Nanoemulsification of Rose ( Rosa damascena ) Essential Oil: Characterization, Anti-Salmonella , In Vitro Cytotoxicity to Cancer Cells, and Advantages in Sheep Meat Application

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Introduction
Lately, there has been a surge of interest in colloidal nanoparticles due to their promising range of potential applications.Te distinctive physicochemical properties of colloidal nanoparticles make them suitable candidates for design and application as pharmaceuticals and foods preservative agents.Te advancement of nanotechnology has led to the emergence of various colloidal nanocarriers within the particle size range of 1-to 100 nm.Among these, polymeric nanoparticles, solid lipid nanoparticles, liposomes, and micelles have established themselves as nanocarriers across diverse industries.However, in contemporary times, even more advanced and innovative nanosystems, including dendrimers, nanoemulsions, nanogels, nanosuspensions, and nanotubes, have demonstrated even greater potential than their predecessors, thanks to the progress achieved through nanotechnological approaches [1,2].A colloidal particle system, known as a nanoemulsion, is made up of two heterogeneous liquids [3].Due to their small particle size, ease of preparation, increased bioavailability, biological efectiveness, and kinetic stability, nanoemulsions are known as a perfect carrier for transferring lipophilic materials.Tey are also referred to as a selfpreserving antimicrobial composition due to the lack of enough water in their structure for microorganism growth [4].Furthermore, nanoemulsions show the stable thermodynamic and kinetic properties that could be characterized by droplet sizes ranging from 20 to 400 nm and a uniform size distribution, setting them apart from other emulsions [5].Due to their numerous physiological, chemical, and biological attributes, nanoemulsions fnd applications in diverse industries, including oil and gas [6], agriculture, chemistry, food, and cosmetics [7].
Foodborne pathogens, i.e., bacteria, fungi, and viruses, pose a global threat by causing food poisoning and intestinal infections.Among these pathogens, S. typhimurium is recognized as a highly dangerous bacterium in the terms of human pathogenicity [14].Gastroenteritis, predominantly caused by Salmonella serotypes, particularly S. typhimurium, is the most common form of Salmonella infection in humans.Tis leads to an annual incidence of 1.4 million cases of nontyphoidal salmonellosis reported in the United States alone [15,16].S. typhimurium is responsible for causing various illnesses, including acute gastroenteritis, typhoid or paratyphoid enteric fever, and systemic infections.Human infections typically occur through the consumption of raw foods, such as meat and eggs, with food contamination from animal sources being the primary route of transmission [17,18].Given that genes play a crucial role in determining the virulence of S. typhimurium, the key genes include spvC, invA, stn, and gyrB.Te spvC gene, located on a plasmid, is essential for the bacterium's survival within the host cell.Te invA gene is responsible for bacterial invasion of epithelial cells, while gyrB encodes the DNA gyrase subunit involved in invasion.In addition, the stn gene produces an enterotoxin and codes for a protein that causes diarrhea [19,20].
In previous studies, various essential oils have been investigated for their biological properties both in vitro and in food models.Dini et al. found that the shelf life of refrigerated meat could be extended more efectively using cumin essential oil nanoemulsion compared to chitosan [21].Keykhosravy et al. demonstrated that chitosan can increase the shelf life of turkey meat by up to 9 days, while nanoemulsions containing a combination of Bunium persicum and Zataria multifora showed an even greater increase of up to 20 days [22].
Terefore, the objective of the present study was to assess the morphology and stability of RDNE, as well as its antibacterial and anticancer properties through in vitro evaluations.Furthermore, the study aimed to investigate the efects of RDNE on the quality, physicochemical characteristics, and sensory attributes of sheep meat during refrigerated storage at a temperature of 4 ± 1 °C.

REO and RDNE Preparation.
Te REO was purchased from Zarin Golab company (Kashan, Iran).To prepare RDNE, 93 µg/mL of REO, 115 µg/mL of Tween 80 (Sigma Aldrich, USA), and 115 µg/mL of Span80 (Sigma Aldrich, USA) were mixed for nanoemulsion formulation.By slowly and continuously adding REO and surfactants to water while shaking at 3000 rpm, we created a coarse emulsion.Ten, a 20 kHz sonicator was utilized to perform ultrasonic emulsifcation on the coarse emulsion (Heshler, Germany).A sonotrode with a 13 mm of diameter piezoelectric crystal was used, and the operation power was changed to 400 W. Finally, 7 mL of the mixture was placed in the sonicator, and ultrasonic waves were delivered for 10 min [23].

Gas Chromatography-Mass Spectroscopy (GC-MS)
Analysis of REO.By using GC-MS analysis, we determined the constituents of the necessary oil.A moderate nonpolar capillary column was integrated with GC-MS (Agilent 6890, USA) (BPX5, 30 m length, 0.25 micrometer flm thickness, 0.25 mm internal diameter).Te injector port temperature was 260 °C.Te sample was injected by splitting, and the split ratio was 1 : 10.Te temperature was programmed from 50 °C to 220 °C at 10 °C per minute and held at 220 °C for 15 min with helium as the carrier gas (fow rate 0.5 mL•min −1 ).Electronic impact, a 70 eV ionization potential, a 220 °C ion source, and a mass range of 30 Da-500 Da included all characteristics of the mass spectra.NIST (2010) used mass spectra and Kováts indexes (KIs) to identify compounds in relation to n-alkanes (Wiley spectral library collection and authentic chemicals) [24,25].

Measurement of RDNE Droplet Size.
After being properly diluted with distilled water, the nanoemulsion was adsorbed in a copper grid covered in carbon.After that, using a Zeiss EM900 transmission electron microscope (TEM) with an 80 kV accelerating voltage, we analyzed the particles' morphology and size and captured them with a camera.For size analysis of RDNE, the dynamic light scattering (DLS) technique was employed using the Zetasizer Nano ZS, model ZEN3500 from Malvern Instruments, UK [26].

Zeta Potential of RDNE.
Trough a Zetasizer, we used electrophoretic light scattering and estimated the zeta potential (mV) of RDNE (SZ-100-Z, Horbia, Japan).20 µL of RDNE was diluted in 1 mL milli-Q water before being deposited in capillary cells with two electrodes.Ten, 30 continuous readings were used to acquire particle charge information [27].200 microliters of 95% ethanol were used to dissolve the labeled bioflm cells, and the samples' optical density (OD) was measured at 570 nm.To minimize the efect of background efects, the positive control wells contained only bacterial cells and BHI (without RDNE), while the negative control wells contained BHI without bacterial cells and RDNE.Te antibioflm index was calculated using the following formula [31,32]: 2.6.Expression of stn Gene.Junior et al. [33] proposed the extraction of S. typhimurium genomic DNA by boiling.All DNA samples were stored at −20 °C, until the S. typhimurium stn gene was detected in them.Te PCR and DNA amplifcation processes were carried out in 25 µL volumes in microtest tubes (PCR tubes), with 5 µL of DNA template, 12.5 µL of 2X PCR Master Mix (Kiagene, Iran), 1 µL of each primer (20 pmol/µL), and nuclease-free water up to 25 µL.PCR amplifcations were carried out using the Mastercycler (Eppendorf, Germany).Table 1 lists the primer sequence, target gene (stn), and PCR conditions.PCR products were analyzed by electrophoresis on 1% agarose gel in 1x TBE bufer at room temperature.Tey were stained with ethidium bromide and visualized using a UV transilluminator (Denazist Asia, Iran) (Ingenious, L. Cambridge, UK) [34].
For target gene expression analysis, frst, S. typhimurium cells in TSB with diferent concentrations (0, 75, 125, and 250 µg/mL) of RDNE were cultured overnight at 37 °C.Ten, we extracted RNAs using an RNA isolation kit (Cinaclon, Iran) following the manufacturer's instructions.cDNA was produced from the total extracted RNA using a special kit (Addbio, Korea).Finally, a StepOnePlus instrument was used to amplify the stn gene.Te fold change in gene expression due to RDNE treatment was calculated using the 2 −∆∆CT method, and the gene expression level was reported in relative units [34].

Te Efect of RDNE on Cancer Cell
Lines.RDNE's antitumor efects on specifc and cervical cancer cell lines (colon HCT 116 and cervix HeLa) were analyzed using an MTTassay.96-well plate was used with a cell-seeding density of 1 × 10 4 cells per well.Cells were cultured for 24 hours in DMEM (Himedia, Bombay, India) supplemented with 10% FBS (HiMedia, Mumbai, India) and allowed to attach.Medium was replaced with suspensions of RDNE that Journal of Food Quality  Journal of Food Quality ranged in concentration from 2.9 to 187 µg/mL (each concentration was seeded into at least three wells) and cells were incubated for 48 hours.Ten, MTT reagent (10 µl per well) was added.Cells were kept in the incubator for additional four hours at 37 °C.After removing the medium, 50 µl of DMSO was added to each well.Te OD of the formazan product was measured at 570 nm using a multiwell spectrophotometer.Te OD readouts were then analyzed, and the cell viability was calculated using the following formula [35]: Y � Mx + C, a linear regression equation, was used to determine the IC 50 value.
In this case, Y � 50 and the values of M and C were obtained from the viability graph.

Preparation of Ground Meat Samples and Treatments.
Te RDNE samples used in this study were obtained from a butcher shop in Kashan and immediately transported on ice to the laboratory for food hygiene at Kashan University of Medical Sciences in Iran.Tey were randomly assigned into four groups of ground meat samples (control: without any coating RDNE, treatment 1: ground meat coating with 75 µg/g of RDNE, treatment 2: ground meat coating with 125 µg/g of RDNE, treatment 3: ground meat coating with 250 µg/g of RDNE).After being kept at 4 ± 1 °C for 12 days, all samples were examined for their microbiological and biochemical characteristics (days 1, 4, 8, and 12).On the other hand, the ground meat samples (10 ± 0.5 g) were gamma irradiated at the Atomic Energy Organization of Iran at a dose rate of 4 KGy/s.Ten, four groups of meat samples were randomly assigned into four groups (control: without any coating RDNE plus 10 5 CFU/mL of S. typhimurium; treatment 1: ground meat coating with 75 µg/g of RDNE plus 10 5 CFU/mL of S. typhimurium; treatment 2: ground meat coating with 125 µg/g of RDNE plus 10 5 CFU/mL of S. typhimurium; treatment 3: ground meat coating with 250 µg/g of RDNE plus 10 5 CFU/mL of S. typhimurium).All samples were kept in storage at 4 ± 1 °C and analyzed on the mentioned days [22,36].

Microbial Analyses.
Microbial analysis was performed using standard methods, including the enumeration of TBC, coliforms, LAB, and S. typhimurium.Te TBC, coliforms, and LAB enumeration were performed using pour plate standard method, while the enumeration of S. typhimurium was carried out using the spread plate method.Incubation of the plates was conducted at 37 °C for 48 hours.Results were shown as log CFU/g [36,37].

Chemical
Analyses.TVB-N and pH parameters were determined using the methods described by Khoshbouy Lahidjani et al. [38], Zhou et al., [39] and other researchers, respectively.

Sensory Analysis.
Every sample was fried in soy oil in the oven for ten minutes.Ten evaluators were selected from the food hygiene and quality control laboratory at Kashan University of Medical Sciences according to their prior experience in initial evaluation trials.Panel members were frst given information about ground meat and its characteristics (taste, odor, color, and overall acceptability).Before the test, they attended a prep session, allowing each panelist to fully discuss and elaborate on each aspect of the fried ground meat.Tree replicates of each sample (0, 75, 125, and 250 µg/g of RDNE) were used for evaluation, and the samples were distributed to panelists in randomized order.All characteristics were scored using a 5-point hedonic scale that was anchored by very nice, 5; good, 4; acceptable, 3; poor, 2; and very poor, 1.A sample of fried ground meat without RDNE was used as the control in the experiment [40].
2.9.Statistical analysis.Tere were three repetitions of each experiment in this study, and mean values were recorded.Te resulting values were then transferred into SPSS software version 21 and compared using one-way ANOVA and the least signifcant diferences (LSD) method.Diferences were considered signifcant at p < 0.05.

RDNE Characterization. Diferent morphological sizes
were detected for the RDNE in the TEM and DLS analysis.Despite the fact that some diferent droplets were seen, the size of the emulsion droplets in the image and the fndings of the particle size analyzer were in good agreement (ranging from 30 to 50 nm).Oil droplets are distinguished by their clearly defned boundaries and smooth surfaces (Figure 1).Te RDNE's stability was confrmed by zeta potential analysis.Based on the observed zeta potential of −47.5 mV (Figure 2), the RDNE has sufcient electrostatic repulsion to keep itself stable in the solution.

Anti-Salmonella Properties of the RDNE.
In the present study, RDNE was shown to have an inhibition efect on S. typhimurium using the Agar disk difusion method, as depicted in Table 3. Te results revealed the anti-Salmonella efects of RDNE on S. typhimurium were lower than that of gentamicin.In addition, the MIC and MBC rates were 375 and 750 µg/mL, respectively.In Figure 3, the bioflm Journal of Food Quality inhibitory activity of RDNE was evaluated at diferent concentrations (0.5, 1, and 2 MICs).

Molecular Analyses.
Te stn gene was successfully amplifed, as the ∼617 bp product was observed with 1% agarose gel (Figure 4).Te RDNE efect on stn gene expression of S. typhimurium was also observed (Figure 5).Te S. typhimurium stn gene showed a greatest decrease in the expression (21.2%) at a dose of 250 µg/mL RDNE.

Cytotoxicity Analysis of RDNE on HCT116 and HeLa Cell
Lines.Te cytotoxicity of RDNE was evaluated on two cancer cell lines.HCT 116 and HeLa cells were exposed to various concentrations of the drug, and a dose-dependent cytotoxicity was observed as shown in Figure 6.With increasing RDNE concentrations, the growth of cancer cells was signifcantly inhibited.Te concentration of RDNE that inhibits cell proliferation by 50% (IC 50 ) was found to be 3.31 µg/mL for HCT 116 and 4.6 µg/mL for HeLa cells.

Sheep Meat Shelf Life
3.6.1.Microbial Changes.At the beginning (Figure 7(a)), the TBC of fresh ground meat samples was 6.4 log CFU/g and slowly raised to 9.4 log CFU/g throughout the 8 th day.Compared to the control group, the TBC in ground meat samples containing RDNE (125 and 250 µg/g) was lower at alltime points.A signifcant diference was found between the RDNE with 250 µg/g concentration and other treatments on days 4 and 8 regarding the inhibitory growth of bacteria.Also, adding RDNE at a concentration of 250 µg/g into fresh ground meat resulted in a TBC below 8 log CFU/g during the storage.
On the frst day, LAB populations were detected at levels ranging from 2.7 log CFU/g in the treated samples to 3.5 log CFU/g, eventually peaking at approximately 4.8 log CFU/g by the 12 th day.Troughout the monitoring period, LAB exhibited a nearly uniform growth pattern across all treatments, except for the samples with a 75 ppm RDNE concentration, which had signifcantly lower fnal counts (3.6 log CFU/g) compared to the other groups (Figure 7(b)).
Te population of coliforms in diferent types of samples after a 12-day storage period is shown in Figure 7(c).At all intervals of time, the control group's coliform count was the higher.As the fgure shows, except for 75 µg/g of RDNE concentration, a steady decrease in the growth of coliforms in treatments was noted from day four to day eight.On the 12 th day of the storage period, the control group had the highest population (∼5 log CFU/g), while samples with a 250 µg/g RDNE concentration had the lowest population (∼4.2 log CFU/g).
Figure 8 depicts how three diferent treatments afected S. typhimurium development over a 12-day storage period.S. typhimurium's initial count was 7.12 log CFU/g; during storage, it rose in all samples.However, in RDNE of 250 µg/g concentration samples (∼6.46 log CFU/g), we found the lowest trend.

Chemical Analysis.
Te comparison of TVB-N and pH during a 12-day period is shown in Table 4. Up to the fourth day, there was no diference between the control and other treatment groups in terms of the TVB-N.As compared to the control at the storage time, TVB-N levels in several treated samples, including samples with RDNE of 250 µg/g concentration, were signifcantly lower after the fourth day (12 days).In all samples, the amount of TVB-N signifcantly increased during the storage period.However, the increase was more prominent in the control sample, raising from 12.5 ± 0.14 mg/100 g to 36.7 ± 0.42 mg/100 g in the refrigerated storage.
Te initial pH values ranged from 4.65 ± 0.07 to 5.40 ± 0.14 among the samples.Troughout the storage period, the pH values increased; however, a slower trend of pH increase was observed in the ground meat samples wrapped with RDNE as compared to the control group.Notably, the treatment with RDNE at a concentration of 250 µg/g demonstrated the most favorable efect on the pH of the ground meat when compared to other treatments (P < 0.05).Based on the panelist's evaluation, the taste was assessed as satisfactory for the ground meat during the procedures and no detrimental impact on the taste ratings was noted.Hence, greater taste scores were obtained when RDNE content was raised (Figure 9).However, there were no discernible variations in color between the control and RDNE-enriched samples.Moreover, RDNE's red hues indicated that the samples of ground meat did not change in color.All samples that included RDNE showed odor values lower than the control, whereas those without RDNE had more pleasant odors.

Discussion
In this study, 84.01% of the total REO was indicated by all the detected chemical compounds.Our results are in agreement with the studies carried out by Alizadeh and Fattahi [41], who identifed heneicosane (30.43%) as the primary component of REO.In contrast, another study showed that the essential components included octadecane (4.70%) and nonadecane (0.32%).Te discrepancy between the two   studies may be due to genetic variances, geographic location, collection timing, plant growth phases, and seasonal and environmental conditions [37,42].Te RDNE was found by visually inspecting the TEM and DLS of the formulation.Tis study successfully tested the droplet size and physical stability of the chosen formulation.Te nanoemulsion's kinetic stability, solubility, and carrier functioning are generally improved with smaller particle sizes [43].Te Ostwald ripening phenomenon, which involves the redeposition of small oil droplets onto larger oil droplets to produce greater sizes, is primarily responsible for the rise in particle size [44].
Te efectiveness of RDNE's anti-Salmonella activity was assessed by the disc difusion, MIC, MBC, and bioflm techniques.Our results revealed that RDNE had anti-Salmonella properties.Te use of naturally occurring antimicrobial compounds in plants with lethal and inhibitive efects on infections has grown in popularity due to the increase in bacterial resistance to several antibiotics [22,45].Bioactive compounds were present in essential oil (e.g., terpenes).Te composition, type, concentration, target microorganism, substrate composition, storage and processing conditions, and antibacterial action of EO show variations across situations [46].Te lipid fraction of the bilayer membrane is disturbed by EO compounds, particularly phenolic compounds, which can interact with cell organelles [47].Tis leads to antibacterial activity that was evidenced by the antibacterial actions of EO derived from R. damascena against S. typhimurium.Nanoemulsions trigger cell death in microorganisms by interacting with their lipids [48].As nanoemulsions join with the microbe, they release some of their internal contents, causing cell lysis.Te likelihood of their interaction with charges on the pathogen surface can be increased by the electrostatic   attraction [49].However, it is likely that their major and minor low-molecular-weight (such as terpenes and terpenoids) components, in combination with the currently used nanoemulsion form, cause REO's potent anti-Salmonella activity [50].Many virulence genes, including stn, have been generated by Salmonella spp. to contribute the pathogenesis and colonization of the bacterium in various host tissues [51].Salmonella's pathogenic symptoms, such as diarrhea, stomach pain, and vomiting, are caused by enterotoxin activity, which is encoded by the enterotoxin gene (stn) [52].Our investigation into the stn production by S. typhimurium demonstrated that RDNE negatively infuenced the inhibition of stn expression, which increased with RDNE concentration.Te increased antibacterial properties of this nanoemulsion formulation are thought to be the result of a number of factors, including an improvement in the penetration of plant-derived components into bacterial cells and a decrease in their susceptibility to bacterial enzyme degradation [53].
One of the main worldwide causes of death today is cancer [54].Many studies have demonstrated that nanotechnology could be benefciary to cancer therapy as an alternative option [55].Natural substances harmful to cancer cells have also been explored for antitumor therapies [56].One of the main herbal substances with broad cytotoxicity against cancer cells is R. damascena [57].In our study, HeLa and HCT 116, respectively, cervix and colon cancer cell lines were signifcantly in a dose-dependent manner responsive to cytotoxic efect of RDNE.Te IC 50 values were approximately 4.6 µg/mL and 3.31 µg/mL for HeLa and HCT 116, respectively.Colon cancer cells were   Using the MTT assay to measure cell viability, the researchers reported that REO had anticancer activity, as it slowed the growth of cancerous cells with an IC 50 of 36.43 ± 3.373 g/mL [59].Te RDNE demonstrated better cytotoxicity against cancer cells than REO and the extract compared to our fndings.TBC was discovered to be efectively regulated, particularly in 250 µg/g of RDNE concentration.A study suggested that when meat and meat products are refrigerated, the amount of TBC can be reduced using plant extracts, EOs, or a combination of the two [60].In a study, safower oil nanoemulsion (SNE) + oxygen absorber (OA) and SNE + 1% cumin essential oil (CEO) were found to be more efective in controlling TBC expansion than the single treatments of SNE and OA.However, the most efective treatment course was SNE + 1% CEO + OA [61].
It is well known that meat with LAB typically has a longer shelf life.LAB might take over as the main bacterial groups after aerobic spoilage bacteria are inhibited.Earlier investigations noted that LAB could develop in a variety of muscle food products both in aerobic and OA packing [62].When RDNE was used in the meat samples, the population of LAB lowered to its lowest levels (3.6 log CFU/g during the 12 th day) compared to the control (4 log CFU/g during the 12 th day).In a study, after 10 days of storage at a refrigerator temperature, the amount of LAB was noticeably decreased (3.21-3.71log) when chicken fllets with PIE (Polylophium involucratum essential oil) integrated wrapped samples (0.5, 1, and 1.5%) [63].
Te results showed that diferent concentrations of RDNE reduced the log of coliform, especially 250, 75, and 125 µg/g concentrations had fewer log efects.Tis fnding is due to the fact that meat contains complex nutritional ingredients, such as proteins and fats [64].Te interactions between the target microorganisms and the antimicrobial agent may be hampered by these food components, necessitating a larger concentration of antimicrobial agents [65].It has also been noted that a high concentration of organic components could be destabilized a nanoemulsion by some mechanisms, including binding impact, depletion, bridging, or electrostatic screening [66].
For S. typhimurium, we could add that the phenolic components of the EO of nanoemulsion interact with the Gram-negative cells' outer membrane.Consequently, this alters the structure of the membrane, results in the loss of the functional permeability barrier, and increases the permeability for RDNE [67].According to our results, diferent concentrations of RDNE are needed to adequately inhibit S. typhimurium.
Over the course of 12 days, the pH values of the control and treated samples increased, and there were no appreciable diferences between the treated samples and the control group.However, RDNE treatment at a concentration of 250 µg/g had the best impact on the pH of the ground meat compared to other treatments.Te increase in pH in all samples could be attributed to the action of microbial enzymes or endogenous enzymes, such as proteases and lipases, which induce a rise in volatile bases during extended storage [68].1% Nacl + lactates and 2% Nacl + lactates treatments in refrigerated and frozen fresh ground pork during 14 days of storage did not signifcantly difer from the control group, according to Tan and Shelef's report [69].
In TVB-N, samples treated with 250 µg/g of RDNE concentration caused a noticeable decrease in TVB-N in comparison to the control group.With time spent at 4 °C, the initial amount of TVB-N in control and coated samples substantially increased, according to our data, which are similar to those of Ojagh et al. [70].At the end of the trial (16 days), fsh samples coated with chitosan-cinnamon had TVB-N that was much lower (14.23 mg/100 g) than fsh samples coated with chitosan or the control, which had high values of 22.86 and 42.93 mg/ 100 g, respectively.Reduced levels of TVB-N in the samples treated with 250 µg/g of RDNE could be the result of a quicker bacterial population decline, a decreased ability of the bacteria to oxidatively deaminate nonprotein nitrogenous compounds, or both [71].
Finally, according to the organoleptic data, there were no appreciable diferences in the control and RDNE-enriched samples' color, odor, taste, or acceptability (p > 0.05).Samples without RDNE had better scores.Tis occurs due to the increased nanoemulsion particle size that alters the ground meat matrix's structure, which impacts organoleptic properties.
While nanoemulsion may ofer a potential mechanism to physically trap the compounds causing these unpleasant tastes or odors, the quality of the raw ingredients and the production process signifcantly impact the end product's quality and acceptability.Consumers are increasingly interested in meals with the fewest processing steps, the most natural ingredients, and additions, which provide health benefts like antimicrobials and naturally occurring antioxidants.Nanotechnology enables the evolution of numerous ingredients' functionality to satisfy this requirement by reducing the concentration of substances, changing their solubility, and improving or regulating their efcacy [42,72,73].

Conclusion
Te size of the RDNE particles was below 50 nm, indicating good stability.RDNE was observed to be highly efective in controlling S. typhimurium in vitro.Our results demonstrated that RDNE exhibited a more pronounced anticancer efect in HCT 116 compared to HeLa cells.In addition, its application in food models resulted in decreased TBC, coliforms, LAB, S. typhimurium, pH, and TVB-N content.RDNE coating also enhanced the favor and overall acceptability of the samples.Considering the biological properties of RDNE, it can be suggested and utilized in the meat industry.

Table 3 :
Anti-S.typhimurium activity of the RDNE by the disk difusion methodletters a, b, c, and d in the column represent signifcant diferences (p < 0.05).

Figure 6 :
Figure 6: Efect of the RDNE on HCT-116 and HeLa cancer cell lines according to the MTT test.Concentration 0 was the positive control well containing untreated cells, MTT solution, and a solubilizing bufer.Te asterisk denotes statistically signifcant differences between groups for each concentration (p < 0.05).

Table 1 :
Primer sequence used for Salmonella enterotoxin (stn) gene amplifcation and PCR conditions.

Table 2 :
Chemical composition of R. damascena essential oil.

Table 4 :
[58]t of the RDNE (75, 125, and 250 µg/g concentrations) on chemical properties ground meat throughout refrigerated storage (4 ± 1 °C).Journal of Food Quality also found to be more sensitive than HeLa cells.In a study, R. damascena extract's cytotoxic efects on the HeLa cell line were assessed.Tis extract greatly slowed proliferation of HeLa cells with IC 50 values of 2135, 1540, and 305.1 µg/mL after 24, 48, and 72 hours[58].Another study examined the cytotoxicity of REO against the lung cancer cell line A549.
Figure 9: Sensory evaluation scores of ground meat with 75, 125, and 250 µg/g of RDNE concentrations and control sample.Te asterisk denotes statistically signifcant diferences between groups for each characteristic (p < 0.05).10