Evaluation of Antioxidant and Antimicrobial Properties of Nanoemulsion of Citrus paradisi Essential Oil Against Pathogenic Microorganisms: In Vitro Study

Oxidation reactions and microorganisms’ activity are considered as the most important factors affecting the quality of food products. Recently, in the light of the inefficiency of some chemical preservatives against microorganisms and the presence of toxic residues in food products, the use of natural antimicrobials and antioxidants has been increased. Natural antimicrobial compounds have the potential to control microbial contamination and reduce the use of antibiotics. Plant essential oils are natural compounds with the potential to be used as active ingredients in the food, pharmaceutical, and cosmetic industries. Various studies have shown that essential oils have antifungal, antibacterial, antiviral, and antioxidant activity. The essential oils are considered as superb preservatives with various biological functions. Essential oils are generally recognized as safe product (GRAS) which can be used as an alternative to synthetic additives.Grapefruit (Citrus paradisi) peel and fruit contain active ingredients such as acids, flavonoids, vitamin C, and potassium, and its essential oil is composed of terpenic hydrocarbons, such as citral, limonene, citronelal, and geraniol. Although plant essential oils have antimicrobial and antioxidant properties, one of the main problems of these natural compounds is their high volatility and instability. In this context, nanoemulsion formulations are frequently used to increase the stability and efficiency of these biologically active compounds. This study is therefore aimed to nanoemulsifying the grapefruit essential oil and evaluate its antioxidant and antimicrobial properties.

β-carotene, linoleic acid, ABTS (2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), and DPPH (2,2-diphenly-1-picrylhydrazyl) were purchased from Sigma-Aldrich Co. (USA). Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) were supplied from Merck Co. (Darmstadt, Germany). Grapefruit peel was dried at ambient temperature and then powdered. The obtained powder was then transferred to a Clevenger device containing 750 ml of distilled water to perform the distillation extraction (3 h). The resulting grapefruit essential oil was stored at 4 °C until use. Grapefruit essential oil was prepared using the hydrodistillation method, and then nanoemulsified. The antioxidant activity of the nanoemulsified essential oil was investigated by DPPH and ABTS  radical scavenging activity and beta-carotene/linoleic bleaching test. The nanoemulsified essential oil or methanolic (control) was mixed with DPPH solution and the mixture was then stored at ambient temperature for 30 min, in a dark place. The control sample was prepared by methanol. The absorbance of the samples was measured at 517 nm. To determine the ABTS-RS activity, the nanoemulsified essential oil was briefly charged with methanolic ABTS radical cation solution and the resulting mixture was left at room temperature for 30 min. Afterward, the absorbance was read at 734 nm. A spectrophotometric method was applied to monitor β-carotene/linoleate solution bleaching in the presence of the nanoemulsified essential oil. To do this, the absorbance of the solution was recorded at 490 nm after 120 min against the control sample at time zero and after 120 min. Antibacterial effect of the grapefruit essential oil nanoemulsion was also evaluated against Escherichia coli ATCC 25922, Salmonella typhi ATCC 6539, Pseudomonas aeruginosa ATCC 27853, Listeria innocua ATCC 33090, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579, Bacillus subtilis ATCC 23857, Streptococcus pyogenes ATCC 19615, and Staphylococcus epidermidis ATCC 12228, based on disk diffusion agar, well diffusion agar, minimum inhibitory concentration, and minimum bactericidal concentration.

The results showed that the nanoemulsion of grapefruit essential oil had a remarkable antioxidant effect of 42.27 mg/ml, 33.27 mg/ml and 54.54%, respectively, based on DPPH, ABTS, and beta-carotene-linoleic acid bleaching tests. According to disk diffusion agar and well diffusion agar results, the lowest inhibition zone was related to E. coli and the highest inhibition zone was observed in L. innocua. The minimum inhibitory concentration for L. innocua and S. aureus (the most sensitive bacteria) was 25 mg/ml, and E. coli, S. typhi, and P. aeruginosa had the highest inhibitory concentration. Also, the lowest bactericidal concentration was related to L. innocua and S. aureus bacteria and the highest concentration was observed for E. coli, S. typhi and P. aeruginosa. The nanoemulsified essential oil generally exhibited greater antibacterial activity against Gram-positive species. This could be mainly due to the difference in the cell wall composition of Gram-positive bacteria in comparison to Gram-negative; Gram-positive bacteria have a thicker mucopeptide layer in their cell wall, while Gram-negative bacteria have only a thin layer of mucopeptide and the wall structure is mainly composed of lipoprotein and lipopolysaccharide, thereby leading to a higher resistant to antibacterial agents According to the results, grapefruit essential oil nanoemulsion can be used as a natural antioxidant and antimicrobial agent to control oxidation reactions and the growth of spoilage and pathogenic microorganisms.