The spices samples were ground using an electric blender (BTC Cast Iron Domestic SS Cup Hand Grinder). About thirty grams of each sample was folded in filter papers and put in extraction timbers. The oil extraction was done using Soxhlet, at 65°C for four hours. The extraction solvent was hexane. After extraction, the oil was dried 45°C to evaporate hexane. It was then stored for further analysis.

Enterococcus hirae OL616073 was isolated from 14 h fermented idli batter and used for the production of glucan EPS. The EPS extraction was carried out as per the method explained in earlier reports and used to develop the spice oil emulsions in this study (32, 36).

A set of preliminary trial experiments were performed to demonstrate the emulsifying activity and efficacy prior to the main study. Spice oil emulsions were prepared in a ratio of 3:1 (EPS: Spice oil), where 1% glucan EPS solution with distilled water and three spice oils (Xae, Mm, and Fl) were involved separately. In first phase, the suspension was stirred homogenously and vortexed for 30 min at 40 Hz (Top mix FB 15024, Fisher Scientific, United Kingdom) to make the coarse emulsion. And in second phase, coarse emulsion was subjected for 10 min sonication (model: Labman Ultrasonicator). Coarse and ultrasonicated emulsions were further analyzed in this study.

Exopolysaccharide based spice oil emulsions were diluted 40 times with phosphate buffer solution and phosphate buffer was used as the blank. Absorbance was measured using an LW-1600FC UV spectrophotometer (Shanghai Jinghua Technology Equipment Company, Ltd., Shanghai, China) at 600 nm. Turbidity (T) was calculated as in Li et al. (37) method, using following formula:

A = Absorbance of diluted emulsion at 600 nm measured against the blank. V = Dilution (40).

I = Optical path length (1 cm).

Before and after sonication, all emulsions were examined at 10, 20 and 40× magnification lenses under Inverted Routine Microscope (Nikon eclipse TS100 microscope) using cavity microscope slides (38).

Spice oil emulsions were subjected for average particle size distribution measurement by using a nanoparticle size analyzer (Zetasizer, United Kingdom) at 25°C.

Antimicrobial assay was performed against Gram-positive (Listeria monocytogenes MTCC 1143) and Gram-negative (Shewanella putrefaciens MTCC 8104 and Salmonella enterica MTCC 950) pathogenic strains. Using aseptic procedures, overnight incubated broth cultures of Listeria monocytogenes, Shewanella putrefaciens and Salmonella enterica, were spread to form microbial growth lawns on Mueller Hinton agar plates (Himedia, India). The sterile disks were evenly spaced on the surface of the inoculated agar plates and impregnated drop by drop with 20 μl of emulsions. Standard antibiotic disks of Cephoxitin (CX30) were considered as positive control for the assay. Disks were allowed to dry for 15 min to initiate uniform diffusion of emulsion into the agar and then inverted and incubated for 24 h at 37°C. The next day zone of inhibition were recorded in mm around the disks in each plate (16). Appropriate spice oil controls for all three emulsions against the reference pathogenic strains were considered in well diffusion assay performed as per the aforementioned protocol.

All the experiments were carried out in triplicates and the results were represented as mean ± SD.

Graphs were plotted using Microsoft Office 2016, GraphPad Prism 8.0.2, and OriginPro 8 software.

Spice oil emulsions were prepared with glucan EPS in two phases. There was a clear difference in the clarity, turbidity and homogeneity of the emulsions before and after sonication (Figure 1). Emulsion turbidity is directly proportional to the emulsion stability and inversely proportional to the size of emulsion droplets (39).

The turbidity of the coarse and sonicated EPS-based spice oil emulsions is recorded in Table 1. It provides insights into the turbidity and stability of various spice oil emulsions across two phases, before and after sonication. Before sonication Xae, Fl, and Mm emulsions showed a turbidity of 902.384 ± 16.2, 2521.457 ± 28.1, and 2463.446 ± 24.8 which increased after the sonication treatment to 1043.573 ± 12.8, 2695.795 ± 24.3, and 3494.742 ± 32.4 respectively. A one-way Analysis of Variance (ANOVA) followed by Tukey’s Significant Difference (HSD) post-hoc test was performed to compare turbidity values among the three emulsions (Xae, Mm, and Fl) within each phase. Superscript letters (a–d) were added to the data table to indicate statistically distinct groups. This moderate increase suggests that the Xae emulsion becomes relatively more stable after sonication, likely due to the breakdown of the emulsion droplets leading to the smaller emulsion droplet size (40).

The morphology of spice oil emulsion droplets was observed before and after the ultrasonication process under an Inverted Routine microscope. Figure 2 reveals that emulsions subjected to ultrasonication exhibit significantly smaller droplet sizes than their pre-sonication counterparts. This reduction in droplet size is evident across all the spice oil emulsions (Xae, Mm, and Fl), indicating the effectiveness of ultrasonication assisted break down of larger droplets into finer and uniform structures. The reduced droplet size and improved homogeneity post-sonication, as seen under magnification lenses are consistent with previous studies which have demonstrated its effectiveness in reducing droplet size increasing the emulsion stability by enhanced dispersion of the oil phase within the aqueous phase (41, 42). The phenomenon can be explained by the cavitation effect, where ultrasonic waves create microscopic vapor cavities in the liquid medium which collapse eventually generating an intense localized energy that disrupts larger droplets into smaller ones (43). The mechanical shear forces generated by ultrasonic waves are known to form uniform structures, thereby enhancing the stability of the emulsion (23).

Colloidal stability, turbidity and other properties of food systems are highly influenced by the size distribution emulsion droplets. Figure 3 highlights the particle size distribution of spice oil nanoemulsions (Xae, Fl, and Mm) before and after sonication, underscoring the transformative impact of sonication on emulsion properties. For the Xae nanoemulsion, the particle size reduced from 874.4 nm before sonication to 608.2 nm after sonication, showcasing a significant improvement in size uniformity. Similarly, the Fl nanoemulsion reduced from 414.8 to 283.6 nm, while the Mm nanoemulsion decreased from 285.2 to 233.9 nm. PDI (Polysidpersivity index) was also revealed in this study. Before sonication, Xae, Fl, and Mm emulsion had PDI of 1, 0.393, and 0.391 which was changed after sonication to 0.669, 0.382, and 0.412 respectively. This consistent reduction in particle size across all emulsions suggest disruption of larger oil droplets into smaller and more stable particles after sonication. The observed trends align with the known effects of sonication, which involves high-energy ultrasonic waves that generate intense shear forces, breaking down oil droplets into nanoscale dimensions. The Mm nanoemulsion showed the smallest particle size both before and after sonication, indicating its inherent stability, potentially due to its composition or initial processing conditions. Sonication also likely contributes to better stabilization by improving the interaction between the oil phase and emulsifying agents, reducing the potential for droplet coalescence and phase separation. The enhanced emulsification, particularly in the Mm nanoemulsion, suggests its suitability for applications where fine particle sizes are critical, such as in targeted drug delivery systems or high-performance food formulations. Smaller particle sizes lead to better surface area-to-volume ratios, which enhance the emulsifying capacity and stability of nanoemulsions, as previously observed in studies (44). These results highlight the effectiveness of sonication in optimizing nanoemulsion characteristics and its potential applications in food, pharmaceutical, and cosmetic based industries which require stable and uniform emulsions (14, 19).

The antibacterial activity of spice oil alone (Figure 4A) and spice oil emulsions (Figure 4B) against Salmonella enterica, Listeria monocytogenes, and Shewanella putrefaciens reveals a significant enhancement when emulsified with EPS. The primary observations from both figures highlight the increased inhibition zone diameters with emulsified spice oils compared to their free oil counterparts, signifying the improved antibacterial efficacy. Fl emulsion showed an average inhibition of 9 mm in Shewanella putrefaciens, 14 mm inhibition for Salmonella enterica and 1 mm inhibition for Listeria monocytogenes. Emulsion of Mm showed inhibition for 17 mm in Shewanella putrefaciens whereas resulted in an enhanced inhibition of 20 mm in Listeria monocytogenes. It did not show any inhibition for Salmonella enterica which may be due to the less antimicrobial activity or low level of diffusion in the agar matrix. Xae emulsion showed clear zone of inhibition of diameter 14 mm against gram negative Shewanella putrefaciens, 15 mm for Salmonella enterica, and maximum inhibition of 18 mm for Listeria monocytogenes. The spice oil emulsions demonstrated significant antibacterial activity that is in agreement with earlier findings (45). The enhanced activity of emulsified spice oils likely results from converging mechanisms like increased surface area diffusion, encapsulation and sustained release, synergistic interface interaction and biofilm interferences. In a previous study, similar oil emulsions and nanoemulsions containing phenolic compounds like thymol and eugenol have shown antimicrobial effects on food pathogens like Listeria monocytogenes, Escherichia coli, and Bacillus subtilis by inducing cell lysis and stimulating bacterial envelope damage (46).

The inhibition zones of spice oils alone (Figure 4A) ranged between 2 and 8 mm, indicating moderate antibacterial activity. In contrast, EPS-based emulsions (Figure 4B) significantly enhanced antibacterial action, with inhibition zones ranging from 9 to 20 mm. For Listeria monocytogenes, the individual spice oils showed relatively weak inhibition (≤ 8 mm), while the emulsions of Xae and Mm spice oils exhibited much higher inhibition zones (18 and 20 mm, respectively). This suggests that emulsification improved the bioavailability and antimicrobial effectiveness against this strain. The spice oil alone showed very weak activity against Shewanella putrefaciens, with inhibition zones not exceeding 4 mm. However, the emulsions resulted in significantly larger inhibition zones, especially with Mm (17 mm) and Xae (14 mm) emulsions, demonstrating that emulsification enhances antimicrobial action even against more resistant strains. The STD disk for Salmonella enterica showed an inhibition zone of 13 mm, while Fl and Xae emulsions exceeded this (14 and 15 mm, respectively). This suggests that EPS-based emulsions can potentially match or even outperform conventional antibacterial agents. Essential oils and spice oils are hydrophobic in nature, limiting their solubility and diffusion in aqueous environments. EPS-based emulsions improve the dispersion of spice oils, leading to better contact with bacterial cells and increased antimicrobial efficacy (47, 48). Emulsification helps in the controlled release of antimicrobial compounds, leading to prolonged activity and reducing the need for high concentrations of spice oil, which can sometimes affect sensory properties in food applications (49, 50).