The essential oil nanoemulsions derived from Eugenia brejoensis were investigated for their chemical composition and antibacterial efficacy against Pseudomonas fluorescens -

The essential oil nanoemulsions derived from Eugenia brejoensis were investigated for their chemical composition and antibacterial efficacy against Pseudomonas fluorescens

Essential oils (EOs) are increasingly recognized for their potential as natural antimicrobial compounds, making them appealing for use as food preservatives without synthetic additives. EOs, including carvacrol, cinnamaldehyde, citral, decanal, eugenol, and thymol, exhibit antimicrobial properties by disrupting microbial cell membranes. Eugenia brejoensis, also known as “cutia," is an emerging plant species with bioactive potential found in Brazilian tropical regions. However, incorporating EOs into food faces challenges like low solubility in water and potential toxicity.

To address these challenges, nanoemulsions, consisting of droplets with diameters between 20 and 500 nm, offer a promising solution. Nanoemulsions enhance the transport of active molecules and exhibit improved functionality due to their small droplet size and increased surface-to-volume ratio. However, antimicrobial activity in food may differ from in vitro observations due to interactions with interfering compounds and factors like electrostatic repulsion.

Processed meat products, such as ham, are prone to microbial growth, particularly by Pseudomonas fluorescens, leading to quality issues and reduced shelf life. This study aims to extract and characterize E. brejoensis EO, produce nanoemulsions using various methods, assess their in vitro antimicrobial effectiveness against P. fluorescens, and apply them to ham slices to investigate microbiological stability.

Material and Method:

Plant Material and Essential Oil Extraction: Leaves of Eugenia brejoensis Mazine were collected in September 2015 from mature trees at Parque Nacional do Catimbau, Pernambuco, Brazil. The leaves were dried, chopped, and subjected to hydrodistillation for 4 hours to obtain the essential oil, which was then stored at -4 °C for further analysis.

Preparation of Bacterial Inoculums: Pseudomonas fluorescens (ATCC 13525) was reactivated and cultured in brain heart infusion broth. The growth of the strain was monitored by measuring optical density at 500 nm.

Essential Oil Characterization: Gas chromatography coupled to mass spectrometry (GC/MS) was employed to analyze volatile compounds in the essential oil. The oil was injected into the GC at specific conditions, and the retention index was calculated based on compound retention times.

Synthesis of Nanoemulsions: Oil-in-water emulsions were prepared using aqueous solutions containing 2% (w/w) of E. brejoensis EO and 1% (w/w) of Tween 80. Homogenization was performed at different speeds using an Ultra Turrax T10.

Droplet Size and Size Distribution: Dynamic light scattering on a Zetasizer Nano ZS was used to determine the size distribution and average diameter of the emulsion droplets.

Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR): The functional groups in E. brejoensis EO and its nanoemulsions were identified using ATR-FTIR.

In vitro Antimicrobial Activity: The antimicrobial activity of nanoemulsions was assessed using agar diffusion methodology. P. fluorescens was cultured on Tryptic Soy Agar, and inhibition zones around nanoemulsion cavities were measured.

Inhibitory Effect on Ham Slices: Ham slices were treated with nanoemulsions, and their inhibitory effect on P. fluorescens was evaluated by daily assessment of colony-forming units (CFU) during storage at 10 °C for 7 days. Control samples were also included for comparison.

Summary: In summary, this study aimed to generate nanoemulsions incorporating 1% (v/v) of Eugenia brejoensis Mazine essential oil (EO) at varying rotation speeds (8000 and 12,000 rpm) and assess the antimicrobial efficacy against Pseudomonas fluorescens (ATCC 13525). The investigation considered droplet size, distribution, surface charge, and functional groups. Higher rotation speeds led to a reduction in droplet size, from 355.63 ± 21.70 to 143.13 ± 1.1 nm, with prevention of aggregation and sedimentation, especially at 8000 rpm, aided by a food-grade emulsifier.

Stable nanoemulsions with appropriate  potential values (around -30 mV) were obtained. Both nanoemulsions exhibited in vitro inhibitory effects against P. fluorescens, although the formulation with larger droplets (approximately 355 nm) demonstrated more substantial inhibition zones. However, when applied to sliced ham preservation, the inhibitory effects diminished, likely due to insufficient EO content in the nanoemulsions and the presence of lipids and proteins in ham. While these nanoemulsions demonstrated promising in vitro antimicrobial efficacy, further studies are essential to optimize synthesis conditions for potential application in real food products.

Reference:

MENDES, J.F., MARTINS, H.H.A., OTONI, C.G., SANTANA, N.A., SILVA, R.C.S., DA SILVA, A.G., SILVA, M.V., CORREIA, M.T.S., MACHADO, G., PINHEIRO, A.C.M. AND PICCOLI, R.H., 2018. Chemical composition and antibacterial activity of Eugenia brejoensis essential oil nanoemulsions against Pseudomonas fluorescens. LWT, 93, pp.659-664.