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

Document Type : Research Article

Authors

• Hossein Zanganeh ¹
• Fakhri Shahidi ¹
• Seyed Ali Mortazavi ¹
• Behrooz Alizadeh Behbahani ²

¹ Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad (FUM), Mashhad, Iran

² Department of Food Science and Technology, Faculty of Animal Science and Food Technology, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran

https://doi.org/10.22067/ifstrj.2021.71660.1074

Abstract

Introduction
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.
 
Materials and Methods
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.
 
Results and Discussions
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.

Keywords

• Antimicrobial activity
• Antioxidant effect
• Grapefruit essential oil
• Nanoemulsion

Main Subjects

• Food Biotechnology

©2023 The author(s). This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source.

References

1. Ahmadi, O., & Jafarizadeh Malmiri, H. (2020). Effectiveness of soluble oxygen in preparation of thyme oil nanoemulsion-simulation and characterization. Iranian Chemical Engineering Journal, 19(110), 42-53. (In Persian with English abstracrt)
2. Alizadeh Behbahani, B., Falah, F., Vasiee, A., Tabatabaei Yazdi, F., & Mortazavi, S.A. (2019). Antimicrobial effect of Citrus aurantium essential oil on some foodborne pathogens and determination of its chemical compounds, total phenol content, total flavonoids content and antioxidant potential. Food Science and Technology, 87(16), 291-304.
3. Alves, J.A., Mantovani, A.L.L., Martins, M.H.G., Abrao, F., Lucarini, R., Crotti, A.E.M., & Martins, C.H.G. (2015). Antimycobacterial activity of some commercially available plant-derived essential oils. Chemistry of Natural Compounds, 51(2), 353-355. https://doi.org/10.1007/s10600-015-1281-0
4. Barzegar, H., Mehrnia, M.A., & Alizadeh Behbahani, B. (2019). Determination of the chemical composition, antioxidant activity and the antimicrobial effect of Heracleum lasiopetalum on infection and food poisoning microorganisms. Journal of Applied Microbiology in Food Industry, 4(4), 15-28.
5. Behbahani, B.A., & Fooladi, A.A.I. (2018). Evaluation of phytochemical analysis and antimicrobial activities Allium essential oil against the growth of some microbial pathogens. Microbial Pathogenesis, 114, 299-303. https://doi.org/10.1016/j.micpath.2017.11.055
6. Behbahani, B.A., Noshad, M., & Falah, F. (2019). Study of chemical, structure, antimicrobial, cytotoxic and mechanism of action of Syzygium aromaticum essential oil on foodborn pathogens. Potravinarstvo, 13(1).
7. Behbahani, B.A., Shahidi, F., Yazdi, F.T., Mortazavi, S.A., & Mohebbi, M. (2017). Use of Plantago major seed mucilage as a novel edible coating incorporated with Anethum graveolens essential oil on shelf life extension of beef in refrigerated storage. International Journal of Biological Macromolecules, 94, 515-526. https://doi.org/10.1016/j.ijbiomac.2016.10.055
8. Behbahani, B.A., Yazdi, F.T., Vasiee, A., & Mortazavi, S.A. (2018). Oliveria decumbens essential oil: Chemical compositions and antimicrobial activity against the growth of some clinical and standard strains causing infection. Microbial Pathogenesis, 114, 449-452. https://doi.org/10.1016/j.micpath.2017.12.033
9. Deng, W., Liu, K., Cao, S., Sun, J., Zhong, B., & Chun, J. (2020). Chemical composition, antimicrobial, antioxidant, and antiproliferative properties of grapefruit essential oil prepared by molecular distillation. Molecules, 25(1), 217. https://doi.org/10.3390/molecules25010217
10. Denkova-Kostova, R., Teneva, D., Tomova, T., Goranov, B., Denkova, Z., Shopska, V., ... & Hristova-Ivanova, Y. (2021). Chemical composition, antioxidant and antimicrobial activity of essential oils from tangerine (Citrus reticulata), grapefruit (Citrus paradisi L.), lemon (Citrus lemon L.) and cinnamon (Cinnamomum zeylanicum Blume). Zeitschrift für Naturforschung C, 76(5-6), 175-185.
11. Durmus, M. (2020). The effects of nanoemulsions based on citrus essential oils (orange, mandarin, grapefruit, and lemon) on the shelf life of rainbow trout (Oncorhynchus mykiss) fillets at 4±2° C. Journal of Food Safety, 40(1), e12718. https://doi.org/10.1111/jfs.12718
12. Erfani, A., Pirouzifard, M., Almasi, H., & Gheybi, N. (2019). Microencapsulation of cinnamon essential oil Nano emulsion stabilized with β-cyclodextrin and sodium caseinate. FSCT, 16(91), 107-117.
13. Filoche, S.K., Soma, K., & Sissons, C.H. (2005). Antimicrobial effects of essential oils in combination with chlorhexidine digluconate. Oral Microbiology and Immunology, 20(4), 221-225. https://doi.org/10.1111/j.1399-302X.2005.00216.x
14. Gharenaghadeh, S., Samadlouie, H.R., Sowti, M., & Gharenaghadeh, S. (2017). Nano emulsion formulation from essential oil of Salvia hypoleuca and investigation of its antimicrobial and physicochemical properties. Food Science and Technology, 14(9), 337-348.
15. Heydari, M., & Bagheri, M. (2018). Synthesis, characterization and investigation of properties of nanoemulsion Persian cumin essential oil. Journal of Advanced Materials and Technologies, 7(2), 49-56.
16. Javanshir, A., Karimi, E., & Homayouni Tabrizi, M. (2020). Investigation of antioxidant and antibacterial potential of Ricinus communis nano-emulsions. Jundishapur Scientific Medical Journal, 19(1), 1-9. (In Persian with English abstracrt)
17. Kaur, H., Pancham, P., Kaur, R., Agarwal, S., & Singh, M. (2020). Synthesis and characterization of Citrus limonum essential oil based nanoemulsion and its enhanced antioxidant activity with stability for transdermal application. Journal of Biomaterials and Nanobiotechnology, 11(4), 215-236. https://doi.org/10.4236/jbnb.2020.114014
18. Li, Z. H., Cai, M., Liu, Y. S., & Sun, P.L. (2018). Development of finger citron (Citrus medica var. sarcodactylis) essential oil loaded nanoemulsion and its antimicrobial activity. Food Control, 94, 317-323. https://doi.org/10.1016/j.foodcont.2018.07.009
19. Lou, Z., Chen, J., Yu, F., Wang, H., Kou, X., Ma, C., & Zhu, S. (2017). The antioxidant, antibacterial, antibiofilm activity of essential oil from Citrus medica var. sarcodactylis and its nanoemulsion. LWT, 80, 371-377. https://doi.org/10.1016/j.lwt.2017.02.037
20. Majdi, B., Mehrnia, M. A., Barzegar, H., & Alizadeh Behbahani, B. (2021). Determination of the structure, chemical composition, antioxidant activity and the cytotoxic effect of Turmeric essential oil. Iranian Food Science and Technology Research Journal, 17(2), 261-271. (In Persian with English abstracrt). https://doi.org/10.22067/ifstrj.2020.39272
21. Masoumi, V.O., Tajik, H., Moradi, M., Forough, M., & Shahabi, N. (2016). Antimicrobial effects of Zataria multiflora Essential oil nanoemulsion against Escherchia coli O157:H7. Studies in Medical Sciences, 27(7), 608-617.
22. Mehrnia, M.A., Alizadeh Behbahani, B., Barzegar, H., & Tanavar, H. (2021). Sclerorhachis platyrachis essential oil: Antioxidant power, total phenolic and flavonoid content and its antimicrobial activity on some Gram-positive and Gram-negative bacteria “in vitro”. FSCT, 18(112), 189-198. https://doi.org/10. 52547/fsct.18.03.16
23. Mesgarpour, B., Adhami, H., & Amanlou, M. (2002). Grapefruit juice effects. Journal of Medicinal of Plants, 1(1), 47-62.
24. Misharina, T.A., & Samusenko, A.L. (2008). Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove, and their mixtures. Applied Biochemistry and Microbiology, 44(4), 438-442. https://doi.org/10.1134/S0003683808040182
25. Moghimi, R., Ghaderi, L., Rafati, H., Aliahmadi, A., & McClements, D.J. (2016). Superior antibacterial activity of nanoemulsion of Thymus daenensis essential oil against coli. Food Chemistry, 194, 410-415.
    https://doi.org/10.1016/j.foodchem.2015.07.139
26. Noshad, M., Alizadeh behbahani, B., & Dehghani, S. (2020). Evaluation of the effect of aqueous and ethanolic extraction methods on extraction yield, phenolic compounds, and antioxidant and antimicrobial activity of Stachys schtschegleevii Food Science and Technology, 17(100), 117-125. https://doi.org/10.52547/fsct.17.100.117
27. Noshad, M., Alizadeh Behbahani, B., & Dehghani S. (2020). Improving oxidative and microbial stability of beef by using a bioactive edible coating obtained from Barhang-e-Sagheerseed mucilage and loaded with Avishan-e-Baghi. Food Science and Technology, 17(4), 1-3. ‎https://doi.org/10.52547/fsct.17.101.1
28. Noshad, M., Alizadeh behbahani, B., Jooyandeh, H., Rahmati-Joneidabad, M., Ebrahimi, M., & Ghodsi Sheikhjan, M. (2021). In vitro investigation of the antimicrobial activity of Dezfuli orange peel essential oil with and without common antibiotics on some pathogenic bacteria. Food Science and Technology, 18(111), 159-167. https://doi.org/10.52547/fsct.18.111.159
29. Özogul, Y., Özogul, F., & Kulawik, P. (2021). The antimicrobial effect of grapefruit peel essential oil and its nanoemulsion on fish spoilage bacteria and food-borne pathogens. LWT, 136, 110362. https://doi.org/10.1016/j.lwt.2020.110362
30. Poorhasan, F., Zokaee, A., & Rahmani, M. (2012). Review on nanoemulsions production and comparison of homogenizers efficiencies. Iranian Chemical Engineering Journal, 11(63), 4-12. (In Persian with English abstracrt)
31. Seibert, J.B., Rodrigues, I.V., Carneiro, S.P., Amparo, T.R., Lanza, J.S., Frézard, F.J.G., ... & Santos, O.D.H.D. (2019). Seasonality study of essential oil from leaves of Cymbopogon densiflorus and nanoemulsion development with antioxidant activity. Flavour and Fragrance Journal, 34(1), 5-14. https://doi.org/10.1002/ffj.3472
32. Shahidi, F., Yazdi F, T., Roshanak, S., Behbahani, B.A., Norouzi, N., & Vasiee, A. (2019a). Antimicrobial activity of Lepidium draba extract on some pathogenic microorganisms “in vitro”. Iranian Journal of Infectious Diseases and Tropical Medicine, 24(85), 1-9. (In Persian with English abstracrt)
33. Shahidi, F., Yazdi, F.T., Behbahani, B.A., Roshanak, S., Norouzi, N., & Vasiee, A. (2019b). Aantibacterial effect of Tragopogon graminifolius extract on Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli and Salmonella typhi “in vitro”. Iranian Journal of Infectious Diseases, 24(84), 1(10). (In Persian with English abstracrt)
34. Tanavar, H., Barzegar, H., Alizadeh Behbahani, B., & Mehrnia, M.A. (2020). Mentha pulegium essential oil: chemical composition, total phenolic and its cytotoxicity on cell line HT29. Iranian Food Science and Technology Research Journal, 16(5), 643-653. (In Persian with English abstracrt). https://doi.org/10.22067/ifstrj.v16i5.84722
35. Tavakkol Afshari, H.S., Homayouni Tabrizi, M., & Ardalan, T. (2019). Evaluation of antioxidant and anticancer effects of nanoemulsions prepared using dill essential oil. Journal Arak University Medical Science, 22(4): 40-51. (In Persian with English abstracrt)
36. Uysal, B., Sozmen, F., Aktas, O., Oksal, B.S., & Kose, E.O. (2011). Essential oil composition and antibacterial activity of the grapefruit (Citrus paradisi) peel essential oils obtained by solvent‐free microwave extraction: comparison with hydrodistillation. International Journal of Food Science & Technology, 46(7), 1455-1461. https://doi.org/10.1111/j.1365-2621.2011.02640.x
37. Yang, S.A., Jeon, S.K., Lee, E.J., Shim, C.H., & Lee, I.S. (2010). Comparative study of the chemical composition and antioxidant activity of six essential oils and their components. Natural Product Research, 24(2), 140-151. https://doi.org/10.1080/14786410802496598
38. Yazdi, F.T., & Behbahani, B.A. (2013). Antimicrobial effect of the aqueous and ethanolic Teucrium polium extracts on gram positive and gram negative bacteria “in vitro”. Archives of Advances in Biosciences, 4(4), 56-62.
39. Yazgan, H., Ozogul, Y., & Kuley, E. (2019). Antimicrobial influence of nanoemulsified lemon essential oil and pure lemon essential oil on food-borne pathogens and fish spoilage bacteria. International Journal of Food Microbiology, 306, 108266. https://doi.org/10.1016/j.ijfoodmicro.2019.108266