of these coated pads. Different lowercase letters a, b, and c indicate significant differences in values (P < 0.05).

3.1.2. Mechanical properties

The mechanical properties of antibacterial pads are critical for determining its suitability in practical applications, as it must possess adequate mechanical strength and flexibility to withstand external stresses (Xi et al., 2024). The thickness of the pads is presented in Table S2. As shown in Fig. 2C, the TS of the cellulose pad exhibits an increasing trend after treatment with PVA and PLA. Compared to the untreated cellulose pad, the TS of the PVA/LZ pads increased by 27.53% when PVA was used to immobilize LZ. Additionally, the application of PLA to the surface of the PVA/LZ layer further enhanced the TS of the pad, reaching 65.52%. This indicates that the layer-by-layer application of PVA and PLA coatings substantially enhances the mechanical properties of the pad. Compared to the cellulose pad, the EAB of PVA/LZ pads and PLA-PVA/LZ pads decreased by 3.95% and 5.26%, respectively, but these reductions were not statistically significant (P > 0.05), demonstrating that the treatment did not significantly influence the EAB of the pads. The negligible change in EAB may be attributed to the structural reinforcement provided by the PVA and PLA coatings, which enhanced TS mainly through hydrogen bonding and polymer chain alignment within the coating layers and at the coating–substrate interface (Bikiaris et al., 2024; Peng et al., 2024).

3.1.3. Microstructure

The microstructure of the cellulose substrate, PVA/LZ pads, and PLA-PVA/LZ pads were analyzed using SEM, with images provided in Fig. 2D. The surface of the cellulose substrate contains numerous interwoven fibers with uniform diameters of approximately 10 nm. The fibers form many pores, which facilitate the immobilization of LZ. In comparison to the cellulose substrate, the microstructures of both PVA/LZ pads and PLA-PVA/LZ pads have changed markedly. The fiber structure is no longer visible, as the fiber outlines are covered by a film-like layer. However, the PVA/LZ pads still contains some larger pores on its surface, whereas the surface of the PLA-PVA/LZ pads appears more compact, with almost no visible pores. The appearance of the exterior structure (Fig. 2E) is also seen in the exterior picture is relatively dense. This also indirectly confirms the successful immobilization of LZ.

3.2. Structural stability considerations of PLA-PVA/LZ pads

Although PLA-PVA/LZ antimicrobial pads exhibit excellent overall performance, the potential for interlayer delamination in their three-layer structure (PLA hydrophobic layer/PVA-LZ antimicrobial layer/cellulose substrate) during long-term applications warrants attention. The pronounced differences in material properties between the layers (hydrophobic PLA vs. hydrophilic PVA/LZ) and the reliance on physical adsorption for interfacial bonding may lead to interlayer separation under variations in temperature and humidity or under mechanical stress. Delamination would compromise the functional integrity of the pad: on one hand, failure of the PLA barrier layer could allow moisture to penetrate into the core layer, accelerating LZ inactivation and promoting microbial growth; on the other hand, exposure or detachment of the PVA/LZ layer would disrupt the controlled release of LZ, significantly reducing antimicrobial efficacy. Furthermore, delamination may result in a decline in mechanical strength and overall durability.

Therefore, to more comprehensively evaluate the material's long-term structural robustness, future studies should include accelerated aging tests (e.g., thermal and humidity cycling) and mechanical stress assessments to validate its resistance to delamination under harsh environmental conditions.

3.3. Antimicrobial activity of PLA-PVA/LZ pads

As shown in Fig. 3A, the inhibition rates of both the cellulose pad and the pure PLA-PVA pads against E. coli and S. aureus were below 15%, confirming that these materials alone lack effective antimicrobial activity. In contrast, the control group LZ pads prepared by directly coating LZ onto cellulose pad substrates exhibited significantly increased antibacterial rates of 44.2% and 49.05% against E. coli and S. aureus, respectively (P < 0.05), but demonstrated clearly insufficient bactericidal capability. This indicates that the direct coating method can achieve a certain amount of LZ loading. However, due to the weak binding force between LZ and the carrier, enzyme detachment readily occurs, ultimately resulting in relatively low antibacterial efficacy of LZ. In contrast, the PLA-PVA/LZ pads exhibited a significantly enhanced antimicrobial effect against both bacteria, achieving inhibition rates of 86.73% for E. coli and 93.93% for S. aureus, respectively. The turbidity of the bacterial suspension in Fig. 3B also exhibits this trend. This excellent bactericidal performance arises from the synergistic effect of the cellulose substrate's porous structure facilitating LZ dispersion, the PVA coating enabling efficient LZ loading, and the PLA coating providing hydrophobic protection.

Fig. 3.

(A) Bacteriostatic activity of antimicrobial pads, (B) appearance of bacterial suspension, ①-④ represent cellulose pad, PLA-PVA, LZ pad, PLA-PVA/LZ respectively, (C) and (D) optimal additions of lysozyme. Different lowercase letters indicate significant differences in values (P < 0.05).

As shown in Fig. 3C and D, the optimal addition amount of LZ for the composite pads was also investigated. As the content of LZ increased, the antimicrobial effect against both E. coli and S. aureus demonstrated an ascending trend. When the LZ addition was 4%, the antimicrobial activity reached its maximum. Beyond this concentration, a slight reduction in antimicrobial performance was observed. This phenomenon could be attributed to the hydrophobic interactions between LZ molecules at elevated concentrations (>4%), which potentially induced the formation of insoluble aggregates. The resulting aggregation may physically shield the active sites of LZ (Ghosh et al., 2021), thereby reducing the proportion of catalytically available molecules. Therefore, the optimal addition amount of LZ was determined to be 4%.

3.4. Antibacterial mechanism of PLA-PVA/LZ pads

3.4.1. Morphological changes of E. coli and S. aureus

The effects of different treatment groups on bacterial morphology are shown in Fig. 4 In the blank and control groups (Cellulose pad and PLA-PVA group), the bacteria exhibited normal cell morphology. E. coli appeared intact, with a rod shape and relatively uniform length. S. aureus showed intact cells, which were spherical or oval in shape, uniform in size, and had smooth surfaces. This suggested that neither the cellulose pad substrate nor the PLA and PVA layers exhibited antimicrobial properties. After treatment with the PLA-PVA/LZ pads, the surface of E. coli showed evidence of shrinkage and wrinkling, while S. aureus exhibited twisting deformation, with pronounced depressions and folds on the surface. Additionally, some bacterial cells were fragmented and formed hollow structures. These results indicated that PLA-PVA/LZ pads could effectively inhibit bacterial growth. The alteration in bacterial surface structure may be due to the interaction between LZ and the bacteria, which disrupted the cell membrane structure.

Fig. 4.

SEM micrographs of E. coli and S. aureus treated with the different cellulose pads, high-magnification images (500 nm scale) show localized morphological damage of E. coli and S. aureus cells.

3.4.2. Fluorescence microscopy analysis

To further investigate the impact of the antimicrobial pad on the cell membrane, bacterial were stained with calcein-AM and PI dyes. Calcein-AM is capable of penetrating the bacterial cell membrane, thereby staining live bacteria and producing green fluorescence. PI cannot penetrate intact bacterial cell but can bind to intracellular DNA or RNA when the cell membrane is compromised or its permeability is increased, resulting in red fluorescence.

The results of fluorescence microscopy analysis are shown in the Fig. 5A and B. The blank and control groups displayed strong green fluorescence with only faint red fluorescence, indicating that the bacterial viability in the untreated samples was not significantly affected. However, the images of the PLA-PVA/LZ group showed extensive red fluorescence, indicating that most of the bacteria were effectively killed. These results demonstrate that the prepared PLA-PVA/LZ pads has good antibacterial activity. Additionally, since propidium iodide is a fluorescent dye that cannot penetrate intact cell membranes, the red staining of dead cells indicates that their cell membranes have been compromised.

Fig. 5.

Live/dead cell plots of (A) S. aureus and (B) E. coli treated with antimicrobial pads. (C) OD_{260 nm} of bacterial supernatant, and (D) protein mass concentration in the supernatant. Effect of inhibitory pads on AKP (E) and ATPase (F) enzyme activities of bacteria. Different lowercase letters indicate significant differences in values (P < 0.05).

3.4.3. Permeability of cell membrane

Macromolecules, including nucleic acids and proteins, are located within the cells. When the cell membrane and cell wall are compromised, these macromolecules are released into the surrounding environment. The permeability of the cell membrane can be assessed by measuring the macromolecule content in the supernatant. As shown in Fig. 5C and D, minimal leakage of nucleic acids and proteins was observed in the blank and control groups, which is a typical phenomenon associated with aging or apoptosis during bacterial growth and reproduction. However, after treatment with the PLA-PVA/LZ pads, the leakage of bacterial nucleic acids and proteins was significantly enhanced (P < 0.05). This indicates that the integrity of the bacterial cell membrane was disrupted by the antimicrobial pad treatment. These results imply that the PLA-PVA/LZ pad targets the bacterial cell membrane, causing irreversible damage to the cells, leading to the release of cellular contents, and ultimately resulting in cell death.

3.4.4. AKP and ATPase activities

AKP plays a direct role in the transfer and metabolism of phosphate groups within microorganisms and is located between the cell wall and the cell membrane. When the cell wall is compromised, AKP leaks from the cell, disrupting the bacterial phosphate metabolism pathway. As shown in Fig. 5E, treatment with the PLA-PVA/LZ pad resulted in a significant increase in bacterial AKP activity compared to the blank and control groups (P < 0.05). This may be due to the antimicrobial pad enhancing the permeability of the bacterial cell wall, facilitating the leakage of AKP from the cell membrane.

ATP is present in the bacterial cell membrane and is generated through the glycolysis process. As shown in Fig. 5F, compared to the blank and control groups, the PLA-PVA/LZ group showed a significant decrease in ATPase activity (P < 0.05). This is because the antimicrobial pad disrupts the cell wall structure, inducing the collapse of the proton-motive force (Ding et al., 2023). The collapse of the PMF across the cell membrane leads to a corresponding decrease in ATPase synthesis. The inactivation of ATPase prevents the cell from transferring energy through ATP breakdown to sustain vital activities, ultimately resulting in bacterial death.

3.5. Application of PLA-PVA/LZ pads for instant cooked rice preservation

Fig. 6A illustrates the morphological changes observed in unpackaged cooked rice compared with cellulose pad packaged samples during a 72 h storage period. At the 48 h, white flocs and slight mold spots were observed on blank group and the cellulose pad group cooked rice sample. Subsequently, the microbial spoilage of the cooked rice gradually became severe, leading to their decay at the 72 h. By comparison, cooked rice packaged in PLA-PVA/LZ pad maintained their appearance during storage, and the gloss of the cooked rice only decreased slightly at 72 h. This result indicates that the PLA-PVA/LZ pad possesses superior antibacterial properties, effectively preserving rice quality and preventing spoilage throughout the storage period.

Fig. 6.

The changes in (A) visual appearance, (B) total colony count, and (C) sensory evaluation of cooked rice packaged with cellulose pads during storage.

3.5.1. Microbiological evaluation

The effect of PLA-PVA/LZ pads on the microbial stability of instant cooked rice samples during storage was evaluated by analyzing the total bacterial count (TBC). As shown in Fig. 6B, no microbial activity was detected in freshly prepared rice samples. After 12 h of storage, the TBC value remained relatively stable without a statistically significant increase. However, beyond 12 h, the TBC value in the blank group (no pads placed) increased rapidly, reaching 5.03 lg (CFU/g) at 48 h. According to China's national standard (GB19295–2021), the acceptable upper limit for TBC in rice and flour products is 5 lg (CFU/g). Consequently, the cooked rice in the blank group exceeded this threshold and was considered non-edible after 48 h of storage. Similarly, rice samples containing only a cellulose pad followed the same trend as the blank group, with TBC values exceeding the edibility threshold at 48 h. This finding suggests that the cellulose pad alone did not exhibit antimicrobial properties. In contrast, the PLA-PVA/LZ pad demonstrated excellent antibacterial performance. Within 48 h, the TBC of instant cooked rice was maintained at nearly zero. Even after 60 h of storage, the TBC of instant cooked rice remained at 4.83 lg CFU/g, which still fell below the threshold established by Chinese national standards. These results indicate that PLA-PVA/LZ pads effectively suppress microbial growth, thereby extending the shelf life of instant cooked rice.

3.5.2. Sensory evaluation

As shown in Fig. 6C, the sensory evaluation curves of the blank group and cellulose pad overlap, indicating that both groups received similar sensory scores for appearance, texture, and odor. After 60 h of storage, both samples displayed slight sourness, a yellowish appearance with visible mold spots on the surface, a hardened texture, and a noticeable reduction in elasticity. This suggests that the cooked rice sample treated solely with cellulose pads does not effectively slow the deterioration of sensory quality. In contrast, the cooked rice samples treated with PLA-PVA/LZ pad received higher sensory scores in terms of appearance, texture, and odor compared to the blank group and cellulose pad. Although the aroma of these samples slightly diminished after storage, their appearance remained normal, showing only slight hardening, while the texture remained relatively soft. This demonstrates that PLA-PVA/LZ pads can effectively inhibit microbial activity, thus delaying the deterioration of sensory quality in rice.

4. Conclusion

The resultant PLA-PVA/LZ pads, fabricated via physical multilayer coating, exhibited significantly improved structural and functional properties. Notably, optimizing the LZ content to 4% achieved the highest antibacterial efficacy, with 93.93% inhibition against S. aureus and 86.73% against E. coli. This antimicrobial activity was mediated through multiple mechanisms, including disruption of bacterial cell walls, induction of membrane permeabilization, and subsequent intracellular enzyme system damage—characterized by suppression of ATPase activity and upregulation of AKP—ultimately leading to bacterial lysis. Furthermore, when applied to cooked rice stored at 25 °C, the pad effectively maintained microbial counts below the detection limit for 48 h, extending the shelf life by at least 12 h according to Chinese national standards. These findings provide critical insights for the design of next-generation functional packaging materials with tailored antibacterial and moisture-resistant properties.

CRediT authorship contribution statement

Qian Han: Writing – original draft, Methodology, Formal analysis. Shihui Liu: Software, Methodology. Xiaoling Huang: Methodology, Investigation. Si Chen: Validation, Data curation. Wei Zhang: Data curation. Pingping Wang: Writing – review & editing. Fang Tang: Software. Xiuying Liu: Writing – original draft, Funding acquisition, Data curation, Conceptualization. Chen Liu: Writing – review & editing. Lijie Zhu: Writing – review & editing.

Declaration of competing interest

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

Appendix A. Supplementary data

Supplementary material

Data availability

No data was used for the research described in the article.