The Open Food Science Journal




ISSN: 1874-2564 ― Volume 10, 2018
RESEARCH ARTICLE

Chemical Composition and Antibacterial Activity of Bergamot Peel Oil from Supercritical CO2 and Compressed Propane Extraction



Marcos Lazarotto1, Alexssandra Valério2, Aline Boligon3, Marcus V. Tres4, Jaqueline Scapinello1, Jacir Dal Magro1, J. Vladimir Oliveira2, *
1 Environmental Sciences Area, Unochapecó, PO Box 1141, Chapecó, SC, Brazil
2 Department of Chemical and Food Engineering, UFSC, Florianópolis, SC, Brazil
3 Center of Health Sciences, UFSM, Santa Maria, RS, Brazil
4 Department of Chemical Engineering, UFSM, Cachoeira do Sul, RS, Brazil

Abstract

Objective:

Essential oils are widely used as flavors and fragrances in the food, cosmetic and pharmaceutical industries, especially the bergamot peel oil due to the high polyphenols content, compared to other citrus species. Two types of polyphenols present in bergamot peel oil, brutieridin and melitidin, are directly related to cholesterol biosynthesis inhibition in a similar way as the statins. In this context, this work reports the extraction yields of bergamot peel oil obtained by supercritical carbon dioxide and compressed propane, together with the antimicrobial activity.

Methods:

The experiments were conducted at 55°C and 350 bar (density 0.881kg/m3) for carbon dioxide and at 55 °C and 40 bar (density 0.441 kg/m3) for propane.

Results:

Regarding the antimicrobial activity, the minimum inhibitory concentrations of bergamot oil were effective for the gram-positive bacteria growth inhibition, Staphylococcus aureus at 31.25 µg.mL-1 of bergamot oil, while 500 µg.mL-1 of oil extract was necessary to afford gram-negative bacterium (Escherichia coli) inhibition.

Keywords: Bergamot peel oil, Compressed solvents, Antibacterial activity, Citric fruits, S. aureus, E-coli.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 10
First Page: 16
Last Page: 23
Publisher Id: TOFSJ-10-16
DOI: 10.2174/1874256401810010016

Article History:

Received Date: 15/8/2018
Revision Received Date: 3/10/2018
Acceptance Date: 13/10/2018
Electronic publication date: 30/10/2018
Collection year: 2018

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© 2018 Lazarotto et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Chemical and Food Engineering, UFSC, Florianópolis, SC, Brazil; Tel: +554837212508; E-mail: jose.vladimir@ufsc.br




1. INTRODUCTION

Research focused on the development of clean technologies, especially on the extraction of natural compounds, has been applied to several areas of science and largely devoted to food, cosmetics, and pharmaceutical industries [1Gurib-Fakim A. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Mol Aspects Med 2006; 27(1): 1-93.[http://dx.doi.org/10.1016/j.mam.2005.07.008] [PMID: 16105678] , 2Chemat F, Rombaut N, Meullemiestre A, et al. Review of Green Food Processing techniques. Preservation, transformation, and extraction. Innov Food Sci Emerg Technol 2017; 41: 357-77.[http://dx.doi.org/10.1016/j.ifset.2017.04.016] ]. Recently, citrus essential oil has gained increasing attention due to the hypoglycemic and hypolipidemic activity as well as anti-inflammatory properties [3Delle Monache S, Sanità P, Trapasso E, et al. Mechanisms underlying the anti-tumoral effects of Citrus Bergamia juice. PLoS One 2013; 8(4): e61484.[http://dx.doi.org/10.1371/journal.pone.0061484] [PMID: 23613861] -6Visalli G, Ferlazzo N, Cirmi S, et al. Bergamot juice extract inhibits proliferation by inducing apoptosis in human colon cancer cells. Anticancer Agents Med Chem 2014; 14(10): 1402-13.[http://dx.doi.org/10.2174/1871520614666140829120530] [PMID: 25173561] ].

Among citric fruits, Citrus deliciosa bergamia (bergamot) has an associated peel essential oil with high commercial value due to the great variety of compounds of interest such as limonene, linalool and linalyl acetate [7Poiana M, Fresa R, Mincione B. Supercritical carbon dioxide extraction of bergamot peels. Extraction kinetics of oil and its components. Flavour Fragrance J 1999; 14: 358-66.[http://dx.doi.org/10.1002/(SICI)1099-1026(199911/12)14:6<358::AID-FFJ841>3.0.CO;2-I] , 8Figoli A, Donato L, Carnevale R, et al. Bergamot essential oil extraction by pervaporation. Desalination 2006; 193: 160-5.[http://dx.doi.org/10.1016/j.desal.2005.06.060] ] that can be applied in different processing industry areas. Thus, bergamot peel obtained by cold extraction cannot be considered a residue of the citrus juice industry as it is a very valuable product, high price, used in perfumery and cosmetic industry. Concerning the biological activity of bergamot peel oil, some studies have reported the anti-inflammatory, antiproliferative, and analgesic effect, including effects on the cardiovascular and central nervous system [9Van Amson G, Haracemiv SMC, Masson ML. Levantamento de dados epidemiológicos relativos à ocorrências/ surtos de doenças transmitidas por alimentos (DTAs) no estado do Paraná Brasil, no período de 1978 a 2000. Cienc Agrotec 2006; 30: 1139-45.[http://dx.doi.org/10.1590/S1413-70542006000600016] -12Navarra M, Mannucci C, Delbò M, Calapai G. Citrus bergamia essential oil: From basic research to clinical application. Front Pharmacol 2015; 6: 36.[http://dx.doi.org/10.3389/fphar.2015.00036] [PMID: 25784877] ].

Among several different techniques available for the extraction of essential peel oils from vegetable matrices, the use of pressurized solvents has demonstrated great potential, as long as physicochemical properties (density, diffusivity, viscosity, and dielectric constant) can be finely tuned by pressure and temperature [13Pronyk C, Mazza G. Design and scale-up of pressurized fluid extractors for food and bioproducts. J Food Eng 2009; 95: 215-26.[http://dx.doi.org/10.1016/j.jfoodeng.2009.06.002] , 14Rodríguez-Pérez C, Mendiola JA, Quirantes-Piné R, Ibáñez E, Segura-Carretero A. Green downstream processing using supercritical carbon dioxide, CO2-expanded ethanol and pressurized hot water extractions for recovering bioactive compounds from Moringa oleifera leaves. J Supercrit Fluids 2016; 116: 90-100.[http://dx.doi.org/10.1016/j.supflu.2016.05.009] ]. Besides, the use of pressurized fluids is considered an environmentally friendly, economic and safety technology demanded by industries and consumers, as it is associated with the use of non-toxic and relatively lower cost solvents [15Knez E Markočič, Leitgeb M, Primožič M, Hrnčič M Knez, Škerget M. Industrial applications of supercritical fluids: A review. Energy 2014; 77: 235-43.[http://dx.doi.org/10.1016/j.energy.2014.07.044] -17Corso MP, Fagundes-Klen MR, Silva EA, et al. Extraction of sesame seed (Sesamun indicum L.) oil using compressed propane and supercritical carbon dioxide. J Supercrit Fluids 2010; 52: 56-61.[http://dx.doi.org/10.1016/j.supflu.2009.11.012] ]. When compared to the conventional methodologies that make use of organic liquid solvents, the use of compressed gases does not require an additional separation step for solvent removal, hence preventing possible degradation of thermo-sensitive compounds.

Although several research works have focused on the development of clean/green technologies for the natural compounds extraction, there is a lack of information in the literature regarding the bergamot peel oil extraction using supercritical carbon dioxide (SCCO2) and compressed propane as solvents, considering the chemical profile and antimicrobial activity of the oils obtained. Though SCCO2 has been the solvent selected for extraction of herbaceous matrices, propane may be advantageous for some applications due to its low dielectric constant, comparable to SCCO2, the ability to extract lipophilic compounds, much reduced working pressure needed, which means lower Capex and Opex (Capex- capital expenditure costs; Opex- operational expenditure costs) [18Fernandes CEF, Scapinello J, Bohn A, et al. Phytochemical profile, antioxidant and antimicrobial activity of extracts obtained from erva-mate (Ilex paraguariensis) fruit using compressed propane and supercritical CO2. J Food Sci Technol 2017; 54(1): 98-104.[http://dx.doi.org/10.1007/s13197-016-2440-4] [PMID: 28242908] ]. Although the use of propane may cause doubts about safe operation, it might be taken into account that much lower pressures than SCCO2 can be employed, typically in the order of 10 bar (as solvent density becomes almost invariant above this value). Additionally, good high precision detectors of light hydrocarbons are available in the market, hence preventing leakage problems - such points have recently drawn attention from the industrial sector [19BR - PI 1020120187523, Sistema e processo para extração de óleos vegetais e essenciais utilizando fluidos pressurizados. Inventors: J.V. Oliveira, M. DiLuccio and M.V. Tres., 20CN102161932-A, Extraction of high-quality soybean germ oil involves extracting rolled soybean germ using liquefied butane as solvent in adverse current, removing solvent under reducing pressure, alkali refining, and decoloring. Inventor(s): LI B; XU B; DONG Y.].

In this context, the main goal of this work was to evaluate the use of carbon dioxide and propane in the oil extraction of Citrus deliciosa bergamia peel (bergamot peel) and compare the results in terms of extraction yield and antimicrobial activity against Escherichia coli and Staphylococcus aureus, as microorganism models. The target here is to use and environmental benign extraction technology combining with the use of a renewable, waste, raw material, as well as replacing the use of organic solvents without residue left in the final product.

2. EXPERIMENTAL

2.1. Materials

Bergamot was harvested in July 2014 from native plants in Descanso (Santa Catarina State, Brazil). After harvesting, the bergamot (Delicious Citrus Bergamia) was washed with abundant water and then peeled. Peels were cut in 1 cm2 pieces and then stored at -18 °C in polypropylene bags. Neither in the peeling step nor in peel cutting, losses of bergamot peel oil were visually observed. Propane and carbon dioxide (CO2) with minimum purity of 99.5% in the liquid phase was purchased from White Martins S.A. Staphylococcus aureus strains (ATCC 25923) and Escherichia coli strains (ATCC 25922) were used for the antimicrobial activity tests. Brain Heart Infusion Broth (BHI), 2,3,5-Triphenyl-tetrazolium chloride (TTC), and dimethylsulfoxide (DMSO, 99%) was purchased from Merck, Sigma-Aldrich and Vetec, respectively.

2.2. Bergamot Peel Oil Extraction With Pressurized Propane and Supercritical CO2

Before extraction, the previously cut bergamot peels were crushed in a slicer and sieved to obtain particles of mean size ≤ 2 mm. Typically, around 30 g (±0.05 g) of raw material was used in all extraction experiments. The equipment consisted of four components: a solvent gas cylinder, a high-pressure pump, an extraction vessel, a micrometric valve connected to collection flask and a temperature and pressure control systems. A detailed discussion about experimental apparatus, methodology and extraction conditions can be found in the works of Capeletto et al. (2016) [21Capeletto C, Conterato G, Scapinello J, et al. Chemical composition, antioxidant and antimicrobial activity of guavirova (Campomanesia xanthocarpa Berg) seed extracts obtained by supercritical CO2 and compressed n-butane. J Supercrit Fluids 2016; 110: 32-8.[http://dx.doi.org/10.1016/j.supflu.2015.12.009] ] and Scapinello et al. (2015) [22Novello Z, Scapinello J, j Dal Magro, et al. Extraction, chemical characterization and antioxidant activity of andiroba seeds oil obtained from pressurized n-butane. Ind Crops Prod 2015; 76: 697-701.[http://dx.doi.org/10.1016/j.indcrop.2015.07.075] ].

After preliminary tests, the extraction conditions for all assays were set as solvent flow rate of 3 mL min-1 and 2 h of extraction time for experimental runs. In fact, preliminary tests showed that extraction times greater than 2 h led to negligible gains of the extract obtained and therefore above this time resulted in practice in solvent losses with additional costs and unfruitful results. Also, preliminary tests and experience from previous works of our research group show that good extraction conditions may be 55 °C and 350 bar for SCCO2 (density 0.881kg/m3) and 55 °C and 40 bar (density 0.441kg/m3) for propane. The purpose for these extraction conditions was to increase the possibility of extraction a larger number of chemical compounds by raising vapor pressure of pure components while improving solvation trough adequate solvent extracting densities. For all extraction conditions using SCCO2 and pressurized propane, bergamot peel oils obtained were collected in amber bottles and then stored under refrigeration (5 ºC ± 2 ºC). The overall extraction yield of bergamot peel oil was obtained by the relation between the total mass oil extracted by the mass of the sample. Based on triplicate experiments carried out for all the experimental conditions for both compressed solvents, the overall average standard deviation of the yields noticed was about 0.2 wt%.

2.3. Bergamot Peel Oil Analysis by Gas Chromatography (GC and GC-MS)

The chemical composition of bergamot peel oil was determined using a Shimadzu gas chromatograph model Varian 3800 equipped with a split-injection port, flame-ionization detector and a HP-Innowax column (25 m x 0.25 mm x 0.5 μm). The chromatograph operating conditions were: split = 1:150; column flux = 0.92 mL.min-1, detector temperature: 250 °C, injector temperature: 250 °C, oven temperature: 60 °C (8 min), 180 °C (4 °C min-1), 180 °C - 230 °C (20 °C min-1), 230 °C (20 min) using helium as carrier gas: split ratio: 50:1, 34 kPa, and injected volume of 1.0 µL. The composition was obtained from electronic integration measurements using flame ionization detection. The GC-MS analysis was performed on a GC-MSD system model HP 5973-6890, operating in EI mode at 70 eV, equipped with a cross-linked column capillary (HP-5, 30 m x 0.25 mm). Helium was used as carrier gas (56 kPa, 1 ml.min-1). The identification of compounds in the bergamot peel oil was based on the retention index, determined as a function of a homologous series of C7-C30 n-alkanes, under identical experimental conditions, to the NBS mass Library (Massada, 1976) described by Felton et al. (2009) [24Adams RP. Identification of essential oil components by gas chromatography/mass spectrometry No 4th ed. 4th ed.2007.]. The relative amounts of the individual components were calculated based on the GC peak area (FID response).

2.4. Minimum Inhibitory Concentration Assays (MIC)

The antimicrobial analyses to determine Minimum Inhibitory Concentration (MIC) were done for the bergamot peel oil extracted with pressurized propane and supercritical CO2 (Table 1). The assays were carried out in triplicate on systems sterile microplates containing 96 cavities shaped bottom “U” containing 100 μL of Brain Heart Infusion Broth (BHI), following the adapted method described by Hentz and Santin (2007) ] [25Hentz SM, Santin NC. Avaliação da atividade antimicrobiana do óleo essencial de alecrim (Rosmarinus officinalis l.) contra Salmonella sp. Evidência Interdisciplinar 2007; 7: 293-100.]. A 200 μL volume of the sample of bergamot peel oil extracted at the concentration from 27 mg.mL-1 (diluted in 10% DMSO) was filtered (0.45 μm Millipore filter) and inoculated in BHI medium. Also, the blank test was done with 100 µL of BHI in 10% DMSO. The bacterial inoculums of Staphylococcus aureus and Escherichia coli with a concentration of 0.5 in the McFarland scale (108 UFC mL-1) were diluted in 0.9% in a sterile saline solution and a volume of 5 µL (104 UFC mL-1) was deposited in each cavity of the microplate utilizing a micropipette. In each line of the microplates containing different concentrations of the bergamot peel oil extract, adjusted with DMSO 10%. The 96-well microplates were incubated in a bacteriological oven at 35 °C for 18 hours. Afterward, 20 µL de TTC 0.5% was added in each cavity of the microplate and again incubated for another three hours. The MIC was defined as the lowest concentration, enough to inhibit the microbial growth (Mann and Markham, 1998).

Table 1
Chemical composition of bergamot peel oil obtained from supercritical CO2 (55 °C and 350 bar) and compressed propane extraction (55 °C and 40 bar).


3. RESULTS AND DISCUSSION

3.1. Extraction Performance

The obtained extraction yields, defined as the weight percentage of the oil extracted with respect to the initial charge of the raw material in the extractor, were 0.48 wt% and 0.37 wt% for SCCO2 and pressurized propane, respectively. Table 2 shows the bergamot peel oil composition obtained by SCCO2 and pressurized propane extraction. It can be observed that more compounds were identified from the SCCO2 (99.8%) extract compared to propane (~ 87%), which may be justified to the fact that in the experimental conditions used the propane has greater power of solvation and in this way extracted less volatile compounds, with higher molecular weight and consequently greater structural complexity that were not identifiable by GC-MS [26Illés V, Daood HG, Perneczki S, Szokonya L, Then M. Extraction of coriander seed oil by CO2 and propane at super- and subcritical conditions. J Supercrit Fluids 2000; 17: 177-86.[http://dx.doi.org/10.1016/S0896-8446(99)00049-2] ].

Table 2
Minimum inhibitory concentration (MIC) of bergamot peel oil extracts.


Comparison of chemical profile obtained from the application of extraction solvents shows that the major constituent in both bergamot peel oils was limonene, an important monoterpene, precursor of many pharmaceuticals and chemicals [27Keasling JD. Manufacturing molecules through metabolic engineering. Science 2010; 330(6009): 1355-8.[http://dx.doi.org/10.1126/science.1193990] [PMID: 21127247] ], with 45% observed for SCCO2 and 21.5% for propane. These results are in accordance with the studies reported by Sawamura et al. (2006) [28Sawamura M, Onishi Y, Ikemoto J, Tu NTM, Phi NTL. Characteristic odour components of bergamot (Citrus bergamia Risso) essential oil. Flavour Fragrance J 2006; 21: 609-15.[http://dx.doi.org/10.1002/ffj.1604] ], in which the authors reported limonene as the major component in bergamot essential oil.

Other important components identified in bergamot peel oil were linalyl acetate, linalool and geraniol with 23.6%, 11.3%, and 0.25% of the total components extracted with CO2 and 19.7%, 15.1%, and 2.03% for propane, respectively. As reported by Elisabetsky et al. (1995) [29Elisabetsky E, Marschner J, Souza DO. Effects of Linalool on glutamatergic system in the rat cerebral cortex. Neurochem Res 1995; 20(4): 461-5.[http://dx.doi.org/10.1007/BF00973103] [PMID: 7651584] ], compounds such as linalyl acetate and linalool may be associated with anxiolytic activity. According to Camargo and Vasconcelos (2014) [30Camargo SB, Vasconcelos DFSA. Atividades biológicas de Linalol: Conceitos atuais e possibilidades futuras deste monoterpeno. Rev Ciênc Méd Biol 2014; 13: 3-381.[http://dx.doi.org/10.9771/CMBIO.V13I3.12949] ], linalool is a secondary metabolite, one of the most important substances in the pharmaceutical industry, used by popular medicine as anti-inflammatory, analgesic, hypotensive, and antimicrobial properties [10Sakurada T, Mizoguchi H, Kuwahata H, et al. Intraplantar injection of bergamot essential oil induces peripheral antinociception mediated by opioid mechanism. Pharmacol Biochem Behav 2011; 97(3): 436-43.[http://dx.doi.org/10.1016/j.pbb.2010.09.020] [PMID: 20932858] , 31Julião LS, Tavares ES, Lage CLS, Leitão SG. Cromatografia em camada fina de extratos de tr{ê}s quimiotipos de Lippia alba (Mill) N.E.Br. (erva-cidreira). Rev Bras Farmacogn 2003; 13: 36-8.[http://dx.doi.org/10.1590/S0102-695X2003000300014] , 32Costa AM, Buglione CC, Bezerra FL, Martins PCC, Barracco MA. Immune assessment of farm-reared Penaeus vannamei shrimp naturally infected by IMNV in NE Brazil. Aquaculture 2009; 291: 141-6.[http://dx.doi.org/10.1016/j.aquaculture.2009.03.013] ].

Results reported in this research work agree with those published by Dugo et al. (2000) [33Dugo P, Mondello L, Dugo L, Stancanelli R, Dugo G. LC-MS for the identification of oxygen heterocyclic compounds in citrus essential oils. J Pharm Biomed Anal 2000; 24(1): 147-54.[http://dx.doi.org/10.1016/S0731-7085(00)00400-3] [PMID: 11108548] ] and Russo et al. (2013) [34Russo R, Ciociaro A, Berliocchi L, et al. Implication of limonene and linalyl acetate in cytotoxicity induced by bergamot essential oil in human neuroblastoma cells. Fitoterapia 2013; 89: 48-57.[http://dx.doi.org/10.1016/j.fitote.2013.05.014] [PMID: 23707744] ], who reported that bergamot peel oil has a non-volatile fraction (4-7%), characterized by coumarins and furocoumarins, and a volatile fraction (93-96%) with hydrocarbons as monoterpenes and sesquiterpenes such as limonene, γ-terpinene, α-β-pinene, β-myrcene, sabinene, β-bisabolene, and oxygenated derivatives, such as linalool, linalyl acetate, neral, geraniol, neryl and geranyl acetate.

3.2. Antibacterial Assays

From the MIC results, it was noticed that the bergamot peel oils were efficient as bacterial growth-inhibitor. However, the antibacterial effect was higher against Gram-positive bacteria (S. aureus) with a determined MIC of 31.2 μg.mL-1. On the other hand, Gram-negative bacteria (E. coli) was more resistant, requiring higher concentrations of bergamot peel oil, 500 μg.mL-1 of oil obtained with SCCO2 and 125 μg.mL-1 of with pressurized propane. The resistance of Gram-negative bacteria such as E. coli may be associated with a complex cell wall compared to Gram-positive bacteria, thereby increasing their resistance to the antibacterial agents. Ashok Kumar et al. (2011) [35Ashok kumar K, Narayani M, Subanthini A, Jayakumar M. Antimicrobial activity and phytochemical analysis of citrus fruit peels -Utilization of fruit waste. Int J Eng Sci 2011; 3(6): 5414-21.], using five different solvent extracts of Citrus lemon and Citrus simensis, analyzed the antimicrobial activity and reported a MIC of 12.5 mg.mL-1 for E. coli and for 25 mg.mL-1 for S. aureus, these results is similar to the strong antimicrobial activity of the bergamot oil obtained in this work.

The difference in the minimum inhibitory concentration for E. coli for the two bergamot peel oils obtained with CO2 (500 μg.mL-1) and propane (125 μg.mL-1) may be related to differences regarding chemical composition of oils obtained, and might be associated with the compounds γ-terpinene (3.25% for CO2 and 6.08% propane), geraniol (0.25% CO2 and 2.03% propane), β-caryophyllene (0.84% CO2 and 1.96% propane), neryl acetate (0.47% CO2 and propane 2.61%), trans- α -bamamotene (0.08% CO2 and 1.17% propane), β-bisabolene (1.27% CO2 and 3.04% propane) and linalool (11.35% CO2 and 15.16% propane).

According to Sokovié et al. (2010) [36Soković M, Glamočlija J, Marin PD, Brkić D, van Griensven LJ. Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model. Molecules 2010; 15(11): 7532-46.[http://dx.doi.org/10.3390/molecules15117532] [PMID: 21030907] ], linalool has higher antimicrobial effects against Gram-positive bacteria compared to Gram-negative ones, with an effect on protein denaturation or dehydration of the cells. Limonene, a cyclic hydrocarbon, can accumulate in the biological membrane and change its structure and function [37Sikkema J, de Bont JAM, Poolman B. Interactions of cyclic hydrocarbons with biological membranes. J Biol Chem 1994; 269(11): 8022-8.[PMID: 8132524] ]. In addition, limonene is reported as an antifungal agent [38Chee HY, Kim H, Lee MH. In vitro Antifungal Activity of Limonene against Trichophyton rubrum. Mycobiology 2009; 37(3): 243-6.[http://dx.doi.org/10.4489/MYCO.2009.37.3.243] [PMID: 23983542] ], bacteriostatic [39Jaroenkit P, Matan N, Nisoa M. In vitro and in vivo activity of citronella oil for the control of spoilage bacteria of semi dried round scad (Decapterus maruadsi). Int J Med Aromatic Plants 2011; 1: 234-9., 40Van Vuuren SF, Viljoen AM. Antimicrobial activity of limonene enantiomers and 1,8-cineole alone and in combination. Flavour Fragrance J 2007; 22: 540-4.[http://dx.doi.org/10.1002/ffj.1843] ], and anti-bactericidal plasma membrane of the bacteria and causes the loss of the bacterial membrane integrity [41Zukerman I. Effect of oxidized d-limonene on micro-organisms. Nature 1951; 168(4273): 517-8.[http://dx.doi.org/10.1038/168517a0] [PMID: 14875145] ]. A study of E. coli inactivation by terpenes and terpenoids (such as carvacrol or citral) demonstrated the sublethal lesions in the cytoplasmic membrane [42Ait-Ouazzou A, Cherrat L, Espina L, Lorán S, Rota C, Pagán R. The antimicrobial activity of hydrophobic essential oil constituents acting alone or in combined processes of food preservation. Innov Food Sci Emerg Technol 2011; 12: 320-9.[http://dx.doi.org/10.1016/j.ifset.2011.04.004] ] suggesting the bactericidal membrane rupture as the inactivation mechanism [43Espina L, Gelaw TK, de Lamo-Castellví S, Pagán R, García-Gonzalo D. Mechanism of bacterial inactivation by (+)-limonene and its potential use in food preservation combined processes. PLoS One 2013; 8(2): e56769.[http://dx.doi.org/10.1371/journal.pone.0056769] [PMID: 23424676] ].

According to Prado et al. (2013) [44Prado ACP, Manion BA, Seetharaman K, Deschamps FC, Barrera Arellano D, Block JM. Relationship between antioxidant properties and chemical composition of the oil and the shell of pecan nuts. Ind Crops Prod 2013; 45: 64-73. [Caryaillinoinensis (Wangenh) C. Koch].[http://dx.doi.org/10.1016/j.indcrop.2012.11.042] ], Wang et al. (2008) [45Wang YS, He HP, Yang JH, Di YT, Hao XJ. New monoterpenoid coumarins from Clausena anisum-olens. Molecules 2008; 13(4): 931-7.[http://dx.doi.org/10.3390/molecules13040931] [PMID: 18463594] ], Sartoratto et al. (2004) [46Sartoratto A, Machado ALM, Delarmelina C, Figueira GM, Duarte MCT, Rehder VLG. Composition and antimicrobial activity of essential oils from aromatic plants used in Brazil. Braz J Microbiol 2004; 35: 275-80.[http://dx.doi.org/10.1590/S1517-83822004000300001] ] and Michielin et al. (2009) [47Michielin EMZ, Salvador AA, Riehl CAS, Smânia A Jr, Smânia EFA, Ferreira SRS. Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods. Bioresour Technol 2009; 100(24): 6615-23.[http://dx.doi.org/10.1016/j.biortech.2009.07.061] [PMID: 19683436] ], it is possible to classify the antimicrobial agents based on MIC values. Michielin et al. (2009) [47Michielin EMZ, Salvador AA, Riehl CAS, Smânia A Jr, Smânia EFA, Ferreira SRS. Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods. Bioresour Technol 2009; 100(24): 6615-23.[http://dx.doi.org/10.1016/j.biortech.2009.07.061] [PMID: 19683436] ] classified the extracts as strong inhibitors with MIC up to 500 μg.mL-1, moderate inhibitor for MIC from 600 to 1500 μg.mL-1, and low inhibitor for MIC over 1600 μg.mL-1, allowing positioning the bergamot peel oils obtained in this work as a strong inhibitors agent against Gram-positive and Gram-negative bacteria.

It should be mentioned that many compounds present in the essential oil from vegetable matrices, e.g. limonene, becomes effective in combination with heat in the inactivation of some bacteria such as E. coli and Listeria monocytogenes [42Ait-Ouazzou A, Cherrat L, Espina L, Lorán S, Rota C, Pagán R. The antimicrobial activity of hydrophobic essential oil constituents acting alone or in combined processes of food preservation. Innov Food Sci Emerg Technol 2011; 12: 320-9.[http://dx.doi.org/10.1016/j.ifset.2011.04.004] ] due to a synergistic effect on pathogens inactivation, making them able for use in food preservation, keeping the organoleptic properties of the fresh food products [43Espina L, Gelaw TK, de Lamo-Castellví S, Pagán R, García-Gonzalo D. Mechanism of bacterial inactivation by (+)-limonene and its potential use in food preservation combined processes. PLoS One 2013; 8(2): e56769.[http://dx.doi.org/10.1371/journal.pone.0056769] [PMID: 23424676] ].

CONCLUSION

This work reports the application of a green technology for the extraction of bergamot peel oil, looking for the green chemistry approach in replacement of traditional organic liquid solvents, positively affecting the quality of the final product. It was possible to identify 99.85% and 86.93% of chemical compounds from bergamot peel oil extract using CO2 and propane, respectively. In general, both extracts presented limonene as the major constituent - 45.13% for CO2 and 21.53% for propane, followed by linalyl acetate - 23.67% for CO2 and 19.74% for propane, and linalool - 11.35% for CO2 and 15.16% for propane. Regarding the antibacterial activity, bergamot peel oil was effective on bacterial growth-inhibitory, especially for the Gram-positive (S. aureus), with MIC of 31.25 μg.mL-1. On the other hand, for Gram-negative (E. coli), results showed a higher microbial resistance, with MIC of 500 μg.mL-1 for bergamot peel oil extract by SCCO2 and 125 μg.mL-1 in the case of propane, allowing to classifying the bergamot peel oil obtained in this work as strong bacteria inhibitors.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors are grateful to Divinut Ind. de Nozes Ltda (Cachoeira do Sul - RS), CNPq, CAPES, FAPERGS/RS, FAPESC/SC and MCTI-FINEP for financial support and scholarships.

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