1 Introduction

The growing demand for sustainability and multifunctionality in contemporary society encourages research to develop active materials that originate from renewable resources [1]. In this context, natural polymers have emerged as potential biopolymers for the synthesis of multifunctional materials owing to the abundance of hydroxyl groups present in their molecular structure, which provide significant opportunities for functionalization [2]. Cellulose, the main component of plant cell walls, is an intriguing biopolymer. It is a linear homopolysaccharide consisting of β-D-anhydroglucopyranose units that are connected by β(1 → 4) ether bonds (glycosidic links) and its chemical formula is (C6H10O5)n [3]. It can be obtained from origins, such as wood, plant cell walls, several bacteria strains, algae, and even tunicate marines, which are the only defined cellulose-producing animals [4, 5]. BC is a substance obtained from bacteria and is capable of being produced in a nearly pure form (up to 90%) without any other components, including hemicellulose and lignin. Its composed of glucose units with the same chemical structure as plant cellulose [6]. BC exhibits several distinctive qualities, including high water retention capacity, ultrafine network purity (high purity), degree of crystallinity, mechanical strength, degree of polymerization, formability, hydrophilicity, biocompatibility, flexibility, lack of toxicity, biodegradability, and useful mechanical properties (Young’s modulus value, tensile strength, compressibility, and elongation) [7,8,9,10]. The functionalization of BC is a widely common challenge reported for application in different fields due to the inherent limitations of BC. To surmount this challenge, there have been numerous attempts to functionalize BC using either an in situ or ex situ approach with the aim of generating a functionalized polymeric composite with distinctive applications. Several studies have focused on the ex situ modification of BC for the purpose of advancing novel applications, like, Silver montmorillonite can be employed as an antimicrobial agent from the perspective of wound healing treatment [11], silver sulfadiazine is employed in the context of diabetic wound healing [12], the utilization of extract of fruit peels for the purpose of antimicrobial applications and in the packaging of strawberries [13], additionally Poly(3,4 ethylenedioxythiophene)/Poly(styrenesulfonate) for optoelectronic applications [14]. Among them, nanomaterials are frequently used as novel antibacterial agents against infectious pathogens [15, 16]. Metal, metal oxide-NPs can inactivate pathogenic microbes by damaging the cell membrane, cell wall, electron transport chain, nucleic acids, proteins or enzymes [17, 18]. Metal oxide-NPs, specifically ZnO-NPs are repeatedly selected due to their nontoxic nature, biological attributes, and remarkable chemical stability. ZnO-NPs are widely recognized NPs that offer numerous therapeutic benefits, including antimicrobial (bacteria and fungi), immunomodulatory, antioxidant, and anticancer effects. Nevertheless, there have been multiple reports highlighting diverse forms of toxicities, such as hepatotoxicity, pulmonary toxicity, neurotoxicity, and mutagenicity associated with use of ZnO-NPs [19]. ZnO-NPs have been utilized in various commercial applications including industrial, medical and agricultural applications [20,21,22]. ZnO-NP plays a crucial role in numerous biomedical applications such as antimicrobial, anticancer, antineoplastic, wound healing, ultraviolet scattering, angiogenic, and antioxidant properties [23, 24]. The nate BC was modified with NPs for the development of antimicrobial agents like BC/ZnO-NPs, which exhibited 90, 87.4, 94.3, and 90.9% activity against E. coli, P. aeruginosa, S. aureus, and Citrobacter freundii, respectively, and the nanocomposite showed significant (66%) healing activity in treated animals [25], BC/Copper oxide-NPs nanohybrids exhibit that the antibacterial activity of ex situ synthesized nanohybrids against S. aureus and E. coli bacteria was greater than both in situ synthesized samples [26], BC/Carbon Quantum Dots-Titanium Dioxide-NPs have antibacterial properties against S. aureus [27], and the bionanocomposite of BC/Magnesium Oxide-NPs showed the highest antifungal activity and prevented the growth of the fungus Aspergillus niger by 85.03% [28]. As per current state of art today is that it is being develop scaffold materials based on the ex situ modification of BC with ZnO-NPs, and other published article related to functionalization of BC with ZnO-NPs for development of nanocomposite as a dressing system for burn wounds treatment [25]. The preparation of BC/ZnO-NPs nanocomposites were reported by in situ technique through the immersing of BC in zinc salt solution, followed by treating with NaOH solution and then dried through appropriate methods [29]. The current study aims to evaluate the BC network to support the immobilization of ZnO-NPs through ex situ technique. The novel aspect of this study was the modification of BC to enhance its medical applications, such as antimicrobial assessment. In this investigation, a BC/ZnO-NPs nanocomposite membrane was developed by reinforcing BC with ZnO-NPs. The pure BC and nanocomposite membranes obtained were analyzed by SEM, TEM, FT-IR, and XRD. The obtained membranes were evaluated as antimicrobial agents against four pathogenic microbes, and the results demonstrated that the BC/ZnO-NPs nanocomposite membrane successfully suppressed the growth of microorganisms.

2 Materials and methods

2.1 Production of BC hydrogel

2.1.1 Strain and standard inoculum

According to the previous investigation, Lactiplantibacillus plantarum AS.6 (L. plantarum AS.6) was identified as a strain that produce BC. This strain was obtained from decaying apples and has the accession number (MW857479.1) that was isolated from decaying apples [30]. For the preparation of standardized inoculum, a freshly and purely single colony of L. plantarum AS.6 was cultivated in the standard Hestrin and Schramm (HS) medium. The medium consisted of 20 g/L glucose, 5 g/L yeast extract, 5 g/L peptone, 2.7 g/L disodium hydrogen phosphate, 1.15 g/L citric acid, and 5 mL/L ethanol at pH 5.5. The cultivation was carried out at 30 °C for 48 h at 150 rpm [31].

2.1.2 Production and purification of BC hydrogel

In accordance with the findings presented in our prior investigation, after conventional and statistical optimization approaches, the final optimized medium was used for BC production by using the following optimal conditions: 15 g/L glucose, 13 g/L yeast extract, 1 g/L magnesium sulphate, 4 g/L potassium dihydrogen phosphate, and 7 mL/L ethanol [30], at pH 7.2. The media were sterilized at a temperature of 121 °C and a pressure of 15 psi for a duration of 20 min, and they were allowed to cool down to room temperature, and subsequently inoculated with 11% standard inoculum of L. plantarum and incubated at 30 °C, for 8 days of cultivation time under static conditions [30]. At the completion of the cultivation period, the BC pellicle observed at the air–liquid interface was gathered and repeatedly rinsed with distilled water in order to eliminate the excess medium ingredients. Following that, the BC hydrogel was treated with 0.5% sodium hydroxide at 90 °C for 30 min to kill the remnant L. plantarum AS.6 or other impurities embedded on the BC hydrogel, and washed with distilled water until it reached the point of a neutral pH value, after which a bright-white BC hydrogel was obtained. The purified BC hydrogel was kept in the refrigerator at 4 °C until used.

2.2 Ex situ preparation of BC/ZnO-NPs nanocomposite membrane

For the preparation of the BC/ZnO-NPs nanocomposite membrane, ZnO-NPs was used to modify the BC hydrogel by ex situ modification in accordance with the method of Khalid et al. [25], with slight changes as shown in Fig. 1. First, the purified BC hydrogel was cut into small fragments, each measuring 2 cm2 in diameter and 3 mm in thickness. Each piece was subjected to compression through the utilization of filter paper, facilitating the expulsion of a significant portion of the water content. About 2% (v/v) of ZnO-NPs (Aldrich chemistry, ≤ 40 nm average particle size (APS), 20 wt.% in H2O) was dispersed by using a very mild water bath sonication (720 W, 50/60 Hz, 230 V, Spain) at 50 °C for 30 min, and then homogenized by ultrasonication (750 wt, 20 KH, pulse 45, Amp 1) for 5 min under an ice-water bath to ensure the complete dispersion of ZnO-NPs. Small fragments of BC hydrogel (2 cm2) were submerged in 100 mL of dispersed ZnO-NPs solution for 1 day at room temperature under shaking conditions (150 rpm) to assure the complete absorption of the dispersed ZnO-NPs on the BC hydrogel. The prepared nanocomposite hydrogel was immersed in distilled water for 10 s and treated with filter paper for the removal of extra-free water from the nanocomposite hydrogel. Finally, the obtained nanocomposite hydrogel was freeze-dried and stored at 4◦C for further use. The experiment was carried out in triplicate.

Fig. 1
figure 1

Schematic illustrating the ex situ modification of BC hydrogel for the formation of a BC/ZnO-NPs nanocomposite membrane

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2.3 Microstructure characterization of BC and BC/ZnO-NPs nanocomposite membrane

2.3.1 SEM analysis

The BC and BC/ZnO-NPs nanocomposite membranes were analyzed through various instrumental techniques. The surface morphology of the obtained membrane was conducted using a Hitachi S-4800 SEM. The average diameter of the nanofiber was calculated from various SEM images using the angle tool of ImageJ with Java 1.8.0 software developed by the National Institute of Health (NIH), USA) [32].

2.3.2 TEM analysis

Ultra-high-resolution TEM (JEOL-JEM 2010, Japan) was utilized to investigate the internal composition structure of the BC/ZnO-NPs nanocomposite membrane.

2.3.3 FT-IR spectroscopy analysis

The FT-IR (model FT-IR-6100 type A) was employed to analyze the chemical functional groups found in both BC and BC/ZnO-NPs nanocomposite membranes. The measurement of the spectra of each sample was conducted within a spectral range spanning from 500 to 4000 cm−1.

2.3.4 XRD analysis

Analyzing the crystallinity of the acquired samples was performed using an X-ray diffractometer (XRD, Philips X’ pert MPD, PANalytical, Netherlands) employing the CuKα line (λ = 1.540 Å) in the diffraction 2θ angular range 10–80°.

2.4 Antimicrobial activity

Mueller Hinton media comprising 0.15% starch, 1.75% acid hydrolysate of casein, and 0.2% beef extract was used as the growth medium for the microbial strains procured from the American Type Culture Collection (ATCC), including Gram-negative bacteria Escherichia coli ATCC 25922 (E. coli) and Salmonella typhimurium ATCC 14028 (S. typhimurium), Gram-positive bacteria Streptococcus mutans ATCC 25175 (S. mutans), and yeast Candida albicans ATCC 10231 (C. albicans). All the microorganisms employed in the study were cultivated for one day at 37°C, and incubated under agitated conditions at 200 rpm. The antimicrobial properties of BC and BC impregnated with ZnO-NPs (BC/ZnO-NPs) membranes were investigated qualitatively using the disc diffusion procedure, according to our previous study with some modifications [33]. Firstly, an amount of approximately 15 mL of sterilized Mueller Hinton media (20 g/L agar) was poured on the Petri dish and allowed to solidify. The microbial suspension (108 CFU/mL) which had a concentration or density approximately equal to 0.5 McFarland standards was evenly spread on the Petri dish along with a 10 mm disc of BC/ZnO-NPs. As a control, a sample without ZnO-NPs was used. The plates were first incubated at 4°C for 2 h to complete the diffusion of the tested sample and momentarily inhibit the model microbes. After that, they were cultivated at 37°C for 1 day. After the period of cultivation, the plates were estimated for their antimicrobial properties by measuring the diameter of the inhibition zone (including the membrane disc). The results represented are the average values of triplicate experiments. All membranes were sterilized for 30 min under UV light in order to ensure aseptic conditions.

3 Results and discussions

3.1 Microstructure characterization

3.1.1 SEM and TEM analysis

The first step in our research investigation involved examining the chemical composition and morphology of pure BC and BC that had been modified with ZnO-NPs. The SEM images of BC show a successful elimination of impurities along the BC network during treatment with 0.5 M NaOH. Furthermore, the nanofibers displayed an uneven distribution, which further confirmed the presence of porous areas within the membranes (Fig. 2A) in accordance with previous literature [34, 35]. In addition, the SEM images shown in Fig. 2B revealed that the average nanofiber diameter of the BC membrane obtained was measured to be 40.49 nm, as illustrated in fiber diameter measurements, which is in line with Zhang et al. and Almasi et al. [36, 37]. To investigate the formation of nanocomposite BC/ZnO-NPs, SEM images of the surface of the BC/ZnO-NPs nanocomposite membrane were taken. Inspection of Fig. 2 (C and D) reveals that the BC nanofibers were consistently covered by ZnO-NPs, showing that the ZnO-NPs regularly covered the BC nanofibers to form coating layers. The ZnO-NPs were able to permeate the BC pellicle individually without direct contact with the nanofibers through the diffusion method. There is no form of interaction observed between ZnO-NPs and BC fibers. Consequently, the NPs would physically deposit (e.g., driven by gravity) onto the BC nanofibers. The proposition was substantiated by the occurrence of random dispersion of the NPs. When the nanocomposite was established, the interior network and the exterior surface of the nanocomposites had ZnO-NPs. These results are in agreement with other reports by Khalid et al. [25]. TEM image of BC/ZnO-NPs nanocomposite membrane at high magnification showed the formation of ZnO-NPs with a size of 30 nm. These regular particles showed uniform size distribution.

Fig. 2
figure 2

A SEM image of pure BC, (B) Nanofiber diameter of BC, (C, and D) SEM and TEM images of BC/ZnO-NPs nanocomposite membrane, respectively

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3.1.2 FT-IR and XRD analysis

The chemical composition of the acquired samples, such as functional groups and molecular bonds, was determined by FT-IR spectroscopy. The FT-IR spectra of the BC sample derived from L. plantarum AS.6 were observed within the wavenumber range of 500 to 4000 cm−1 as depicted in Fig. 3A. A strong area of absorption in the BC spectrum at 3330.3 cm−1 corresponds to the intra-molecular hydrogen bond of the hydroxyl (OH) of cellulose type I, which aligns with [38, 39]. While the band at 2885 cm−1 was assigned to aliphatic C-H stretching vibration, the cellulose absorption spectrum includes a band at 1635.2 cm−1 which has been designated for the carboxyl (C = O) functional group. Bands were also detected at 1431.2 cm−1 signifying the bending of CH2 bending; 1340.5 cm−1 (symmetric angular deformation of C H bonds), 1161.4 cm−1 indicating asymmetrical stretching of C–O–C, 1109.5 cm−1 and 1045.3 cm−1 (stretching of C–OH and C–C-OH bonds in secondary and primary alcohols). These findings are consistent with [30, 40]. Finally, the FT-IR result ultimately demonstrates the existence of a crystalline region and the exceptional purity of BC. The characteristic absorption bands for both BC and BC/ZnO nanocomposite were presented in the “fingerprint” region (400–1650 cm−1). Some apparent changes appeared in the FT-IR spectrum of the BC/ZnO nanocomposite compared to pure BC. A new peak at 435 cm−1 assigned to the stretching mode of the Zn–O bond was recorded for the obtained BC/ZnO-NPs nanocomposite membrane [25]. The Zn–O vibrations were not sharp in the prepared composite due to the low ZnO content which was reported in previous study [41]. XRD was employed to reveal the crystal structure of the materials that were obtained. In Fig. 3B, XRD patterns of pure BC and BC/ZnO nanocomposite membranes are displayed. Both BC and BC/ZnO-NPs exhibit two strong, wide peaks at 16.3 and 22.2°, as well as a less intense and sharp reflection at 34.4°. These signals correspond to the characteristic 110, 200, and 004 planes assigned to the typical cellulose-I structure. Additional peaks attributed to the presence of the ZnO-NPs were identified in the pattern of BC/ZnO-NPs. The high intensity may be attributed to the abundant presence of NPs covering the surface of BC. Several sharp and intense Bragg reflections were detected at 31.56°, 34.22°, 36.11°, 47.35°, 56.43°, 62.63°, 67.72°, and 68.92°. These reflections well corresponded with the described peak positions for bulk ZnO (in accordance with standard PDF card no. 01–079-0207) demonstrating the existence of a wurtzite hexagonal crystal structure of ZnO. Moreover, these results indicate that ZnO was well crystallized in the composite. Moreover, the diffraction peaks of cellulose in the composite were much weaker. This might be affected by ZnO-NPs which cover the cellulose surface, making hard to collect the diffraction data of BC through the XRD study [42].

Fig. 3
figure 3

FT-IR spectra (A) and XRD analysis (B) for pure BC and BC/ZnO-NPs nanocomposite membrane

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3.2 Antimicrobial assessment

The disc diffusion technique was used to investigate the antimicrobial activities of BC and BC/ZnO-NPs nanocomposite membranes against pathogenic strains. According to the results illustrated in Table 1 and Fig. 4, the data indicated a significant antimicrobial activity against all employing pathogens through the BC/ZnO-NPs nanocomposite membrane, which aligns with [25, 43, 44]. The BC/ZnO-NPs nanocomposite membrane showed higher antimicrobial activity against S. typhimurium (21 ± 1.21 mm), with no significant effects against E. coli (18 ± 1.15 mm) or S. mutans (19 ± 1.19 mm), while the lowest antimicrobial activity was shown against C. albicans (14 ± 1.06 mm). In contrast, the BC membrane free from NPs showed no antimicrobial activity against any of the tested microbes, which is supported by existing literatures [13, 33, 45]. We can conclude that the antimicrobial activity existed only due to ZnO-NPs impregnated inside the BC membrane and was not attributed to individual BC. To overcome this problem, the BC membrane was loaded or fabricated with NPs for the development of an antimicrobial membrane, for example, ZnO-NPs [46], Ag-NPs [47], CuO-NPs [33, 48], and MgO-NPs [28]. The antimicrobial efficacy of BC/ZnO-NPs nanocomposite membrane has been extensively documented; however, the primary mode of action has yet to be completely understood. The mechanism of action of the ZnO-NPs is based on their morphology, concentration, and composition [49]. The primary mechanisms of the antimicrobial properties of ZnO-NPs are the generation of reactive oxygen species (ROS), the release of Zn+2, internalization of ZnO-NPs into microbes, and electrostatic interactions. These mechanisms modify the external membrane, disrupt the cell wall, induce protein denaturation, oxidize the lipid membrane, and cause oxidative DNA damage in microorganisms [50,51,52]. Other metal oxide-NPs, specially CuO-NPs based on their binding with the lipid layer of the bacterial cell membrane, which interferes with membrane permeability and nutrient uptake, were proposed to elaborate such activity [53], while the antibacterial activity of MgO-NPs is attributed to the production of ROS, which induce lipid peroxidation in bacteria [54], while the antimicrobial activity of Ag-NPs is based on the release of Ag+1 ions into the solution and then bind the bacterial membrane and proteins, causing cell lysis. The Ag+1 ions can originate from the solution but may also be transferred directly from the surface-exposed Ag+1 ions to the bacteria without being dissolved in the medium [55]. We can summarize the BC functionalized with NPs as having antimicrobial activity, as presented in Table 2.

Table 1 Antimicrobial activity of BC and BC/ZnO-NPs nanocomposite membranes against four pathogenic microbes
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Fig. 4 
figure 4

Disc diffusion method for antimicrobial activity expressed as halo zones of BC and BC/ZnO-NPs nanocomposite membranes against four pathogenic microbes

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Table 2 Comparison of BC impregnated with NPs with previously reported composite membrane
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4 Conclusion

BC was prepared and decorated with ZnO-NPs for preparing nanocomosite membranes as antimicrobial agents for wound healing applications. The formed nanocomposite membranes were characterized and evaluated by chemical analysis and biological assessments. The FT-IR and XRD analayses proved the decoration process, while SEM and TEM results exhibited the formation of BC/ZnO-NPs nanocomposite membrane with a homogenous distribution of the ZnO-NPs which are regularly covered by the BC nanofibers to form coating layers. The BC/ZnO-NPs nanocomposite exhibits high antibacterial properties, as displayed by the formation of the inhibition zone for S. typhimurium (21 mm), E. coli (18 mm), and S. mutans (19 mm), while the lowest antimicrobial activity was shown against C. albicans (14 mm). The antimicrobial activity of the nanocomposite is related to the presence of ZnO-NPs. Therefore, BC/ZnO-NPs nanocomposite membranes have significant potential for application as an antimicrobial biomembrane for wound healing activity.