Oxidation is one of the most common spoilage mechanisms of foods; in fact there is a widespread use of antioxidants in foods. Some of them are obtained by chemical synthesis, and as consumers prefer natural products, there is a growing demand for natural sources of antioxidants. Several plant essential oils (EOs) have a long history of being used in foods, and are considered GRAS, when such oils are obtained from organic grown plants by approved procedures they can also be incorporated into organic foods. The European Commission [1
] has strategic plans to promote organic farming in the European Union. Nowadays, EOs are mainly used in the food industry as flavoring agents, and are used as well by the hygienic, pharmaceutical, cosmetic, and perfume industries [2
The food industry also benefits from EOs preservative properties [2
]. Meat products, soups, dairy products (cheeses, creams), flavored oils and vinegars, and fermented vegetables, among others, usually contain EOs or other plant parts or extracts. In order to enhance food safety it is of major interest to investigate the antimicrobial properties of EOs, especially on food spoilage and pathogenic microorganisms, as well as the interactions among food-EOs-microorganisms and possible combinations of antimicrobial agents.
Limiting factors of the use of EOs in foods as preserving agents are (i) EOs are potent flavoring agents and are acceptable, from a sensory point of view, for specific foods; (ii) EOs addition into foods is common at reduced concentrations, sometimes below effective antimicrobial concentrations; (iii) they are not usually effective antimicrobials by themselves and rather need combination with other antimicrobial agents.
Most antimicrobial studies of EOs have been carried out on bacteria, and to a lesser extent on molds and yeasts. As a common trend, gram-negative bacteria have lower susceptibility to EOs than gram-positive ones [4
], mainly due to their membrane characteristics that act as barriers against macromolecules and hydrophobic compounds. Given that EOs are hydrophobic compounds, gram negative bacteria are somehow protected against them [5
Antioxidant properties of EOs have also been reported. Antioxidant compounds pose the ability to delay or inhibit the oxidation of lipids and other molecules by inhibiting the initiation or propagation of oxidation chain reactions [6
]. The association of the myriad of compounds present in EOs provides higher antioxidant activity than the summed activity of the individual components [7
]. According to Zeng and Wang [12
], EOs may be used as food preserving agents mainly due to the presence of phenolic compounds as main components, which are responsible for the antioxidant properties and may be an alternative to the use of synthetic antioxidants.
Given the potential of EOs as antimicrobials and antioxidants, it is of great interest to study in vitro properties of organic EOs. Their knowledge may allow their proper use in organic foods, and also pose an alternative to synthetic antioxidants for conventionally produced foods. The present study is focused in three EOs from widely used species of mild taste and flavor: fennel (Foeniculum vulgare), parsley (Petroselium crispum), and lavender (Lavandula officinalis), obtained from organic grown plants cultivated in Spain. EOs composition, phenolic content, antioxidant properties, and antimicrobial properties against two psycrotrophic bacteria, one responsible of food spoilage (Pseudomonas fluorescens CECT 844) and the other indicator of the presence of Listeria spp. (Listeria innocua CECT 910) are investigated.
2. Experimental Section
Chemicals: Ascorbic acid, butylated hydroxytoluene (BHT), 2,2′-diphenyl-1-picrylhydrazyl (DPPH), ferrozine, iron(III) chloride, iron(II) chloride, trichloroacetic acid (TCA), pentane, and Trolox were from Sigma Chemical Company (Germany). Potassium hydrogen phosphate, anhydrous sodium sulphate, 2-thiobarbituric acid (TBA), and disodium hydrogen phosphate were from Merck (Darmstadt, Germany). Potassium ferricyanide was from Fluka BioChemika (Neu Ulm, Germany). The solvent used for preparing standard solutions was methanol of HPLC grade, supplied by Merck. Solutions were freshly prepared, all flasks and vials were of amber glass and were kept in darkness.
Plant materials: Fennel (Foeniculum vulgare), parsley (Petroselium crispum), and lavender (Lavandula officinalis) commercial essential oils from organic grown plants were purchased from Herbes del Molí (Benimarfull, Alicante, Spain). The plantation and company are certified for organic agriculture by CAECV (Comité de Agricultura Ecológica de la Comunitat Valenciana). EOs were extracted from fennel plants, parsley plants, and lavender flowers and plants by hydrodistillation. The company reported an extraction yield for lavender of 3.45 mL/100 g dry weight, no yield data was recorded by the company for fennel and parsley EOs.
GC-MS and GC-FID Analytical Conditions
: The volatile compounds were isolated, identified and quantified as described in a previous work [13
]. A Shimadzu GC-17A gas chromatograph (Shimadzu Corporation, Kyoto, Japan), coupled with a Shimadzu mass spectrometer detector (GC-MS QP-5050A, Shimadzu, Kyoto, Japan) was used for peaks identification. The GC-MS system was equipped with a TRACSIL Meta X5 column (Teknokroma S. Coop. C. Ltd, Barcelona, Spain; 30 m × 0.25 mm i.d., 0.25 μm film thickness). Analyses were carried out using helium as carrier gas at a flow rate of 1.0 mL/min, at a split ratio of 1:10 and the following temperature programme: 40 °C for 5 min; rising at 3.0 °C/min to 200 °C and held for 1 min; rising at 15 °C/min to 280 °C and held for 10 min. Injector and detector were held at 250 °C and 300 °C, respectively. Diluted samples (1/10 pentane, v/v
) of 0.2 μL of the extracts were always injected. Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of 45–450 m/z
. Most of the compounds were identified by simultaneously using two different analytical methods [14
]: (a) KI, Kováts Index in reference to n-alkanes (C8
); and (b) mass spectra (authentic chemicals and Wiley spectral library collection). Identification was considered tentative when it was based on mass spectral data only. Semi-quantification of compounds was run in a Shimadzu GC-2100 equipped with an FID detector and the same column previously described and the same flow and oven conditions. 0.2 μL were injected manually in the split mode (split ratio 1/44). Quantitative data were obtained electronically from FID area data without using correction factors. All the tests were performed in triplicate.
Total phenolic content
: Total Phenolic Content was assessed by Folin-Cicalteau method [15
′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method:
The antioxidant activity of fennel, parsley, and lavender essential oils was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical DPPH [16
]. The amount of sample necessary to decrease the absorbance of DPPH (IC50
) by 50% was calculated graphically. Each assay was carried out in triplicate; (2) Ferric reducing antioxidant power:
The ferric reducing power (FRAP) of the essential oils was determined by using the potassium ferricyanide-ferric chloride method [16
]. The FRAP of a sample is estimated in terms of Trolox equivalent antioxidant capacity (TEAC) in mmol/L Trolox. Each assay was carried out in triplicate; (3) Inhibition of lipid peroxidation of buffered egg yolk by essential oils:
The method of Daker et al.
] was modified, to determine the thiobarbituric acid reactive substance (TBARS), a secondary product of lipid peroxidation. Each assay was carried out in triplicate.
Microbial strains: The essentials oils were individually tested against Listeria innocua CECT 910 and Pseudomonas fluorescens CECT 844 from the Spanish Type Culture Collection (CECT) of the University of Valencia.
Agar disc diffusion method:
The agar disc diffusion method described by Tepe et al.
] with some modifications used to determine the antibacterial capacity of the essential oils. Briefly, a suspension (0.1 mL of 106
) of Listeria innocua
was spread on the solid medium plates (BHI agar; Sharlab, Sharlab SL, Barcelona, Spain). Sterile filter paper discs, 9 mm in diameter (Schlinder & Schuell, Dassel, Germany) were impregnated with 40 μL of the oil and placed on the inoculated plates; these plates were incubated at 37 °C for 24 h. Pseudomonas fluorescens
, was cultured in nutritive agar II (Oxoid, Basingstoke, Hampshire, England), and incubated at 26 °C for 48 h. The diameters of the inhibition zones were measured in millimeters. All tests were performed in triplicate.
Determination of volume effect:
The volume effect (VE) was studied to ascertain which amounts of essential oil had an inhibitory effect on bacterial growth in the disc diffusion assay. The culture techniques used were those described in the previous paragraph (Agar disc diffusion method), but adding 40, 20, 10, 5, and 2.5 μL of essential oil [19
]. All tests were performed in triplicate.
: Data on antioxidant and antibacterial activities were analyzed by means of multivariate procedure GLM (General Lineal Model). For comparison among means Tukey’s test was used (p
< 0.05) [20
]. Antioxidant activity was studied by means of ANOVA test with two factors (EO type: fennel, parsley, and lavender and concentration: 20, 15, 10, and 5 g/L). For antibacterial activity one-way ANOVA for each EO was applied being the factor oil volume in de discs (40, 20, 10, 5, and 2 μL). All determinations were run on SPSS®
Statistics 188.8.131.52. software (International Business Machines Corp., Armonk, New York, USA).