The sesame seed coats (SSC) were obtained from a Lebanese industry that processes sesame paste (tahini). It should be noted that the sesame seeds are imported from Gadarif State-Sudan. Specifically, the SSC used in our study were obtained by mixing and processing together the coats from three different lots. The following chemicals and reagents were obtained from Sigma Aldrich (Steinheim, Germany): methanol (99.8%), Folin–Ciocalteu reagent, sodium carbonate (Na₂CO₃), gallic acid (3,4,5-trihydroxybenzoic acid), DPPH (2,2-diphenyl-picrylhydrazyl), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), and dimethylsulfoxide (DMSO; HPLC grade), chitosan (low molecular weight), glacial acetic acid, tween80 along with all HPLC standards (gallic acid, catechin, caffeic acid, p-coumaric acid, and quercetin). Polyethylene glycol was purchased from MERCK-Schuchardt.

Brain Heart Infusion (BHI), Tryptone Soya Broth (TSB), Mueller-Hinton Broth (MHB), Tryptone Soya Agar (TSA), Listeria Selective Agar Base, Sorbitol MacConkey Agar, Modified Listeria Selective Supplement, Cefixime Tellurite Selective Supplement, and peptone water were purchased from Oxoid (Basingstoke, UK). All media were prepared according to the manufacturer's instructions, except peptone water, which was prepared by dissolving 1 g in 1 liter of distilled water to obtain a 0.1% solution.

WB extraction was carried out using a digital water bath (JSR JSWB-22T, Gongju City, Korea). Before extraction, the SSCs were sieved to obtain a particle size of < 500 μm and stored in the dark at room temperature. Ten g of SSCs were placed in 100 ml of 70% methanol solution (ratio 1/10), covered with aluminum foil, at 74°C for 17.5 min in the water bath, based on a previous study (25). The extracts were filtered through Whatman No. 1 filter paper and centrifuged for 10 min at 4,500 g/min. The supernatant was filtered through a 0.22 μm polyvinylidene fluoride (PVDF) syringe filter and stored at −80°C.

SSC extracts were concentrated using a rotary evaporator and then lyophilized (Edwards freeze-dryer, UK) for 48 h. Freeze-dried samples were converted into powder with the help of a pestle and mortar (26). For all subsequent analyses, SSC was utilized in its lyophilized form. In order to calculate the yield of the lyophilized extract, the weight of the extract after lyophilization was divided by the initial weight of the plant material.

TPC was determined according to the Folin–Ciocalteu method described previously in the literature (27). A volume of 200 μl of the SSC extract (100, 50, 25, and 12.5 mg/ml) and 1,000 μl of the Folin-Ciocalteu reagent (diluted 1/10 v/v) were added to 800 μl of Na₂CO₃ 7.5% (w/v). The mixture was then incubated for 10 min at 60°C, followed by an additional 10 min at 4°C. The absorbance was measured at 750 nm using a UV-vis spectrophotometer (GENESYS 10 UV, Thermo Electron Corporation, Waltham, MA, USA). A calibration curve using gallic acid as the standard compound was employed for quantification. The TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry matter (mg GAE/g of DM).

The free radical scavenging activity was evaluated by measuring the ability of phenolic compounds to reduce the DPPH radical (28). Briefly, 1.45 ml of DPPH (0.06 mM) was added to 50 μl of SSC extract (100, 50, 25, and 12.5 mg/ml) or Trolox (positive control) or methanol (negative control). After 30 min of incubation at room temperature in the dark, the absorbance was measured at 515 nm. Pure methanol was used as the blank, and a calibration curve was made using Trolox as the standard. The antioxidant activity of the extracts was calculated according to the following formula:

Phenolic compounds in the extracts obtained from SSC were analyzed using HPLC-DAD. The HPLC system used for this analysis was an Agilent 1,100 series system equipped with an autosampler, a Zorbax column oven, and a diode array detector. To separate phenolic compounds, a C18 column (250 × 4.6 mm; 5 μm) was used. The following standards were used for identification and quantification: gallic acid, catechin, caffeic acid, p-coumaric acid, and quercetin. The mobile phase consisted of acetonitrile (A) and water/phosphoric acid pH 2 (B) of HPLC grade. The flow rate was set at 1 ml/min and the gradient program was as follows: 95% B to 65% B in 20 min, 65% B to 60% B in 10 min, 60% B to 50% B in 10 min, 50% B to 30% B in 12 min, and 30% B to 95% B in 8 min. The injection volume was 50 μl. The phenolic compounds were identified by comparing the retention times of the observed peaks with those of the standard compounds. To determine the concentration of these phenolic compounds, standard curves for each specific compound were established by employing various concentrations of the corresponding standards (Supplementary Figure 1).

The chemical profile of the SSC was assessed following the methodology described by Qadir et al. (29). A gas chromatograph (GC) (SHIMADZU QP2010, Japan) coupled with mass spectrometry (MS) featuring a fused-silica capillary column was employed, with a mobile phase carrier gas (helium) set at a flow rate of 1.69 ml/min. The oven temperature was set at 50°C for 1 min followed by an increase of 3°C/min to 280°C. The total running time was 27 min.

Bacterial cultures used in this study included Listeria monocytogenes (ATCC 7644), Salmonella Typhimurium (02:8423 strain), and Escherichia coli O157:H7 (02:0627 strain). These cultures were individually preserved in BHI broth containing 20% glycerol (v/v) at −20°C. For each working culture, the stock bacteria were streaked on TSA and then incubated at 37°C for 24 h to obtain a single colony. Subsequently, subculturing was performed on TSB, except for L. monocytogenes, where the TSB was supplemented with 0.6% yeast and incubated at 37°C for 24 h. The final concentration of bacterial cells was adjusted to 10⁶ CFU/ml.

The antibacterial activity against each of the three bacterial strains was determined using the microdilution method. The highest concentration of lyophilized extract was obtained by dissolving 100 mg in 1 ml of 0.5% DMSO and then serially diluted twice to obtain concentrations of 50 mg/ml and 25 mg/ml. In a 96-well microtiter plate, 100 μl of each bacterial culture was mixed with 100 μl of extract. A 0.5% DMSO solution mixed with each bacterial strain served as a positive control, while DMSO containing the extracts was used as a negative control. The sealed microplate was then incubated for 24 h at 37°C. One hundred μl aliquots of each sample were taken and decimal dilutions with 0.1% peptone water were prepared. To determine the minimal inhibitory concentration (MIC) of each extract, the lowest concentration at which no visible growth is observed compared to the negative and positive controls should be identified. However, because of the dark color of the extract and the difficulty of detecting growth, the minimal bactericidal concentration (MBC), which is the lowest concentration of extract that kills the entire bacterial population, was also determined.

For this, 100 μl from each of the appropriate dilutions was spread on the surface of TSA and incubated for 24 h at 37°C (30).

An aqueous solution was first prepared by dissolving 0.5 g of chitosan in 1,000 ml of 2% glacial acetic acid by stirring at 750 rpm for 30 min. Then, this solution was centrifuged at 4,500 g for 10 min (31). To 25 ml of the upper solution, 0.125 g of polyethylene glycol was added and stirred for 20 min. To prepare the nanoemulsion, the SSC extract was first mixed with 0.1% of Tween 80. Then, this phase was gradually added dropwise to the chitosan solution at a rate of 0.1 ml/min while on a magnetic mixer. The solution was stirred for 30 min to ensure the formation of the SSC-chitosan nanoemulsion (NE-SSC). The control solution (NE) was obtained by the same procedure but without the SSC for comparison.

The experiments were carried out three times (n = 3), and the mean value ± standard error of mean (SEM) value was used to present the results. Both control and experimental groups were analyzed and p-values < 0.05 were considered statistically significant, indicating a confidence level >95%. Statistical analysis of the data involved independent t-test (unpaired 1-tailed) and Analysis of Variance (ANOVA) followed by Duncan Multiple Range Test (DMRT) using IBM SPSS Statistics for Windows, Version 25.0 (Released 2017. IBM Corp., New York, USA).

The extraction yield in this study was 10.62 ± 0.42 %, which is consistent with the study by Elleuch et al. (13) on extracting sesame seed coat using 70% methanol, which yielded almost 10.5%. The extraction yields in this study were greater than those reported by Chang et al. (34) (8.19%) for 100% methanol extraction. This suggests that increasing the water content of the solvent may lead to an increase in the yield (34).

The total phenolic content and antioxidant activity at different concentrations of the SSC extract are presented in Figure 1. At a concentration of 12.5 mg/ml, the total phenolic content was measured at 3.95 mg GAE/g DM (Figure 1A). This value increased significantly to 7.24 mg GAE/g DM at 25 mg/ml. An additional increase was observed at higher concentrations, with values reaching 14.22 mg GAE/g DM at 50 mg/ml and 22.91 mg GAE/g DM at 100 mg/ml. As is evident, the increasing trend in the total phenolic content suggests a positive correlation between the concentrations of the SSC lyophilized extract and its phenolic richness. In another study, the polyphenol content in the sesame coat was approximately 9.45 mg GAE/g DM when extracted with 70% methanol (13). Besides, a study by El-Roby et al. (11) demonstrated a polyphenol yield of 7.233 mg GAE/g DM from sesame coat when extracted with 80% ethanol. These results are in accordance with the values obtained in this study.

The free radical scavenging activity of the SSC extract was assessed by measuring its antioxidant activity using the DPPH assay. It was assessed in terms of percentage of inhibition (Figure 1B). At a concentration of 12.5 mg/ml of SSC extract, the antioxidant activity was 10.38%. This activity increased significantly to 24.36% at 25 mg/ml. Subsequent elevations were observed at higher concentrations, with antioxidant activities reaching 51.97% at 50 mg/ml and 83.53% at 100 mg/ml. The increasing trend in antioxidant activity suggests a positive correlation between the concentration of the lyophilized extract of SSC and its ability to reduce the DPPH radical. In a comparable study, the inhibition percentage was recorded at 81.29% at a concentration of 180.8 μg/ml for the SSC extract (11). In addition, Elleuch et al. (13) tested the scavenging effect of aqueous 70% ethanol and 70% methanol SSC extracts at a dose of 0.5 mg and it was approximately 94.4% (13). While the antioxidant activity in these studies is closely aligned with our findings, it is important to note that the concentrations of extracts employed in those studies were different. This variation can be attributed to differences in the origin of SSC and the extraction methods used.

Further increasing the concentration of the SSC extract will result in reaching a point of maximum TPC and antioxidant activity, leading to stabilization. These results suggested using the highest concentration of SSC (100 mg/ml) for subsequent methods in this study to achieve optimal outcomes.

The method was used to detect and quantify phenolic compounds in the SSC extract. The analysis revealed the presence and quantification of five different types of polyphenols (Table 1 and Supplementary Figure 2). The main compounds were catechin, gallic acid, quercetin, p-coumaric acid, and caffeic acid. Catechins, members of the flavanol family, have numerous health benefits, such as anticancer, antiobesity, antidiabetic, anticardiovascular, antiinfectious, neuroprotective, and hepatoprotective effects (35). It was the most abundant (2.41 ± 0.45 ppm) and significantly higher than all the other compounds found in the SSC extract (p < 0.05). Many studies have proven that the key factors behind the strong effects, such as fighting radicals and bacteria, in sesame seed coat extracts are flavonoids (36, 37). Gallic acid, which belongs to the hydroxybenzoic acid family, has almost the same health benefits as catechins. It is classified as the second most abundant compound in the SSC extract at 1.41 ± 0.07 ppm. However, its concentration was not significantly higher than that of quercetin and p-coumaric acid (p < 0.05). Quercetin is a highly abundant polyphenolic bioflavonoid, specifically classified as a flavonol. It is known for its various beneficial biological activities, including antioxidant, anti-inflammatory, anticancer, and antiviral properties (38). It was detected at a concentration of 1.03 ± 0.15 ppm in the SSC, with no significant difference among gallic acid, p-Coumaric acid, and caffeic acid (p > 0.05). p-Coumaric and caffeic acids, classified as hydroxycinnamic acids, have preventive effects against chronic diseases and cancer (39). p-Coumaric acid and caffeic acid were detected in the SSC extract (0.99 ± 0.06 ppm and 0.37 ± 0.00 ppm; respectively), with no significant differences between them (p > 0.05). El-Roby et al. (11) found similar results, analyzing polyphenols in SSC; however, they were detected at different concentrations (11). This could be due to differences in the sources of the sesame coat. According to the study's findings, the presence of polyphenols in the SSC extract was confirmed by quantifying various phenolic compounds. This discovery suggests that the antibacterial activity of the SSC extract may be positively influenced.

GC-MS analysis of the SSC extract in the current study showed 7 peaks, including siloxanes, acrylate monomers, fatty acids, and alkanes (Supplementary Figure 1). Table 2 lists the retention times, compound names, and peak areas (%) of the detected compounds. The chemical components identified in the SSC extract include 1.68% dodecamethylcyclohexasiloxane, 1.43% tridecyl acrylate, 5.70% palmitic acid, 64.73% oleic acid, 13.31% stearic acid, 5.52% 2-methyltetracosane, and 7.62% 2-linoleoylglycerol. GC-MS analysis revealed that the SSC extract is predominantly composed of saturated and unsaturated fatty acids, accounting for a combined 83.74%. Oleic acid stands out as the major fatty acid, comprising the highest percentage at 64.73%. This is in accordance with a study conducted by Elleuch et al. (10) on sesame by-products, where the most abundant fatty acid was oleic acid (43%).

The primary objective of conducting GC-MS on the SSC extract was to detect the presence of fatty acids, which provided the oily texture necessary for the formulation of the nanoemulsion. By confirming the presence of fatty acids through GC-MS analysis, it was possible to support the rationale behind the formulation of SSC extract within the nanoemulsion. Additionally, the detected fatty acids may play a role in the antibacterial effect. Fatty acids demonstrate detergent properties that elucidate their adverse impact on bacterial cells. This characteristic allows them to engage with the cell membrane, creating temporary or permanent pores of various sizes. At high concentrations, these interactions can cause the lipid bilayer to release components such as membrane proteins or larger segments (40, 41). According to a study conducted by Dilika et al. (42), oleic acid (C18:1), obtained from Helichrysum pedunculatum leaves, has shown antibacterial properties against Gram-positive bacterial strains.

Our current study is the first to assess the antibacterial potential of SSC, using the microdilution method. The effect of the SSC extract on the viability of L. monocytogenes, E. coli, and S. Typhimurium at increasing concentration is presented in Figure 2. A clear trend emerges as the concentration of SSC extract increases, resulting in a corresponding increase in antibacterial capacity. The highest antibacterial effect was observed for L. monocytogenes treated with SSC extract at 100 mg/ml, where no viable bacteria were detected. At 100 mg/ml of SSC, there was no significant difference between E. coli and S. Typhimurium (p < 0.05). However, both Gram-negative bacteria had a significant effect with a log reduction of 1.5 (p < 0.05). At 50 mg/ml of SSC extract, the antibacterial effectiveness of L. monocytogenes (3.2 log reduction) was still significantly higher than E. coli and S. Typhimurium (p < 0.05). However, at a concentration of 25 mg/ml, there was no significant difference between its effect on L. monocytogenes (8.65 ± 0.05) and the control (8.75 ± 0.07). Therefore, the MBC of L. monocytogenes was below 100 mg/ml, while for E. coli and S. Typhimurium, the MBC exceeded 100 mg/ml. Most plant extracts show a more potent antibacterial effect against Gram-positive bacteria compared to Gram-negative bacteria (43). This discrepancy may be attributed to the presence of a lipopolysaccharide outer membrane in Gram-negative bacteria, along with porins that regulate the diffusion of substances into the cytosol, contributing to their resistance to antibacterial compounds (44). On the other hand, the Gram-positive bacteria lack an outer membrane.

Bacterial imaging was carried out using atomic force microscopy (AFM), a potent tool for studying microbial surfaces and structural alterations on the nanometer scale (47). In this study, the non-contact mode was employed, where the cantilever maintained a distance of tens to hundreds of angstroms (Å) away from the sample, only vibrating in proximity to it (48). This approach protects both the sample and the tip from potential damage, making it advantageous over contact mode (49). The main purpose of incorporating AFM in this study was to analyze the morphological changes that occur in bacterial cells. The AFM images of control and treated bacterial cells are shown in Figure 5. The control images revealed smooth bacterial surfaces with typical rod-shaped morphology for L. monocytogenes and Gram-negative bacteria (i.e., E. coli O157:H7 and S. Typhimurium). However, when treated with SSC (100 mg/ml), the bacteria showed surface protrusions, stiffness, and swelling, while S. Typhimurium only showed some roughness on the surface. Upon treatment with NE-SSC, the bacteria had distorted shapes and indentations. This observation may imply a synergistic effect for the antibacterial activity of SSC when it is encapsulated within a nanoemulsion.

Considering that food matrices consist of diverse food ingredients, the addition of an antibacterial can lead to various molecular interactions. These interactions may influence antibacterial behavior within the food matrix differently than in microbiological media (50, 51). In this study, sterilized milk stored at 4 and 10°C for 7 days was employed as a food model system (in situ) to assess the effectiveness of SSC at a concentration of 100 mg/ml. Table 4 illustrates the impact of SSC against L. monocytogenes and E. coli O157:H7 on days 0, 1, 3, and 7 in milk subjected to incubation at both 4 and 10°C, comparing control samples with those enriched with SSC extract.

The results revealed a significant inhibitory effect of SSC extract when added to milk against L. monocytogenes at 4 and 10°C (p < 0.05). At incubation temperatures of 4 and 10°C, reductions of 4.2 and 4.5 log CFU/ml, respectively, were observed for L. monocytogenes at the end of the storage period (Table 4 and Supplementary Table 1). Additionally, SSC exhibited a significant inhibitory effect against E. coli O157:H7 at 4°C, resulting in a log CFU/ml 3.3 after 7 days (p < 0.05). However, at 10°C, a relatively lower reduction of approximately 0.9 log CFU/ml was observed for E. coli O157:H7 (Table 4).

Clearly, the inhibitory effects of the SSC extract were more pronounced against L. monocytogenes when subjected to the optimal growth temperature of 37°C, except for E. coli O157:H7 at 4°C. This phenomenon can be attributed to the increased metabolic activity exhibited by bacterial cells at their optimal growth temperatures. Specifically, at 37°C, the presence of SSC extract at a concentration of 100 mg/ml effectively inhibited the growth of L. monocytogenes (Figure 2). On the contrary, at lower temperatures of 4 and 10°C, the impact of the SSC extract was limited to a reduction in bacterial cell count. This increased efficacy can be associated with the increased antibacterial activity observed at elevated temperatures (52, 53), which is likely related to the increased active state of bacterial cells, resulting in elevated growth and death rates at elevated temperatures (54). In contrast, lower temperatures tend to slow the growth rate of bacterial cells, making them less susceptible to the action of the antimicrobial agent (55). Furthermore, the inhibition effect of SSC against L. monocytogenes in milk was lower compared to that in broth. This discrepancy is attributed to the ability of milk to supply ample nutrients, which promotes bacterial growth (56). Typically, in antimicrobial challenge assessments, achieving a 2-log reduction in the target microbial strain holds significant practical implications. However, variables such as the initial inoculum level can affect the ultimate reduction outcome (57).

Table 5 shows a comparison of the effect of different concentrations of NE and NE-SSC on L. monocytogenes and E. coli O157:H7 in milk stored at 4 and 10°C. The antibacterial impact of 3% NE on L. monocytogenes and E. coli O157:H7 was found to be statistically insignificant compared to 0% NE on day 7 at both 4 and 10°C (p > 0.05), except for a minimal reduction of 0.33 log CFU/ml observed for E. coli O157:H7 at 10°C (Table 5). However, using 7% NE, a significant effect against both bacteria was observed at 4 and 10°C on day 7, ranging between 0.12 and 2.19 log CFU/ml reductions compared to 0% NE (p < 0.05). The antibacterial efficacy of NE also increased at 10% concentration at both incubation temperatures on day 7, with reductions ranging between 0.58 and 3.76 log CFU/ml (p < 0.05).

The antibacterial influence of 3% NE-SSC on both L. monocytogenes and E. coli O157:H7 was statistically insignificant compared to 0% NE-SSC at the end of the storage period at 4 and 10°C (p > 0.05). However, a notable reduction of 0.64 log CFU/ml was observed for E. coli O157:H7 at 10°C (Table 5). In contrast, at 7% NE-SSC, a significant antibacterial effect against both bacteria were evident at 4 and 10°C on day 7, resulting in reductions ranging from 0.43 to 3.67 log CFU/ml compared to 0% NE-SSC (p < 0.05). The antibacterial efficacy of NE-SSC was further increased at a 10% concentration at both incubation temperatures on day 7, yielding reductions ranging between 1.81 and 4.73 log CFU/ml (p < 0.05).

In summary, concentrations of 7% and 10% for both NE and NE-SSC demonstrated significant efficacy at 4 and 10°C in reducing viable counts of L. monocytogenes and E. coli O157:H7 after incubation for 7 days.

Table 6 outlines the differences in the antibacterial effect between NE and NE-SSC against L. monocytogenes and E. coli O157:H7 in milk over 7 days of storage at 4 and 10°C. At 3% of both NE and NE-SSC, there were no significant differences in logarithmic count between them after 7 days at 4 and 10°C (p > 0.05) (Table 6 and Supplementary Table 2). However, at a concentration of 7%, there was a significant difference in effectiveness between NE (7.92, 8.01 log CFU/ml) and NE-SSC (7.61, 5.20 log CFU/ml) against L. monocytogenes and E. coli O157:H7 at 10°C at the end of the incubation period, respectively (p < 0.05). This resulted in a difference of 0.31 and 2.81 log CFU/ml between the effects of 7% NE and NE-SSC on L. monocytogenes and E. coli O157:H7 at 10°C, respectively.

At a concentration of 10%, there was a significant difference between NE and NE-SSC against L. monocytogenes and E. coli O157:H7 at 4 and 10°C after 7 days (p < 0.05). The variation in counts ranged between 0.53 and 1.42 log CFU/ml for the effects of 10% NE and NE-SSC against both bacteria at the end of the study period at both incubation temperatures. In all instances of significant differences at all concentrations, the addition of NE-SSC to milk exhibited lower bacterial counts than that of NE. At a 10% concentration of NE-SSC, the reduction in the number of L. monocytogenes was 2.6 and 1.81 log CFU/ml at 4 and 10°C, respectively. Regarding E. coli O157:H7, the reductions were 3.94 and 4.73 log CFU/ml at 10% NE-SSC at 4 and 10°C, respectively. The SSC extract exhibited a capacity to reduce bacterial counts of L. monocytogenes and E. coli O157:H7, achieving reductions ranging from 0.9 to 4.5 log CFU/ml under identical conditions on day 7. In contrast, NE-SSC at a concentration of 10% demonstrated effectiveness in reducing bacterial counts for both strains at the end of the incubation period, achieving reductions in the range of 1.81 to 4.73 log CFU/ml. This suggests that the inclusion of SSC into the NE enhanced its antibacterial efficacy, demonstrating a synergistic effect and affirming the effectiveness of nanoemulsion formation.