Essential Oils from Fruit and Vegetables, Aromatic Herbs, and Spices: Composition, Antioxidant, and Antimicrobial Activities

biology Article Essential Oils from Fruit and Vegetables, Aromatic Herbs, and Spices: Composition, Antioxidant, and Antimicrobial Activities Soumi De-Montijo-Prieto 1, María del Carmen Razola-Díaz 2,3 , Ana María Gómez-Caravaca 4,*, Eduardo Jesús Guerra-Hernandez 2, María Jiménez-Valera 1, Belén Garcia-Villanova 2, Alfonso Ruiz-Bravo 1 and Vito Verardo 2,3   Citation: De-Montijo-Prieto, S.; Razola-Díaz, M.d.C.; Gómez-Caravaca, A.M.; Guerra-Hernandez, E.J.; Jiménez-Valera, M.; Garcia-Villanova, B.; Ruiz-Bravo, A.; Verardo, V. Essential Oils from Fruit and Vegetables, Aromatic Herbs, and Spices: Composition, Antioxidant, and Antimicrobial Activities. Biology 2021,10, 1091. https://doi.org/ 10.3390/biology10111091 Academic Editor: Francesca Mancianti Received: 3 August 2021 Accepted: 20 October 2021 Published: 25 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Microbiology, Campus of Cartuja, University of Granada, 18071 Granada, Spain; [email protected] (S.D.-M.-P.); [email protected] (M.J.-V.); [email protected] (A.R.-B.) 2 Department of Nutrition and Food Science, Campus of Cartuja, University of Granada, 18071 Granada, Spain; [email protected].es (M.d.C.R.-D.); [email protected] (E.J.G.-H.); [email protected] (B.G.-V.); [email protected] (V.V.) 3 Institute of Nutrition and Food Technology ‘JoséMataix’, Biomedical Research Center, University of Granada, Avda del Conocimiento sn., Armilla, 18100 Granada, Spain 4Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Avd. Fuentenueva s/n., 18071 Granada, Spain *Correspondence: [email protected] Simple Summary: The use of essential oils (EOs) in the food industry is a popular research topic, as they have antioxidant and antimicrobial activity and could be used as ingredients directly in food or as bioactive component in food coating and food packaging. Thus, the study of their antioxidant and antimicrobial activity is a crucial step to evaluate their use in food packaging/coating. In this work, we evaluate the antioxidant and antimicrobial activities of 13 EOs from herbs, spices, fruits, and vegetables. Briefly, the EOs from aromatic herbs and spices showed the highest antioxidant and antimicrobial activity. Fennel essential oil reported the lowest antioxidant activity, however it showed very good antimicrobial activity against Botrytis cinerea, one of the post-harvest pathogen microorganisms in fruits and vegetables. Abstract: In the field of food preservation, encapsulated Essential Oils (EOs) could be the best non-toxic and eco-friendly tool for food preservative applications substituting the chemicals ones that have several disadvantages for the environment and health. Thirteen commercial EOs from plants, fruits, and vegetables were characterized by GC-MS. The antioxidant activity was measured by DPPH and ABTS techniques. Antimicrobial activity was assessed by agar well-diffusion method and the Minimum Inhibitory Concentration (MIC) by agar dilution method against six bacteria, Candida albicans, and Botrytis cinerea. All the EOs tested have demonstrated antioxidant activity in the range of IC 50 0.01–105.32 mg/mL. Between them, cinnamon EOs were the best, followed by oregano and thyme EOs. Fennel EO showed the lowest radical scavenging. MIC values ranged from 0.14 to 9 mg/mL. C. cassia, thyme, and oregano EOs were the most effective against the bacterial species tested, and the yeast C. albicans. On the contrary, citric fruit EOs showed low or no inhibition against most bacterial strains. The percentages of inhibition of mycelia growth of B. cinerea ranged from 3.4 to 98.5%. Thyme, oregano, mint, and fennel EOs showed the highest inhibition. Keywords: essential oils; DPPH; ABTS; food spoilage; antiradical activity 1. Introduction Food security is supported in four main pillars: food access, food utilization, food stability, and food preservation. The latter mainly consists of the degradation and microbial contamination that can affect food. Food spoilage is one of the problems that should be avoided. A lot of chemical preservatives have been developed and proved to have a Biology 2021,10, 1091. https://doi.org/10.3390/biology10111091 https://www.mdpi.com/journal/biology Biology 2021,10, 1091 2 of 21 significant contribution in controlling this degradation. However, they have often raised negative concerns to the consumers as they are not from a green source. They need long term degradation cycles, they are environmental toxicology and have potential risks of carcinogenesis and teratogenesis in humans and animals. Due to this, essential oils (EOs) and their active components are under study for their potential use as preservatives due to their wide antibacterial, antifungal, antimycotoxigenic spectrum, and antioxidant properties [ 1 ]. Therefore, the use of EOs in food industry is growing, as they could be directly added to edible products or used for active packaging and edible coatings [ 2 ]. Thus, the use of essential oil in food industry has a double action due to their antioxidant and antimicrobial properties [ 3 ]. Moreover, the bioactive compounds contained in the EOs could also be used for pharmaceutical and cosmetic applications [4]. Overall, EOs are products that can be isolated from leaves, bark, seeds, fruit peels, roots, flowers, buds, and stems, namely, from agro-industrial by-products in the majority of cases. The antioxidant activity of the EOs is related to the complex mixture of terpenes, terpenoids, and phenylpropanoids that compose them. Among others, it was noticed that carvacrol, thymol, and eugenol are able to inhibit the oxidation processes [5]. The same happens with the antimicrobial activity of the EOs, but in this case the mechanism depends on the specific chemical components. The most common mechanism seems to be related with the alteration in the membranes, modifying their dynamicity and permeability, and in consequence releasing the cytoplasmic constituents. However, the effect is different for each microorganism depending on the variability of the membrane thickness, composition, and cellular metabolic activities [1]. The limitations that could carry the use of EOs as preservatives in food can be due to the intense aroma, high reactivity, hydrophobicity, reduced solubility, and possible negative interaction with the matrices, leading to changes in the intestinal absorbance and organoleptic properties. However, nowadays they are avoidable due to the new mechanisms studied and developed for encapsulating those EOs. These methods can improve the stability and solubility of the EOs, protecting them from the environmental interactions [6]. Therefore, for agriculture and food fields, encapsulated EOs could be the best non-toxic and eco-friendly option for preservative applications as active food packaging, enhancing food shelf life. In this context, one of the goals of the SHEALTHY project is to find essential oils that could be incorporated in food packaging in order to obtain active packaging/coating for fruit and vegetables. In this way, it is important to identify essential oils with antibacterial and anti-fungus properties that can act against foodborne pathogens, e.g., Botrytis cinerea, a necrotrophic fungus that affects several fruits, causing the production of high quantities of fruit wastes. Thus, the aim of this work is to evaluate and compare the antioxidant and antimicrobial activities of 13 commercial EOs from plants and fruit and vegetables. To achieve this, the EOs were characterized by GC-MS. The antioxidant activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2 0 -Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assays. The antimicrobial activity was tested against microorganisms that usually spoil food and are dangerous for human health. 2. Materials and Methods 2.1. Reagents and Samples All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solvents were from Honeywell (Wabash, Lafayette, IN, USA) except ethanol, which was from PanReac (Barcelona, Spain). Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA ). Thirteen essential oils were purchased from a local supplier; seven of them hail from aromatic plants and spices, and others were from fruits and vegetables (Table 1). All samples were obtained by hydrodistillation (purity 100%). Selection of the essential oils took the literature data and the availability in the Spanish market into account. Biology 2021,10, 1091 3 of 21 Table 1. List of the EOs used in this work. Common Name Scientific Name Part Used Aromatic herbs and spices True cinnamon Cinnamomum zeylanicum J. Presl Leaf Cinnamon Cinnamomum cassia J. Presl Bark Oregano Origanum vulgare L. Leaf Thyme Thymus vulgaris L. Flower/leaf Rosemary Rosmarinus officinalis (L.) Schleid. Leaf Peppermint Mentha piperita L. Leaf Sage Salvia lavandulifolia Vahl Leaf Fruits and vegetables Celery Apium graveolens L. Seed Fennel Foeniculum vulgare Mill. var. dulce Not reported Mandarin Citrus reticulata Blanco Peel Sweet orange Citrus sinensis Osbeck Peel Lemon Citrus limon L. Peel Grapefruit Citrus paradisi Macfad. Peel 2.2. DPPH Free Radical-Scavenging Capacity DPPH radical scavenging activity was assayed with a method proposed by several authors [ 7 , 8 ]. Briefly, 2.9 mL of 100 µ M DPPH (in methanol or n-hexane depending on the polarity of the essential oil) was mixed with 100 µ L of each essential oil at different concentrations. They were incubated during 30 min at 25 ◦ C and were measured at 517 nm . The antioxidant activity of the EOs was expressed as the concentration of extract that inhibited the DPPH radical formation by 50% (IC 50 ). IC 50 for each sample was calculated by elaborating a curve where is represented the concentration (mg/mL) and the percentage of inhibition calculated as in the following Equation (1): Inhibition (%) = (blank −sample)/blank ×100 (1) where “blank” is the absorbance of DPPH with the sample replaced with methanol and “sample” is referred to the absorbance of the DPPH mixed with the essential oil or the control. Ascorbic acid was used as a reference of positive control from 0.0001 to 0.5 mg/mL. 2.3. ABTS Acid Cation Radical-Scavenging Capacity This technique was developed by Re at al. [ 9 ] in which the monocation ABTS •+ is generated by oxidation of the ABTS with potassium persulfate in the dark at room temperature for 12–24 h. For the analyses, 2 mL of 7 mM ABTS solution was added to 20 µ L of different concentrations of each sample, and they were measured at 734 nm after 30 min of incubation at 30 ◦ C. The antioxidant activity of the EOs was expressed as the concentration of extract that inhibited the ABTS radical formation by 50% (IC 50 ). The IC 50 for each sample was calculated by elaborating a curve where is represented the concentration (mg/mL) and the percentage of inhibition calculated as in the Equation (1). Ascorbic acid was used as a reference of positive control from 0.0001 to 0.5 mg/mL. 2.4. Antimicrobial Activity 2.4.1. Test Microorganisms The antimicrobial activity of essential oils was tested against Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Salmonella enterica serovar Typhimurium (S. Typhimurium), Listeria monocytogenes ( L. monocytogenes) ,Candida albicans (C. albicans), and Botrytis cinerea (B. cinerea). All bacterial strains and C. albicans were isolated from various clinical samples in the Microbiology Service of the Virgen de las Nieves Hospital (Granada, Spain) and were stored as glycerol stocks and reactivated by incubation in tryptic soy agar at 37 ◦ C for 24 h. B. cinerea was Biology 2021,10, 1091 4 of 21 obtained from the Spanish Type Culture Collection (CECT 2100) and was maintained and grown at 25 ◦C in Sabouraud dextrose agar. 2.4.2. Agar-Well-Diffusion Method For bacteria and C. albicans, antimicrobial activity was assessed following the method described by Hayes and Markovic [ 10 ] with modifications, as follows: 15 mL of molten Mueller Hinton agar were poured into sterile Petri dishes and allowed to set to form a base layer. An 8 mm diameter stainless steel cylinder was placed over the base layer, and 10 mL of molten Mueller Hinton agar containing the inoculum were poured over the surface of the base layer and left to set. For the preparation of inoculate, cultures of the strains were suspended in buffered saline solution until reaching a turbidity corresponding to 0.5 McFarland standard and were inoculated in molten Mueller Hinton agar to obtain a final concentration of approximately 1 × 10 6 CFU/ mL. After solidification of the upper layer, the cylinders were carefully removed and 25 µ L of essential oils were added into the resulting holes. As a positive control for antimicrobial activity, ciprofloxacin was used at 2 mg/mL for MRSA, 0.1 mg/mL for L. monocytogenes, and 0.01 mg/mL for the remaining bacteria, and ketoconazole at 0.01 mg/mL for C. albicans. After incubation at 4 ◦ C for 30 min to allow extracts to diffuse into the medium, the plates were incubated at 37 ◦ C for 24 h. The inhibition zone diameters were measured (mm) and recorded as the mean ±standard deviation (SD). Three replicates were conducted. For B. cinerea, Sabouraud dextrose plates were prepared following the previous procedure but testing one extract per plate. After 30 min incubation to allow the extract to diffuse, a 4-mm plug of mycelium was placed at a 3 cm distance from the essential oil. The plates were incubated for 7 days at 25 ◦ C. The growth control consisted of Sabouraud dextrose plates inoculated with mycelium alone. Ketoconazole at 20 µ g/mL was used as antimicrobial control. The percentage of mycelium inhibition for each essential oil was calculated by measuring the area of fungal growth and comparing it to the control using the ImageJ software [11]. Three replicates were conducted. 2.4.3. Determination of Minimum Inhibitory Concentration (MIC) MIC values were assessed by agar-dilution method as follows: from 288 mg/mL solutions in 98% ethanol of each essential oil, serial dilutions in sterile water supplemented with 0.5% Tween 80 were prepared (144–0.56 mg/mL). A total of 5 mL of the essential oil’s dilutions were added in 15 mL of molten Mueller Hinton or Sabouraud agar, which was prepared previously with three-quarters of its volume of water, and plated onto sterile Petri dishes. The essential oils were tested in concentrations ranging from 72 to 0.14 mg/mL for bacteria and C. albicans, and from 72 to 0.07 mg/mL for B. cinerea. Bacterial suspensions corresponding to 0.5 McFarland’s standard were adjusted to obtain a concentration of approximately 1 × 10 6 CFU/mL, and a spot of 10 µ L of each bacterial suspension was added onto the plates. B. cinerea was inoculated placing a 4-mm plug of mycelium at the center of each plate. A plate containing 5 mL of sterile water supplemented with 0.5% Tween 80 in 15 mL of molten Mueller Hinton or Sabouraud agar prepared and inoculated as before, was used as a growth control. All experiments were performed in duplicate. The MIC was defined as the lowest concentration of the essential oils that completely inhibited microbial growth after incubation at 37 ◦ C for 24 h for bacteria and C. albicans, and 25 ◦ C for 7 days for B. cinerea. 2.5. Determination of Essential Oil Compounds by GC-MS Essential oils were diluted in trichloromethane and analysed by GC-MS according to Ben Lajnef et al. [ 12 ]. Separation was achieved using an Agilent 7890A GC coupled to a Waters QUATTRO micro TM GC mass spectrometer. The compounds were separated on a capillary column DB-5MS (30 m × 0.25 mm; f.t. 0.25 µ m) purchased from Agilent Technologies (J&W Scientific, Folsom, CA, USA). Oven temperature was set at 40 ◦ C for Biology 2021,10, 1091 5 of 21 2 min , after that the temperature increased from 40 to 250 ◦ C at 3 ◦ C min −1 , and remained at 250 ◦C for 10 min. MS detector parameters were set at: scan range: 40–450 m/z; solvent delay time: 3.0 min ; transfer line temperature 250 ◦ C; ion source 230 ◦ C; and ionisation energy 70 eV. Carrier gas was (He) at a flow of 1.0 mL min −1 . Samples were injected in splitless mode. GS-MS chromatograms are showed in Supplementary Figure S1. 2.6. Statistical Analysis Pearson’s correlations between antioxidant methods were evaluated using Statistica 6.0 (2001, StatSoft, Tulsa, OK, USA). 3. Results and Discussion 3.1. Composition of the Essential Oils Essential oils were analyzed by GC-MS and a total of 56 compounds were detected. Table 2reported the relative amounts (%) of each compound determined in aromatic plant EOs. According to the literature [ 13 ], cinnamon samples presented different composition according to the part of the plant that was used for the extraction. Briefly, eugenol was the main compound in C. zeylanicum that was extracted from leaves; on the other hand, cinnamaldehyde was the first compound in C. cassia sample that was extracted from bark. Oregano EO showed carvacrol as the main compound (84.5%) followed by p-cymene, γ - terpinene and thymol. Similar composition has been reported by Diniz do Nascimento et al. [4] ; as remarked by the same authors, the oregano essential oil composition is highly influenced by the part of the plant and the agronomic and technological processes. Thymus EO composition is also affected from the different parts of plant that are used for extraction (among agronomical and processing factors). The samples that were analyzed in this work proceed from a mix of flowers and leaves; thymol was the first compound accounting for more than 60%, followed by p-cymene, γ -terpinene, carvacrol, and linalool, respectively, accounting for about the 90% of total compound. The same compounds were described by Diniz do Nascimento et al. [4] in thymus EOs. β -myrcene was the first compound in Rosmarinus EO, the second one was camphor, followed by eucalyptol and α -pinene; the present composition is in the same order of magnitude than that reported by Diniz do Nascimento et al. [4]. Mentha EO reported the typical compounds previously found in this matrix [ 4 ] and are characteristic of this EO. Briefly, menthol and menthone accounted for 69.5% of the total compounds, followed by menthyl-acetate, eucalyptol, isomenthone, isomenthol, and mentho-furan. Other minor compounds that are usually found in this plant are piperitone and neomenthol acetate. Finally, according to Porres-Martínez et al. [ 14 ], 1,8-cineole (eucalyptol) followed by camphor, camphene, and βand α-pinene were the main compounds of sage EO. Table 3reports the composition of essential oil obtained from fruits and vegetables. Biology 2021,10, 1091 6 of 21 Table 2. Composition (expressed as %) of aromatic plant EOs determined by GC-MS. Compound Cinnamomum cassia Cinnamomum zeylanicum Mentha piperita Origanum vulgare Rosmarinus officinalis Salvia lavandulifolia Thymus vulgaris 1 Tricyclene n.d. n.d. n.d. n.d. 0.2 ±0.02 0.2 ±0.01 0.08 ±0.00 2 Thujene n.d. 0.04 ±0.00 0.04 ±0.00 0.3 ±0.01 0.2 ±0.00 0.2 ±0.01 0.6 ±0.03 3α-pinene 0.05 ±0.00 0.2 ±0.03 0.7 ±0.07 1.0 ±0.10 11.9 ±0.90 5.5 ±0.30 0.7 ±0.06 4 Camphene n.d. 0.08 ±0.00 0.06 ±0.00 0.2 ±0.00 5.0 ±0.30 7.7 ±0.40 0.7 ±0.01 5 Sabinene n.d. n.d. 0.2 ±0.00 n.d. n.d. 1.1 ±0.30 0.004 ±0.00 6β-pinene 0.03 ±0.00 0.07 ±0.00 0.9 ±0.02 0.07 ±0.01 2.5 ±0.30 5.1 ±0.20 0.1 ±0.01 7β-myrcene n.d. 0.008 ±0.00 n.d. 0.5 ±0.30 30.7 ±0.40 2.0 ±0.08 0.7 ±0.03 8α-phellandrene n.d. 0.2 ±0.01 0.01 ±0.00 0.07 ±0.00 0.7 ±0.02 0.04 ±0.00 0.1 ±0.00 9α-terpinene 0.02 ±0.00 0.01 ±0.00 0.1 ±0.00 0.5 ±0.02 0.6 ±0.02 0.3 ±0.01 1.2 ±0.10 10 p-cymene 0.05 ±0.00 0.1 ±0.01 0.3 ±0.03 5.3 ±0.20 1.6 ±0.10 0.3 ±0.02 16.5 ±0.30 11 D-limonene 1.5 ±0.20 0.09 ±0.00 2.1 ±0.20 0.2 ±0.01 2.9 ±0.30 4.7 ±0.40 0.3 ±0.02 12 1,8-cineole (Eucalyptol) n.d. n.d. 4.9 ±0.30 0.07 ±0.00 14.8 ±0.50 38.6 ±0.80 n.d. 13 cis-ocimene n.d. n.d. n.d. n.d. 0.1 ±0.00 0.06 ±0.00 n.d. 14 γ-terpinene n.d. n.d. 0.1 ±0.03 2.4 ±0.20 0.7 ±0.02 0.4 ±0.01 7.1 ±0.30 15 Terpinolene n.d. 0.02 ±0.00 0.05 ±0.00 0.03 ±0.00 0.3 ±0.03 0.2 ±0.00 0.05 ±0.00 16 Linalool 1.8 ±0.10 0.4 ±0.01 n.d. 0.7 ±0.04 0.5 ±0.06 0.7 ±0.00 2.5 ±0.08 17 Pinone n.d. n.d. 0.004 ±0.00 n.d. n.d. 0.3 ±0.01 n.d. 18 Camphor n.d. n.d. 0.09 ±0.00 0.07 ±0.00 20.7 ±0.30 23.6 ±0.50 0.6 ±0.02 19 Menthone n.d. n.d. 29.8 ±0.50 n.d. n.d. n.d. n.d. 20 Menthofuran 0.002 ±0.00 n.d. 1.9 ±0.30 n.d. 0.004 ±0.00 n.d. n.d. 21 Isomenthone 0.002 ±0.00 0.002 ±0.00 4.2 ±0.20 n.d. 0.01 ±0.00 n.d. n.d. 22 Isomenthol n.d. n.d. 3.6 ±0.10 n.d. n.d. n.d. n.d. 23 Borneol n.d. 0.004 ±0.00 n.d. 0.3 ±0.04 0.9 ±0.10 2.0 ±0.20 1.3 ±0.10 24 Menthol n.d. n.d. 39.7 ±0.30 n.d. n.d. n.d. n.d. 25 Terpinen-4-ol n.d. 0.02 ±0.00 n.d. 0.3 ±0.00 0.4 ±0.01 0.4 ±0.02 1.0 ±0.1 26 α-terpineol n.d. 0.04 ±0.00 0.2 ±0.00 n.d. 1.0 ±0.06 0.4 ±0.01 0.1 ±0.01 27 Estragole n.d. n.d. n.d. n.d. n.d. n.d. 0.01 ±0.00 28 Verbenone 0.006 ±0.00 n.d. n.d. n.d. 0.3 ±0.00 n.d. 0.01 ±0.00 29 Methyl thymyl ether n.d. n.d. n.d. n.d. n.d. n.d. 0.1 ±0.02 30 Pulegone n.d. n.d. 0.8 ±0.10 n.d. n.d. n.d. n.d. 31 Linalyl anthranilate n.d. n.d. n.d. n.d. n.d. 2.2 ±0.30 n.d. 32 Piperitone n.d. n.d. 0.5 ±0.03 n.d. n.d. n.d. n.d. 33 Neomenthol acetate 0.003 ±0.00 n.d. 0.3 ±0.01 n.d. n.d. n.d. n.d. 34 Cinnamaldehyde 91.9 ±1.70 n.d. n.d. n.d. n.d. n.d. n.d. 35 Bornyl acetate n.d. n.d. n.d. n.d. 0.8 ±0.02 0.8 ±0.01 n.d. 36 Myrtenyl acetate n.d. n.d. n.d. n.d. n.d. 1.1 ±0.02 n.d. Biology 2021,10, 1091 7 of 21 Table 2. Cont. Compound Cinnamomum cassia Cinnamomum zeylanicum Mentha piperita Origanum vulgare Rosmarinus officinalis Salvia lavandulifolia Thymus vulgaris 37 Menthyl acetate n.d. n.d. 6.1 ±0.70 n.d. n.d. n.d. n.d. 38 β-isosafrole n.d. 0.1 ±0.00 n.d. n.d. n.d. n.d. n.d. 39 Thymol n.d. n.d. n.d. 1.6 ±0.20 n.d. n.d. 61.2 ±1.10 40 Carvacrol n.d. n.d. n.d. 84.5 ±1.60 n.d. n.d. 2.6 ±0.5 41 Terpinyl acetate n.d. n.d. n.d. n.d. n.d. 0.6 ±0.02 0.06 ±0.00 42 Eugenol 4.2 ±0.30 95.2 ±1.40 n.d. n.d. n.d. n.d. n.d. 43 β-caryophyllene 0.5 ±0.02 1.6 ±0.20 2.7 ±0.10 1.6 ±0.10 2.2 ±0.20 0.7 ±0.10 1.9 ±0.20 44 α-caryophyllene 0.03 ±0.00 0.3 ±0.04 0.2 ±0.00 0.1 ±0.00 0.7 ±0.08 0.4 ±0.03 0.05 ±0.00 45 β-eudesmene n.d. n.d. 0.08 ±0.00 n.d. n.d. n.d. 0.07 ±0.00 46 Eremophilane n.d. n.d. 0.03 ±0.00 n.d. n.d. 0.2 ±0.01 0.01 ±0.00 47 Bisabolene n.d. n.d. n.d. 0.12 n.d. n.d. 0.07 ±0.00 48 Isoeugenol n.d. 0.4 ±0.10 0.2 ±0.00 n.d. 0.3 ±0.03 0.1 ±0.00 0.07 ±0.00 49 2-(2-propenyl)-furan n.d. 0.007 ±0.00 n.d. n.d. n.d. n.d. n.d. 50 Benzyl benzoate n.d. 1.0 ±0.30 n.d. n.d. n.d. n.d. n.d. Monoterpenes 3.4 1.3 39.9 11.4 58.2 28.9 30.8 Oxygenated monoterpenes n.d. n.d. 5.0 0.1 35.5 62.2 0.6 Alcohols n.d. 0.1 43.6 0.5 2.3 2.8 2.4 Ethers n.d. n.d. n.d. n.d. n.d. n.d. 0.1 Esthers n.d. 1.0 6.4 n.d. 0.8 2.5 0.1 Sesquiterpene hydrocarbons 0.5 1.8 3.1 1.8 2.9 1.3 2.1 Aldehydes 91.9 n.d. n.d. n.d. n.d. n.d. n.d. Phenols 4.2 95.7 0.2 86.1 0.3 0.1 63.9 Others n.d. 0.2 1.9 n.d. n.d. 2.2 n.d. n.d. = not detected. Table 3. Composition (expressed as %) of fruit and vegetable EOs determined by GC-MS. Compound Apium graveolens Citrus limon Citrus paradisi Citrus reticulata Citrus sinensis Foeniculum vulgare 1 Tricyclene n.d. n.d. n.d. 0.003 ±0.00 n.d. 0.004 ±0.00 2 Thujene 0.009 ±0.00 0.1 ±0.02 n.d. 0.01 ±0.00 n.d. n.d. Biology 2021,10, 1091 8 of 21 Table 3. Cont. Compound Apium graveolens Citrus limon Citrus paradisi Citrus reticulata Citrus sinensis Foeniculum vulgare 3α-pinene 0.3 ±0.04 0.8 ±0.05 0.1 ±0.00 0.2 ±0.02 0.2 ±0.03 1.2 ±0.10 4 Camphene n.d. 0.03 ±0.00 n.d. n.d. n.d. 0.01 ±0.00 5 Sabinene 0.06 ±0.00 0.8 ±0.10 0.2 ±0.01 0.2 ±0.00 0.2 ±0.00 0.03 ±0.00 6β-pinene 2.4 ±0.20 6.0 ±0.30 0.08 ±0.00 0.05 ±0.00 0.05 ±0.00 0.02 ±0.00 7β-myrcene 0.4 ±0.02 0.4 ±0.10 0.3 ±0.03 0.4 ±0.02 0.4 ±0.01 0.01 ±0.00 8α-terpinene n.d. 0.08 ±0.00 n.d. n.d. n.d. 0.003 ±0.00 9 Cymene 0.06 ±0.00 0.1 ±0.02 0.02 ±0.00 0.03 ±0.00 0.02 ±0.00 0.01 ±0.00 10 D-limonene 71.4 ±1.50 87.0 ±1.20 99.0 ±1.60 99.0 ±1.10 99.2 ±1.50 1.2 ±0.40 11 1,8-cineole (Eucalyptol) n.d. n.d. n.d. n.d. n.d. 0.01 ±0.00 12 γ-terpinene n.d. 3.8 ±0.07 0.04 ±0.00 0.06 ±0.00 n.d. 0.01 ±0.00 13 Terpinolene n.d. 0.1 ±0.02 n.d. n.d. n.d. 0.03 ±0.00 14 Fenchone 0.003 ±0.00 n.d. n.d. 0.002 ±0.00 n.d. 0.3 ±0.01 15 Linalool n.d. n.d. n.d. n.d. n.d. 0.001 ±0.00 16 Pinone 0.01 ±0.00 0.001 ±0.00 n.d. 0.002 ±0.00 n.d. 0.0005 ±0.00 17 Camphor n.d. n.d. n.d. n.d. 0.005 ±0.00 0.003 ±0.00 18 5-undecen-3-yne, (E)- 1.2 ±0.30 n.d. n.d. n.d. n.d. n.d. 19 Menthofuran n.d. n.d. 0.001 ±0.00 0.002 ±0.00 0.002 ±0.00 0.001 ±0.00 20 Isomenthone 0.002 ±0.00 0.008 ±0.00 0.001 ±0.00 n.d. n.d. n.d. 21 Isomenthol n.d. n.d. 0.01 ±0.00 n.d. n.d. 0.001 ±0.00 22 Borneol n.d. 0.004 ±0.00 n.d. n.d. n.d. n.d. 23 Menthol 0.01 ±0.00 n.d. n.d. n.d. n.d. n.d. 24 Terpinen-4-ol n.d. 0.04 ±0.00 n.d. n.d. 0.005 ±0.00 0.001 ±0.00 25 α-terpineol n.d. 0.05 ±0.01 0.06 ±0.01 0.01 ±0.00 n.d. 0.002 ±0.00 26 Estragole 0.01 ±0.00 n.d. n.d. n.d. n.d. 0.3 ±0.02 27 Verbenone n.d. n.d. n.d. n.d. 0.003 ±0.00 0.002 ±0.00 28 Anethol n.d. n.d. n.d. n.d. n.d. 96.8 ±1.80 29 β-caryophyllene 0.4 ±0.02 0.1 ±0.00 0.1 ±0.01 n.d. n.d. n.d. 30 Bergamottin n.d. 0.2 ±0.01 n.d. n.d. n.d. n.d. 31 α-caryophyllene 0.04 ±0.00 n.d. n.d. n.d. n.d. n.d. 32 β-eudesmene 9.8 ±0.20 n.d. n.d. n.d. n.d. n.d. 33 Eremophilane 1.2 ±0.04 n.d. n.d. n.d. n.d. n.d. 34 Bisabolene n.d. 0.2 ±0.00 n.d. n.d. n.d. n.d. 35 Isoeugenol 0.05 ±0.00 n.d. 0.03 ±0.00 n.d. n.d. n.d. 36 1-(2,4-Dimethylphenyl)propan-1-one 2.5 ±0.30 n.d. n.d. n.d. n.d. n.d. Biology 2021,10, 1091 9 of 21 Table 3. Cont. Compound Apium graveolens Citrus limon Citrus paradisi Citrus reticulata Citrus sinensis Foeniculum vulgare 37 Allyl phenoxyacetate 8.2 ±0.40 n.d. n.d. n.d. n.d. n.d. 38 2-(2-propenyl)-furan 1.9 ±0.20 n.d. n.d. n.d. n.d. n.d. Monoterpenes 74.6 99.4 99.8 100 100 2.6 Oxygenated monoterpenes n.d. n.d. n.d. n.d. n.d. 0.4 Alcohols n.d. 0.1 0.1 n.d. n.d. n.d. Phenylpropanoids n.d. n.d. n.d. n.d. n.d. 97.0 Esthers 8.2 n.d. n.d. n.d. n.d. n.d. Sesquiterpene hydrocarbons 11.5 0.4 0.1 n.d. n.d. n.d. Phenols 0.1 n.d. n.d. n.d. n.d. n.d. Others 5.6 0.2 n.d. n.d. n.d. n.d. n.d. = not detected. Biology 2021,10, 1091 16 of 21 growth of B. cinerea from 72 h of contact with the EOs, whereas thyme EO achieved the same from 120 h [ 83 ]. According to our results, Palfi et al. [ 84 ] reported inhibition of the mycelial growth of B. cinerea in the presence of thyme, fennel, peppermint, rosemary, and sage essential oils, whereas lemon oil lacked inhibitory activity. Table 7. Percentage of mycelium inhibition of B. cinerea and minimal inhibitory concentration (MIC) of essential oils. Essential Oils Mycelium Inhibition (%) MIC (mg/mL) Thymus vulgaris 98.2 1.125 Origanum vulgare 98.5 0.56 Rosmarinus officinalis 79.0 9 Apium graveolens 48.8 9 Salvia lavandulifolia 79.8 2.25 Cinnamomum zeylanicum 70.4 1.125 Cinnamomum cassia 81.5 0.14 Citrus sinensis 3.4 9 Citrus reticulata 3.6 9 Citrus limon 9.7 4.5 Citrus paradise 18.4 9 Foeniculum vulgare 93.1 1.125 Mentha piperita 93.8 0.56 In addition to the dose-dependent effect observed in the MIC assay, some EOs inhibited the fungus by more than 90% at concentrations much lower than their minimum inhibitory concentration. Such is the case of the fennel EO that inhibited 95% of the fungus at a concentration as low as 0.07 mg/mL and thyme EO that inhibited the mycelia growth in 97.6% at 0.14 mg/mL, a concentration 8 times lower than its MIC value (Figure 2). Similarly, only 0.56 mg/mL of the celery EO inhibited 90.2% of mycelia growth, 16 times lower than its MIC value. This result is comparable with those of other researchers, such as the case of Grul’ováet al. [ 85 ] that reported complete inhibition of B. cinerea with 500 ppm oregano EO, and more than 80% with 100 ppm of oil incorporated into Potato Dextrose Agar. Biology 2021, 10, x FOR PEER REVIEW 15 of 20 ppm oregano EO, and more than 80% with 100 ppm of oil incorporated into Potato Dextrose Agar . Figure 2. Inhibition of B. cinerea mycelium growth by thyme EO at 0.56 mg/mL (A), 0.28 mg/mL (B), 0.14 mg/mL (C), 0.07 mg/mL (D); and fennel EO at 0.28 mg/mL (E), 0.14 mg/mL (F), and 0.07 mg/mL (G). Control of mycelium growth (H) after 7 days of incubation at 25 °C. B. cinerea is a phytopathogen that causes the grey mould disease in more than 200 crop species worldwide such as grapes, cucumbers, tomatoes, strawberries, and leading to vast economic losses due to the severe damage in pre-and post-harvest [86,87]. Control strategies are carried out including chemical control, resistance induction and biological control. Benzimidazoles, dicarboximides, phenylpyrroles, aromatic hydrocarbons, and phenylcarbamates are the main chemical fungicides used to control it [88]. In addition to the toxicological risk presented by their residues, B. cinerea has developed resistance to most of these substances [89–91]. Essential oils have a significant interest as an alternative to chemical treatments as they are bio-sourced products and therefore more ecological [92]. Essential oils can act on fungus via inhibition of sporulation or producing cell damage [93]. Hydrophobic character enables EOs to break through lipids of cell membranes and mitochondria increasing fungal membranes permeability [92]. The changes in the fluidity may leak electrolytes or cellular contents resulting in protein metabolism alteration and calcium ion concentration [93]. Moreover, permeabilization of out and inner mitochondrial membrane leads to cell death by apoptosis and necrosis [94]. Although most of the antimicrobial activities of essential oils have been attributed to their major components, the total antimicrobial effect is the result of the synergism of all their components [95]. Therefore, the antimicrobial activity is not related to a single mechanism of action, as the essential oils have different bioactive compounds, and each one of them has different structural groups in their composition [96]. 4. Conclusions EOs from herbs and spices showed the highest antioxidant activity. Essential oils obtained from aromatic plants showed higher antibacterial and antifungal activity than those obtained from citric fruits. The most effective essential oils were those of C. cassia, T. vulgaris, and O. vulgare. Although fennel essential oil reported the lowest antioxidant activity, it showed very good antimicrobial activity against B. cinerea, one of the postharvest pathogen microorganisms in fruits and vegetables, thus it could be considered in active packaging production or food coating. The strong antimicrobial activity of essential Figure 2. Inhibition of B. cinerea mycelium growth by thyme EO at 0.56 mg/mL ( A ), 0.28 mg/mL ( B ), 0.14 mg/mL (C), 0.07 mg/mL ( D ); and fennel EO at 0.28 mg/mL ( E ), 0.14 mg/mL ( F ), and 0.07 mg/mL ( G ). Control of mycelium growth (H) after 7 days of incubation at 25 ◦C. Biology 2021,10, 1091 17 of 21 B. cinerea is a phytopathogen that causes the grey mould disease in more than 200 crop species worldwide such as grapes, cucumbers, tomatoes, strawberries, and leading to vast economic losses due to the severe damage in pre-and post-harvest [ 86 , 87 ]. Control strategies are carried out including chemical control, resistance induction and biological control. Benzimidazoles, dicarboximides, phenylpyrroles, aromatic hydrocarbons, and phenylcarbamates are the main chemical fungicides used to control it [ 88 ]. In addition to the toxicological risk presented by their residues, B. cinerea has developed resistance to most of these substances [ 89 – 91 ]. Essential oils have a significant interest as an alternative to chemical treatments as they are bio-sourced products and therefore more ecological [ 92 ]. Essential oils can act on fungus via inhibition of sporulation or producing cell damage [ 93 ]. Hydrophobic character enables EOs to break through lipids of cell membranes and mitochondria increasing fungal membranes permeability [ 92 ]. The changes in the fluidity may leak electrolytes or cellular contents resulting in protein metabolism alteration and calcium ion concentration [ 93 ]. Moreover, permeabilization of out and inner mitochondrial membrane leads to cell death by apoptosis and necrosis [94]. Although most of the antimicrobial activities of essential oils have been attributed to their major components, the total antimicrobial effect is the result of the synergism of all their components [ 95 ]. Therefore, the antimicrobial activity is not related to a single mechanism of action, as the essential oils have different bioactive compounds, and each one of them has different structural groups in their composition [96]. 4. Conclusions EOs from herbs and spices showed the highest antioxidant activity. Essential oils obtained from aromatic plants showed higher antibacterial and antifungal activity than those obtained from citric fruits. The most effective essential oils were those of C. cassia, T. vulgaris, and O. vulgare. Although fennel essential oil reported the lowest antioxidant activity, it showed very good antimicrobial activity against B. cinerea, one of the postharvest pathogen microorganisms in fruits and vegetables, thus it could be considered in active packaging production or food coating. The strong antimicrobial activity of essential oils and the broad spectrum they showed provide evidence that they may be used for prolonging the shelf life of food products, developing functional foods, and for protecting plants and crops. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biology10111091/s1, Figure S1: GS-MS chromatograms of fruit and vegetables, and aromatic herbs and spices essential oils. Author Contributions: Conceptualization, V.V. and A.M.G.-C.; formal analysis, M.d.C.R.-D., S.D.-M.-P. and V.V.; investigation, M.d.C.R.-D., S.D.-M.-P. and V.V.; data curation, E.J.G.-H., M.J.-V. and A.R.-B.; writing—original draft preparation, M.d.C.R.-D., S.D.-M.-P. and V.V.; writing—review and editing, M.J.-V., A.R.-B., B.G.-V. and V.V.; supervision, V.V.; and funding acquisition, V.V. ands A.M.G.-C. All authors have read and agreed to the published version of the manuscript. Funding: This study is part of the project SHEALTHY that has received funding from European Union’s Horizon 2020 research and innovation programme under grant agreement No 817936. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: Vito Verardo thanks the Spanish Ministry of Economy and Competitiveness (MINECO) for “Ramon y Cajal” contract (RYC-2015-18795). 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