Biogenic synthesis of Zinc oxide nanostructures from Nigella sativa seed: Prospective role as food packaging material inhibiting broad-spectrum quorum sensing and biofilm

Bacterial spoilage of food products is regulated by density dependent communication system called quorum sensing (QS). QS control biofilm formation in numerous food pathogens and Biofilms formed on food surfaces act as carriers of bacterial contamination leading to spoilage of food and health hazards. Agents inhibiting or interfering with bacterial QS and biofilm are gaining importance as a novel class of next-generation food preservatives/packaging material. In the present study, Zinc nanostructures were synthesised using Nigella sativa seed extract (NS-ZnNPs). Synthesized nanostructures were characterized hexagonal wurtzite structure of size ~24 nm by UV-visible, XRD, FTIR and TEM. NS-ZnNPs demonstrated broad-spectrum QS inhibition in C. violaceum and P. aeruginosa biosensor strains. Synthesized nanostructures inhibited QS regulated functions of C. violaceum CVO26 (violacein) and elastase, protease, pyocyanin and alginate production in PAO1 significantly. NS-ZnNPs at sub-inhibitory concentrations inhibited the biofilm formation of four-food pathogens viz. C. violaceum 12472, PAO1, L. monocytogenes, E. coli. Moreover, NS-ZnNPs was found effective in inhibiting pre-formed mature biofilms of the four pathogens. Therefore, the broad-spectrum inhibition of QS and biofilm by biogenic Zinc oxide nanoparticles and it is envisaged that these nontoxic bioactive nanostructures can be used as food packaging material and/or as food preservative.

Scientific RepoRts | 6:36761 | DOI: 10.1038/srep36761 All the analytical grade chemicals were procured without further purification from the standard brand of E. Merck Limited, India. The common solvent medium used throughout the synthesis was double distilled water (DDW). The glassware's utilized during the reactions were received from Borosil, India.
Preparation of Nigella sativa (NS) seed extract. Nigella sativa seed extract was prepared by crushing seed in a grinder. Resultant seed powder was thoroughly washed in distilled water and 10 g of this powder was homogenized completely in 50 ml double distilled water and incubated with constant stirring (100 rpm) at 80 °C for 20 min. The resultant mixture then filtered using Whatman filter papers No. 1 to remove debris. This extract was used for generating green zinc nanoparticles. The extract was stored at 4 °C for future uses.
Zinc nanoparticle synthesis. All of the reagents involved in the experiments were of analytical grade purity and utilized as received without further purification. Zinc nitrate (99.999%) was purchased from Sigma Aldrich, and the seed extract was collected. The synthesis was carried out in a domestic microwave oven (Samsung, 750 W). In a typical experiment, a 0.05 M aqueous solution of zinc nitrate in 100 ml distilled water was prepared in which 10 ml Nigella sativa seed extract was added to obtain a mixture solution in a round-bottom flask, and then put into a domestic microwave oven. Microwave irradiation proceeded at 100% power for 20 min. After microwave processing, the solution was cooled to room temperature. The resulted precipitate was separated by centrifugation, then washed with deionized water and absolute ethanol for several times, and dried in an oven at 80 °C for 24 h. Finally, the product was calcined at 800 °C for 2 h.
Characterization. The phase purity of the as-obtained product was characterized by X-ray diffraction using Rigaku (Miniflex-2) X-ray diffractometer with Cu Kα radiations (λ = 1.5406 Å) operated at voltage of 40 kV and current of 15 mA. The particle size and surface morphology of the synthesized powders were carried out using field emission transmission electron microscope (FE-TEM; JEOL/JEM-2100F version) operated at 200 kV. Fourier transmission infrared (FTIR) spectra of the powders (as pellets in KBr) were recorded using a Fourier transmission infrared spectrometer (Perkin Elmer) in the range of 4000-400 cm −1 with a resolution of 1 cm −1 . Room temperature optical absorption spectrum was recorded in the range of 200-800 nm using a UV-Vis spectrophotometer (Perkin Elmer). The thermal study of the synthesized product was carried out using thermo gravimetric analyzer (TGA-DTA; Thermo Scientific) up to 1000 °C in air at the heating rate of 10 °C/min, after purging N 2 gas.
Screening for anti-QS activity. A standard disc diffusion assay was used to determine the anti-QS activity of NS-ZnNPs 28 . Overnight grown culture of C. violaceum 12472 (100 μ l) was mixed with 5 mL of molten LB agar (0.3%) and immediately poured over the surface of pre-poured LB agar plate. After solidification, sterilized paper discs were placed on the surface of medium. Various concentrations of the synthesized NS-ZnNPs were applied on the discs and incubated overnight at 30 °C. Zone of inhibition of violacein pigment around the disc was considered as anti-QS activity.

Determination of Minimum inhibitory concentration (MIC). The MICs of synthesized NS-ZnNPs
against the bacterial pathogens were determined using the CLSI macrobroth dilution method 29 . MIC is defined as the minimum concentration of ZNPs at which there was no visible growth of the test strains. Concentrations below the MICs were considered sub-inhibitory and were further used to study the anti-QS and biofilm inhibitory properties.
Violacein inhibition assay. Biosensor strain Chromobacterium violaceum CV026 was incubated for 16-18 h (OD 600 nm = 0.1) and inoculated to in Erlenmeyer flasks containing Luria broth (LB), LB supplemented with C6-HSL (10 μ M/l) and LB supplemented with C6-HSL and test agent. The flasks were incubated at 27 °C with 150 rev/min agitation for 24 h in a shaking incubator 30 .
Violacein production by Chromobacterium violaceum (CVO26) in presence of NS-ZnNPs was studied using method described by Husain et al. 30  Assay for biofilm inhibition. The effect of NS-ZnNPs on biofilm formation was measured using the polyvinyl chloride biofilm formation assay 37 . Briefly, overnight cultures of test pathogens were re-suspended in fresh LB medium in the presence and the absence of NS-ZnNPs and incubated at 30 °C for 24 h. The biofilms in the microtiter plates stained with a crystal violet solution and quantified by solubilizing the dye in ethanol and measuring the absorbance at OD 470 .
In situ visualization of the biofilms. Briefly, 1% of overnight cultures of the test pathogens (0.4 OD at 600 nm) were added into 1 ml of fresh LB medium containing cover glass of 1 cm 2 along with and without NS-ZnNPs at respective sub-MICs. After 24 h of incubation, the cover glasses were rinsed thrice with distilled water to remove the planktonic cells and biofilms in the cover glasses were stained with 0.2% crystal violet (CV) solution. Stained cover glasses were placed on slides with the biofilm pointing up and visible biofilms were visualized by light microscope at magnifications of 40X (Nikon Eclipse Ti 100, Japan).
For CLSM analysis, biofilms of various bacterial strains were allowed to form on the glass coverslips as described in light microscopic analysis. After 24 h, biofilm formed in the glass slides were stained with 20 μ l of 1% acridine orange (Sigma Aldrich, Switzerland). The excess stain was washed out and the stained cover glasses were visualized with CLSM (Zeiss Spinning disk confocal microscope, Zeiss, Germany) equipped with an excitation filter 515-560 and magnification at 40× .

Inhibition of mature biofilms.
To ensure the potential of NS-ZnNPs in disturbing the mature biofilm of pathogenic bacterial organisms (P. aeruginosa, E. coli, L. monocytogenes, and C. violaceum), biofilms of bacterial pathogens were initially allowed to develop in microtitre plates for 16 h. Mature biofilms were then treated with respective sub-MICs of NS-ZnNPs for 8 h, stained with a crystal violet and quantified by solubilizing the dye in ethanol and measuring the absorbance at OD 470 .
Antibacterial activity of NS-ZnNPs in food model media. Lettuce leaf model media (L) was prepared as described by Gutierrez et al. 38 but with some modifications. 50 g of lettuce (Lactuca sativa sp.) were added to 100 ml of sterile deionized water and shaken for 1 min. The suspension was filtered using 18.5 cm Whatman filters and pH was adjusted from 5.6 to 7.2 by mixing two parts of lettuce media with one part 0.3 M potassium phosphate buffer, giving a final concentration of 0.1 M phosphate buffer, pH 7.2. The buffered medium was then autoclaved at 121 °C for 15 min. To investigate the NS-ZnNPs efficacy in meat-based model media, experiments were performed with autoclaved beef extract (BE, 12% protein). Beef extract model media was adjusted to pH 7.2 to rule out pH effects.
The effect of NS-ZnNPs (2 × MIC) on the growth of P. aeruginosa, E. coli, L. monocytogenes, and C. violaceum in Lettuce leaf and beef extract food model media was assessed by optical density measurements. Bacteria were grown in NS-ZnNPs treated lettuce leaf model media, beef extract media, and growth was monitored in a microplate reader (Thermo Scientific Multiskan FC) at 600 nm over 24 h at 4 h intervals.

Statistical analysis.
All experiments were performed in triplicate and the data obtained from the experiments were presented as mean values and the differences between control and test were analyzed using Student's t test.

Results
XRD measurements were performed to determine the crystalline phase of the samples. Figure 1 shows the XRD patterns of ZnO sample, which was indexed using POWDER-X software as the ZnO wurtzite structure and well matched with the standard data (JCPDS, . It can be clearly seen that it showed a single phase nature with hexagonal wurtzite structure. Refined value of the lattice parameters a and c of as prepared ZnO are found to be a = 3.253 Å and c = 5.246 Å, which are in good agreement with the standard data base [JCPDS, . Moreover, all diffraction peaks of the product show stronger peak intensities, indicating that the obtained ZnO nanoparticles have high crystallinity. The crystallite size of the synthesized powder was estimated from x-ray lines broadening using Scherer's equation 39 : where λ is the wavelength (Cu Kα ), β is the full width at the half-maximum and θ is the diffraction angle. The average grain size of ZnO nanoparticles was found ~26 nm.
The detailed morphology of ZnO nanostructures was investigated by TEM analysis. Figure 2 shows the TEM image of ZnO nanoparticles which are homogeneous and agglomerated with a particle size ~24 nm. Due to the uniform distribution of oxidized metal anions in the three-dimensional polymeric network structure, the agglomeration could be induced by densification resulting from the narrow space between particles. It is clear from the TEM micrographs that the particles have mixed morphology of spherical and elongated rod shape. Inset of Fig. 2 shows high resolution transmission electron microscopy (HRTEM) image of the ZnO nanoparticles. The HRTEM image shows clear lattice fringes indicating the highly crystalline ZnO wurtzite structure, and d spacing is well matched with the standard d values of ZnO.
UV-vis absorption spectrum as shown in Fig. 3, is carried out to evaluate the potential optical properties and to understand the electronic structure of the as-prepared ZnO nanoparticles. For the UV-vis absorption measurement, the as-prepared ZnO product is ultrasonically dispersed in absolute ethanol before examination, using absolute ethanol as the reference. The spectrum was corrected for the solvent contribution. The absorption spectrum of ZnO nanoparticles show well-defined exciton band at ~402 nm (calculated band gap of ~3.08 eV) which is red shifted by ~29 nm relative to the bulk exciton absorption (373 nm) 40 . The reason of the shifting of absorption band could be due to the oriented attachment of the nanoparticles by microwave irradiation, may lead to defect formation in the nanoparticles. The absorption in the visible range of wavelength implies that there exist  more defect energy levels in the synthesized ZnO nanostructures that are due to the specific experimental synthesis conditions 41 . Similar observations for shifting of absorption bands of ZnO towards visible region were also reported earlier 42 . Surface area and surface defects play an important role in the functional properties of metal oxides, which affects the optical and electronic properties 43 , due to which the optical absorption shifts towards the visible region. One of the strategies adopted for tuning the band gap is to introduce intentional defects in the crystal lattice by which the electronic structure of ZnO can be altered. In this method, defects in ZnO nanoparticles might be generated by the microwave irradiation.
To study the change in Zn-O bonding, FTIR measurement of ZnO nanoparticles has been carried out. FTIR measurements were performed in the wave number range 4000-400 cm −1 using KBr method at Room temperature as shown in Fig. 4. The FTI spectrum shows main absorption bands near 3400 cm −1 represent O-H mode, band arising from the absorption of atmospheric CO 2 on the metallic cations at 2300 cm −1 and 1400-1600 cm −1 are the C= O stretching mode. The absorption band at 431 cm −1 is the stretching mode of ZnO 44 as shown in the inset of Fig. 4.
TGA and DTA surveys were conducted to investigate the formation of ZnO during the calcinations process. Figure 5 shows the TGA/DTA curves for decomposition of bio-synthesized ZnO. TGA showed a weight loss in two steps at around 220 and 650 °C and corresponding DTA showed two endothermic peaks at these temperatures. It is considered that the weight loss around 220 °C results from the removal of the bio-products by heating process to burn out the organics in air. Furthermore, the following weight loss up to 700 °C occurs due to the thorough decomposition of those charred products and the formation of ZnO. As seen from the differential curve, the peak at around 220 °C is due to the removal of absorbed water when the product is heated from room temperature to 220 °C. Upon prolongation of temperature, the major weight loss was attributed toward the loss of moisture and carbon. Herein the bio-synthesis of ZnO can be separated into two steps: one is the decomposition of bio-product, and the other is the crystallization of hexagonal ZnO by the complete removal of the bio-product during the calcinations process up to 700 °C.   MIC level were considered as Sub-MICs and were used throughout the study to assess the anti-QS and biofilm inhibitory properties of the NS-ZnNPs.

QS (Violacein) inhibition assay.
Anti-quorum sensing potential of NS-ZnNPs was screened using disc diffusion assay with bio-indicator strain Chromobacterium violaceum 12472, which produces the AHL-regulated violet-colored 'violacein' pigment. Concentration dependent violacein inhibition effect of NS-ZnNPs was recorded. Highest inhibition was recorded at 400 μ g/ml followed by 200, 100 and 50 μ g/ml while no pigment inhibition was observed at lower concentrations (Fig. 6). Azithromycin (2 μ g/ml) was used as positive control.
Findings of the assay with Chromobacterium violaceum 12472 were further confirmed by colorimetric determination of violacein production in Chromobacterium violaceum CVO26. NS-ZnNPs at all tested concentration exhibited a statistically significant decrease in violacein content without inhibiting bacterial growth. At the concentration of 50 μ g/ml NS-ZnNPs reduced violacein production up to 41% in comparison to untreated control (p ≤ 0.05). Dose dependent increase in the inhibitory activity was observed with increasing concentration of NS-ZnNPs to a maximum of 91% (p ≤ 0.001) at 400 μ g/ml concentration of (Fig. 6).

Inhibition of PAO1 virulence by sub-MICs of NS-ZnNPs. Effect of sub-MICs of synthesized NS-ZnNPs
on the QS regulated virulence factors (elastase, total protease, pyocyanin production and alginate production) in PAO1 was assessed. Dose dependent decrease in the production of elastase (35-82%), total protease (20-77%) and pyocyanin production (48-93%) was recorded (Table 1). Alginate extracted from untreated and treated cultures of PAO1 was quantified. Gradual drop in alginate production was observed with increasing concentration of nanoparticle concentration. NS-ZnNPs demonstrated 20-73% reduction in alginate production of PAO1 at sub-MICs ranging from 10-80 μ g/ml (Table 1).
Effect on β-galactosidase activity. Effect of 10-40 μ g/ml concentrations of NS-ZnNPs on lasB and pqsA transcriptional activity was examined using β -galactosidase assay. NS-ZnNPs at 10, 20, 40 and 80 μ g/ml concentrations significantly (p ≤ 0.001) reduced the lasB transcriptional activity by 35, 55, 78 and 85%, respectively. Moreover, 41-84% down regulation in pqsA was also recorded at concentration ranging from 10-80 μ g/ml concentration of NS-ZnNPs (Fig. 7a,b). the reduction in transcriptional pqsA activity was significant at all tested concentrations.   (Fig. 10). Statistically significantly impaired biofilm formation was recorded at all tested concentrations against all pathogens. The results of biofilm inhibition correlated positively with swarming and EPS reduction as these play an important role in adhesion and maturation of biofilms.

In-situ visualization of biofilm inhibition. Untreated biofilms grown on glass coverslips showed a thick
layer of biofilm, stained easily with CV, and visualized under the light microscope. However, NS-ZnNPs treated coverslips exhibited dose dependent impairment of biofilm formation of L. monocytogenes, P. aeruginosa PAO1, C. violaceum and E. coli. Results of microscopic analysis revealed the maximum level of reduction in number of microcolonies at the 1/2 × MIC against all tested food pathogens (Fig. 11A). Further, CLSM images also showed loose biofilm architecture and reduced biofilms of all foodborne pathogens after treatment with respective sub-inhibitory concentration in comparison to untreated control (Fig. 11B).         1000, 256, 1000 and 512 μ g/ml concentration of NS-ZnNPs, respectively. Figure 13(A-D) shows the reduction in growth rate for all the microbes tested after incubation with respective concentrations of NS-ZnONPs in beef extract media. Similarly, in the lettuce leaf model media significantly impaired growth of the test pathogens was recorded after treatment with NS-ZnNPs as compared to the untreated control ( Fig. 14A-D). Antibacterial activity of the synthesized NS-ZnNPs was similar in both the model food mediums and it is envisaged that these zinc nanostructures have broad-spectrum antibacterial property.

Discussion
Excessive food is lost due to quorum sensing regulated microbial spoilage and/or contamination of food by human pathogens 1 . Hence, disrupting the quorum-sensing circuit and biofilm formation can play a major role in controlling food spoilage and ensuring food safety. Quorum sensing inhibitory activity of synthesized NS-ZnNPs was assessed using Chromobacterium violaceum CV12472 and CVO26 biosensor strains. Production of pigment violacein is regulated by CviIR-dependent quorum sensing system. Therefore, inhibition of pigment is suggestive of interference with AHL-regulated QS 45 . Concentration dependent reduction in pigment production (opaque, non-transparent halo zones) was observed around the disc loaded with NS-ZnNPs (Fig. 6). Further, for quantitative assessment of violacein inhibition an engineered biosensor strain Chromobacterium violaceum CVO26 was used. CVO26 does not produce its own its AHL but responds to exogenous AHL. The findings of colorimetric assay revealed significant reduction in violacein at all tested concentrations (50-400 μ g/ml) of NS-ZnNPs. Our findings on QS inhibition in Chromobacetrium violaceum biosensor strain are well supported by those reported with silver nanowires (80% inhibition), mycofabricated silver nanoparticles (100% violacein inhibition) and AgCl-TiO 2 nanoparticles 4,46,47 .

Inhibition of PAO1 virulence by sub-MICs of NS-ZnNPs.
To ascertain the QS inhibitory of NS-ZnNPs, effect of these nanoparticles was assessed on QS regulated virulence factors of P. aeruginosa PAO1. QS signaling in Pseudomonas aeruginosa is N-acyl homoserine lactones (AHLs) based and comprises of three QS systems namely, the las, rhl and pqs systems that regulate the production of virulence factors, including elastase, exoproteases, siderophores, exotoxins, rhamnolipid, pyocyanin, pyoverdin and participate in the development of biofilms 48,49 .
Proteases and elastase enzymes contribute greatly to the pathogenesis of P. aeruginosa by degrading host tissues enhancing the growth and invasiveness of the organism 50 . Sub-inhibitory concentrations of NS-ZnNPs inhibited elastase (35-82%) and protease (20-77%) significantly in dose dependent manner. Pyocyanin is a secondary metabolite of P. aeruginosa that causes severe toxic effects by damaging the neutrophil-mediated host defense 51 . Effect of NS-ZnNPs on pyocyanin was assayed and statistically significant decrease in production was recorded over untreated control (Table 1). P. aeruginosa biofilm matrix is composed of an expolysaccharide called alginate, its production is also known to be controlled by QS. The effect of sub-MICs of NS-ZnNPs on alginate production wasstudied and alginate production o was reduced significantly with increasing concentration of NS-ZnNPs. Results of alginate inhibition points towards successful disruption of biofilm matrix of P. aeruginosa. Our findings on inhibition of virulence factors find support from the observations on silver nanoparticles, Zinc nanoparticles, ceftazidime, menthol and caffeine 29,47,[52][53][54] . Effect on β-galactosidase activity. Findings of the β -galactosidase activity showed that sub-MICs of NS-ZnNPs reduced both the elastase activity of PAO1 and the transcriptional activation of lasB in E. coli, indicating that synthesized NPs inhibits the las QS system. There is a positive co-relation between AHL concentration and lasB-lacZ expression as demonstrated by Pearson et al. 33 Our findings correlate well with the above observations, as reduced β -galactosidase activity is indicative of reduced AHL levels and, therefore, reduced expression of the lasB gene. Since the production of pyocyanin production is regulated by pqs system, NS-ZnNPs inhibits the transcriptional activation of pqsA in E. coli, which indicates that NPs inhibits the pqs system. Results of the present assay demonstrates the broad-spectrum anti-QS property of these NPs as it inhibits the las and pqs systems of P. aeruginosa.
Inhibition of swarming motility and EPS production. Flagella-driven swarming motility is a QS-dependent virulence function that play an important role in the initiation of cell/surface attachment during biofilm development 55 . Present investigation examined the ability of sub-inhibitory concentrations of synthesized NS-ZnNPs to impair the migration of test pathogens in a dose-dependent manner (Table 2, Fig. 8). Reduction in the swarming migration is indicative of the inhibition in the flagellar synthesis by the NS-ZnNPs. Thus, NS-ZnNPs indirectly effects the biofilm formation of all the target food pathogens in part by interfering with the ability of the pathogen to reach the substratum and subsequent biofilm formation by disturbing AHL-regulated QS system.
EPS plays a key role in the maintenance of biofilm architecture and provides increased resistance to the cells to antibiotics as well as to osmotic and oxidative stresses 48 . Therefore, any interference with EPS synthesis or production is bound to effect the biofilm architecture and bacteria in the biofilm mode of growth will become more susceptible to the administered antibiotics. Hence, resistance to drugs will also be reduced. In the present study, EPS was reduced significantly when bacterial pathogens were treated with sub-MICs of NS-ZnNPs (Fig. 9).

Biofilm inhibition.
Biofilm inhibitory properties of NS-ZnNPs were examined against foodborne pathogens L. monocytogenes, E. coli, C. violaceum, and P. aeruginosa PAO1. NS-ZnNPs reduced the biofilm biomass significantly in a dose-dependent manner (Fig. 10) without affecting the bacterial growth against all pathogens tested. Light microscopic images (Fig. 11A) revealed that NS-ZnNPs reduced the number of microcolonies during the biofilm formation of test bacterial pathogens. Therefore, it is envisaged that treatment of bacterial pathogens with NS-ZnNPs resulted in the formation of weak biofilms possibly by reducing the surface adhesion and subsequent microcolony formation. Further, CLSM analysis of NS-ZnNPs treated biofilms displayed poor and weakened architecture and reduced thickness than that of untreated biofilms of all pathogens tested (Fig. 11B). Our findings are agreement with the report on AgCl-TiO 2 nanoparticles, where in, biofilm formation in C. violaceum was completely inhibited at 100 μ g/ml 4 . In another study, silver nanowires arrested biofilm formation significantly at 4 mg/ml concentration without affecting viability of microbial cells 45 .
Although further research is necessary to unearth the plausible mechanism of biofilm inhibition by the ZnO nanoparticles. Considering P. aeruginosa PAO1 as a model organism, Lee et al. 56 reported that two-component response regulator CzcR is required for the inhibition of biofilm and pyocyanin. They demonstrated that ZnO nanoparticles inhibit pyocyanin production and biofilm formation via the czcRS system and rhlR quorum sensing system and also repress the production of the PQS autoinducer. In addition, increased cell surface hydrophobicity after treatment with ZnO NPs was another major reason for the impaired biofilm formation in PAO1 56 . Our findings on the inhibition of pyocyanin and biofilm formation by sub-MICs of NS-ZnNPs, correlates well with the findings of Lee et al. 56 Therefore, it is predicted that the synthesized NS-ZnNPs inhibit biofilm via response regulator CzcR and increased hydrophobicity in P. aeruginosa PAO1.
Disruption of mature biofilm. Since quorum sensing is helps in the maturation of biofilms, therefore, interference with QS mechanism is deemed to impair maturation of biofilms. Figure 12 shows significant reduction in preformed biofilms of all tested bacterial pathogens upon treatment with sub-inhibitory concentrations of NS-ZnNPs. Our results are in accordance with the previous report where Silver(I) complexes with phthalazine and quinazoline demonstrated marked ability to disrupt mature biofilms of P. aeruginosa 57 . Extract of Capparis spinosa also showed significant reduction in the preformed biofilms of Escherichia coli, Proteus mirabilis, Serratia marcescens and PAO1 58 .

Antibacterial activity of NS-ZnNPs in food model media.
To assess the effectiveness of the synthesized NS-ZnNPs as antimicrobial agents in food. We studied the antibacterial action of these nanostructures in two food model media namely, lettuce leaf model media and beef extract media. The NS-ZnNPs demonstrated significant reduction in the growth pattern of the tested pathogens in both the food models as shown in Figs 13 and 14. Our results find support from the observations made with natural products, alone or in combination with other preservative, against food-borne pathogens when applied to meat 38,59 , vegetable 38,60 or milk 61 . Results obtained in food model media are important as they may provide the leads for further studies on food directly and help in the optimization of the test agents before final application. These model media are better tool than the commonly used standard laboratory media.

Conclusion
In conclusion, our result demonstrates the development of a rapid, cost-effective, eco-friendly and safe method of zinc oxide nanoparticle synthesis exploiting the reducing and capping potential of Nigella sativa (black seed). Study further highlights the broad-spectrum attenuation of AHL mediated quorum sensing and QS regulated functions of C. violaceum and P. aeruginosa by synthesized NS-ZnNPs. Biogenic nanoparticles (NS-ZnNPs) impaired the biofilm formation of four food pathogens viz. L. monocytogenes, P. aeruginosa PAO1, and E. coli considerably. Vital factors like swarming motility and EPS production, contributing in the initial attachment and maturation of biofilm were also reduced significantly. Moreover, significant reduction in preformed biofilms of all tested bacterial pathogens upon treatment with sub-inhibitory concentrations of NS-ZnNPs is a key finding. This is probably the first report on the broad-spectrum inhibition of QS and biofilm by biogenic Zinc oxide nanoparticles. The study projects the synthesized Zinc nanostructures to be a potential QS and biofilm inhibitor that can not only be exploited as an antipathogenic but nontoxic bioactive material that can be used as food packaging material and/or as food preservative.