A Bio-Mimetic Approach: Non-adverse Effects of Zinc Oxide Nanoparticles with Probiotic Isolate (Lactiplantibacillus plantarum)

2.1. Biosynthesis of Zinc Nanoparticle

Lactiplantibacillus plantarum was cultured in De Man, Rogosa, and Sharpe (MRS) broth at 37°C for 24 h under static conditions. The culture was centrifuged at 10,000 rpm for 15 min, and the supernatant was collected as cell-free supernatant (CFS). A 0.1 M zinc acetate dihydrate [Zn(CH₃COO)₂·2H₂O] (Himedia, India; GRM692) solution was prepared using deionized water and used as the zinc precursor. The biosynthesis reaction was carried out by mixing CFS and zinc acetate solution in a 1:1 (v/v) ratio (50 mL each), giving a total reaction volume of 100 mL. The pH of the reaction mixture was measured using a calibrated digital pH meter and adjusted to 8.5-9.0 by the dropwise addition of 0.1 M sodium hydroxide (NaOH) under continuous magnetic stirring at 500 rpm. The reaction mixture was incubated at 80°C for 24 h with constant stirring, and nanoparticle formation was indicated by the development of white turbidity [3]. After incubation, the mixture was cooled to room temperature and centrifuged at 10,000 rpm for 15 min to collect the precipitate, which was washed three times with deionized water and ethanol to remove impurities. The product was dried at 60°C overnight and calcined at 400°C for 2 h to obtain zinc oxide nanoparticles (ZnONPs). The percentage yield was calculated using the formula (1):

Resulting in a final yield of 2.8 mg (approximately 70%). The synthesized nanoparticles were stored in an airtight container at room temperature under dry conditions for further use.

2.2. Characterisation Study

The crystallographic structure was examined using a powder X-ray diffraction (XRD) machine, specifically the D8 Advance ECO XRD system with an SSD160 1D detector, employing CuKα radiation (λ = 0.15406 nm). The FTIR (Fourier Transform Infrared Spectroscopy) spectrum was recorded on a Shimadzu IR-Trace 100 spectrometer. Perkin-Elmer LAMBDA-325 was used for optical analysis. To assess the surface morphology of the produced sample, a FE-SEM (Field Emission Scanning Electron Microscopy) was utilised, and the elemental composition was determined by recording the EDAX spectrum using the Carl Zeiss - Sigma 300 apparatus. The structural morphology of the synthesised material was analysed using a JEOL JEM-2100 HR-TEM (High-Resolution Transmission Electron Microscopy) instrument.

2.3. Antibacterial Activity

The antibacterial efficiency of probiotics must confer beneficial effects, and viable count generally assesses the effective probiotic dose at 1×10⁸ CFU/mL, which is the minimum threshold for producing measurable biological and functional effects. For using concentration evaluated (Supplementary Table 1) by the agar well diffusion method against selected aquatic pathogens, including Aeromonas hydrophila, Escherichia coli, and Vibrio parahaemolyticus, at concentrations of 25, 50, 75, and 100 µg/mL. The plates were incubated at 37°C for 24 hrs, and the zone of inhibition was measured in each well, using sterile distilled water as a negative control to evaluate the results. As a positive control, 10 µg of amoxicillin was used. This experimental procedure was conducted in triplicate [6, 7].

2.4. Cytotoxicity Assay

The cytotoxicity of the ZnO: LP NPs was evaluated using the MTT assay [8]. The monolayer culture was trypsinized, and the cell count was adjusted to 1.0 × 10^5 cells/mL with complete culture medium containing 10% FBS. Cells were seeded at 1 × 10^4 cells/well in 96-well plates and incubated for 24 h to form a monolayer. The treatment solutions were then discarded, and 100 µL of MTT solution (0.5 mg/mL in PBS) was added to each well, followed by incubation for 4 h. The resulting formazan crystals were dissolved by adding 100 µL of DMSO, and absorbance was measured at 590 nm using a microplate reader. Cell viability was expressed as a percentage relative to untreated control cells, and dose-response curves were used to assess cytotoxicity in L929 fibroblast cells.

2.5. Statistical Analysis

Statistical analysis has been added to evaluate the concentration-dependent antibacterial activity. Results are expressed as mean ± standard deviation, and statistical significance was assessed using one-way ANOVA with an appropriate post hoc test.

3.1. Anti-bacterial Activity of ZnO-based Lactiplantibacillus plantarum

The antibacterial activity of ZnO: LP NPs was evaluated against the three pathogens A. hydrophila, E. coli, and V. parahaemolyticus using the agar well diffusion method. Fresh overnight bacterial cultures were adjusted to the 0.5 McFarland standard (approximately 1×10⁸ CFU/mL) and uniformly swabbed onto sterile Muller-Hinton agar plates. Wells of 6 mm diameter were aseptically punched into the agar and loaded with ZnO: LP NPs suspensions at concentrations of 25, 50, 75, and 100 μg/mL (Fig. 2). Sterile distilled water served as the negative control to ensure that inhibition was not due to solvent effects. The plates were incubated at 37°C for 24 h, after which the zones of inhibition were measured in millimetres. The positive control (Amoxicillin) has been included. The zone of inhibition values is: A. hydrophila (3.005 ± 0.004 mm), E. coli (3.572 ± 0.012 mm), and V. parahaemolyticus (3.274 ± 0.027 mm). All experiments were conducted in triplicate, and results were expressed as mean ± standard error.

To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), both assays were performed according to CLSI guidelines. Twofold serial dilutions of ZnO: LP NPs (12.5 μg/mL-100 μg/mL) were prepared in Muller-Hinton broth and inoculated with bacteria suspensions adjusted to approximately 1×10⁶ CFU/mL. After incubation at 37°C for 24 h, the MIC (expressed in μg/mL) was defined as the lowest concentration of ZnO: LP NPs that inhibited visible bacterial growth. Aliquots from wells showing no visible growth were subcultured onto fresh Muller-Hinton agar plates and incubated for 24 h. MBC (expressed in μg/mL) was defined as the lowest concentration resulting in complete absence of colony formation (≥99.9% bacterial killing) (Table 2).

To confirm that the antibacterial activity was specifically attributable to the synthesized ZnO NPs, appropriate controls were included. The sterile cell-free supernatant from a L. plantarum culture (without a zinc precursor) was tested separately to evaluate the contribution of probiotic metabolites, and only minimal inhibition was observed. Similarly, zinc acetate solution at equivalent concentrations was tested independently and showed negligible antibacterial activity compared to ZnO: LP NPs. These control experiments demonstrate that the observed antibacterial effect primarily resulted from the synthesized ZnO NPs rather than residual bacterial metabolites or unreacted zinc precursor. The concentration-dependent increase in inhibition zones, along with MIC (25 μg/mL) and MBC (50 μg/mL) values, confirms the effective bacteriostatic and bactericidal properties of the ZnO: LP NPs formulation. According to Rajivgandhi et al. [15], biosynthesized ZnO nanoparticles were more effective against S. aureus than against E. coli, a trend also observed in our studies. Khatami et al. [16] reported that biosynthesized ZnO nanoparticles demonstrated enhanced inhibitory activity against both S. aureus and E. coli, with MIC and MBC values of 20.0 μg/mL. This activity is likely due to bacteriocins and organic acids produced by the probiotic strain, underscoring the formulation's dual role in pathogen control. Such properties are particularly beneficial for enhancing biosecurity in aquaculture and offer potential applications in food safety and human health.

3.2. Cytotoxicity Assay

The calculated IC₅₀ value was 38.75 µg/mL, indicating moderate cytotoxicity. At concentrations ≤20 µg/mL, cell viability remained above 40%, demonstrating partial cytocompatibility at lower doses (Fig. 3AC). However, a pronounced dose-dependent decline in viability was observed at concentrations above 30 µg/mL, with complete inhibition occurring at 50 µg/mL and higher [17, 18]. These findings suggest that the cellular response is strongly concentration-dependent and highlight the importance of dose optimization in potential applications.

Numerous in vitro studies have shown that ZnO nanoparticles may pose risks to various cell types [14]. Notable harmful effects include the induction of oxidative stress and cellular damage associated with the release of free Zn²⁺ ions from ZnO nanoparticles, leading to cellular injury. Nevertheless, findings regarding the cytotoxic effects of ZnO nanoparticles on cell lines are not always consistent. According to Fouda et al. [19], nanoparticle cytotoxicity is influenced by factors such as size, shape, and dosage. This aligns with earlier findings showing that ZnO-NPs exhibit selective cytotoxicity depending on size, surface charge, and capping agents. Hypothetically, ZnO nanoparticles release Zn²⁺ ions and generate reactive oxygen species (ROS), leading to oxidative stress, membrane and cellular disruption, mitochondrial dysfunction, and DNA damage, resulting in bacterial cell death or eukaryotic cell apoptosis [1]. However, the biosynthesis of probiotic-mediated ZnO nanoparticles involves biomolecule-assisted nucleation, biological capping, and controlled growth, resulting in nanoparticles that exhibit enhanced antibacterial effects through ROS generation and membrane disruption while maintaining dose-dependent biocompatibility in mammalian cells. Future studies should incorporate complementary assays, such as intracellular ROS quantification, caspase activation analysis, membrane integrity assays (e.g., LDH release), mitochondrial membrane potential measurements, and evaluation of apoptosis markers, to provide a more comprehensive assessment of nanoparticle-induced cellular responses. Furthermore, although probiotic-mediated synthesis may influence nanoparticle growth and surface characteristics during formation, calcination at elevated temperatures likely reduces the persistence of surface biomolecules. Therefore, the nanoparticles produced in this study are best described as biologically initiated rather than definitively biologically capped after calcination.