[['Article', 'Comparative Analysis of the Composition and Active', 'Property Evaluation of Certain Essential Oils to', 'Assess their Potential Applications in Active', 'Food Packaging'], ['Cornelia Vasile 1,*, Morten Sivertsvik 2,*, Amalia Carmen Mitelu¸t 3, Mihai Adrian Brebu 1,', 'Elena Stoleru 1, Jan Thomas Rosnes 2, Elisabeta Elena T˘anase 3, Waqas Khan 4, Daniela Pamﬁl 1,', 'C˘alina Petru¸ta Cornea 3, Anamaria Irimia 1 and Mona Elena Popa 3', '1'], ['Physical Chemistry of Polymers Department, “Petru Poni” Institute of Macromolecular Chemistry', 'Romanian Academy, 41A, Gr. Ghica Voda Alley, Iasi 700487, Romania; bmihai@icmpp.ro (M.A.B.);', 'elena.paslaru@icmpp.ro (E.S.); pamﬁl.daniela@icmpp.ro (D.P.); anamaria.sdrobis@icmpp.ro (A.I.)'], ['2 Noﬁma AS, Department of Processing Technology, Muninbakken 9-13, Tromsø 9291, Norway;'], ['3'], ['thomas.rosnes@noﬁma.no', 'Faculty of Biotechnology, University of Agronomic Sciences and Veterinary Medicine of Bucharest,', '59 M˘ar˘a¸sti Blvd, District 1, Bucharest 011464, Romania; amaliamitelut@yahoo.com (A.C.M.);', 'elena.eli.tanase@gmail.com (E.E.T.); pccornea@yahoo.com (C.P.C.); pandry2002@yahoo.com (M.E.P.)'], ['4 Department of Biological Chemistry, University of Stavanger, Stavanger 4036, Norway;'], ['khanwaqas2006@gmail.com'], ['* Correspondence: cvasile@icmpp.ro (C.V.); Morten.Sivertsvik@noﬁma.no (M.S.);'], ['Tel.: +40-232-217-454 (C.V.); +47-5184-4637 or +47-9059-7998 (M.S.); Fax: +40-232-211-299 (C.V.)'], ['Academic Editor: Jung Ho Je', 'Received: 28 October 2016; Accepted: 3 January 2017; Published: 7 January 2017'], ['Abstract: The antifungal, antibacterial, and antioxidant activity of four commercial essential oils', '(EOs) (thyme, clove, rosemary, and tea tree) from Romanian production were studied in order', 'to assess them as bioactive compounds for active food packaging applications. The chemical', 'composition of the oils was determined with the Folin–Ciocâlteu method and gas chromatography', 'coupled with mass spectrometry and ﬂame ionization detectors, and it was found that they', 'respect the AFNOR/ISO standard limits. The EOs were tested against three food spoilage fungi—', 'Fusarium graminearum, Penicillium corylophilum, and Aspergillus brasiliensis—and three potential', 'pathogenic food bacteria—Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes—using', 'the disc diffusion method. It was found that the EOs of thyme, clove, and tea tree can be used', 'as antimicrobial agents against the tested fungi and bacteria, thyme having the highest inhibitory', 'effect. Concerning antioxidant activity determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and', '2,2’-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) methods, it has been established that', 'the clove oil exhibits the highest activity because of its high phenolic content. Promising results', 'were obtained by their incorporation into chitosan emulsions and ﬁlms, which show potential for', 'food packaging. Therefore, these essential oils could be suitable alternatives to chemical additives,', 'satisfying the consumer demand for naturally preserved food products ensuring its safety.'], ['Keywords: essential oils; antifungal; antimicrobial; antioxidant; spoilage fungi'], ['1. Introduction'], ['Consumers demand high quality foods with minimal changes in nutritional properties. A minimal', 'amount of synthetic additives combined with a suitable packaging technology that retains or creates', 'desirable food qualities or reduces undesirable changes in food due to microbial activity is therefore a'], ['Materials 2017, 10, 45; doi:10.3390/ma10010045'], ['www.mdpi.com/journal/materials'], ['materials\x0cMaterials 2017, 10, 45'], ['2 of 24'], ['goal of food manufacturers [1]. New processes must be designed to meet the required food product', 'safety or shelf-life demands, and additional hurdles for microorganisms should be introduced. World', 'Health Organization (WHO) reports [2] in recent years estimate that 30% of people in industrialized', 'countries suffer from a food-borne disease each year. Reducing or eliminating food-borne pathogens', 'via “green” consumerism concomitantly with low salt consumption to diminish the incidence of', 'cardiovascular diseases is increasingly becoming a public health concern. On the other hand,', 'antimicrobial resistance affects all areas of health, as many medicinal procedures are related to', 'antibiotics [3].'], ['Therefore, new methods and additives should be found to prolong service life and to improve the', 'safety of foods. Recently, effective preventive measures and intelligent preservation methods have', 'been put into place to reduce food spoilage, increase safety, and prolong food shelf-life. One of these', 'methods is bioactive packaging by the use of natural compounds with multifunctional properties both', 'to achieve the protection of food and to improve the health of consumers. The concern regarding', 'safety issues of the synthetic antimicrobial agents has led to the use of essential oils (EOs), which', 'represent eco-friendly alternatives to chemicals. Essential oils (also called volatile oils) are oily liquids', 'obtained from plant materials (ﬂowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, and', 'roots). Plant-derived essential oils are complex mixtures of natural volatile compounds resulting', 'from the plant secondary metabolism and extracted from vegetable materials by expression (i.e.,', '“cold pressing”), fermentation, enﬂeurage, or extraction, but the method of distillation with water', 'or steam is the most commonly used for the commercial production of EOs. Essential oils contain', 'important classes of compounds such as monoterpenes (C10 hydrocarbons based on 2 isoprene units),', 'phenylpropanoides (C6 aromatic compounds with C3 side chains), sesquiterpenes (C15 hydrocarbons', 'based on 3 isoprene units), diterpenes (C20), triterpenes (C30) and their oxygenated derivatives, and', 'phenolic compounds (such as thymol and carvacrol). Due to their versatile content, essential oils', 'constitute a rich source of biologically active compounds possessing antimicrobial, antibacterial,', 'antifungal, antioxidant, antiviral, antimycotic, antitoxigenic, antiparasitic, antibiotic, and antiseptic', 'properties and insecticidal activities; therefore, they are useful in a wide range of applications [4,5].'], ['Each of the above-mentioned constituents contributes to beneﬁcial or adverse effects; therefore,', 'it is very important to know as much as possible about the composition and properties of EOs, with', 'each study bringing new highlights on the advantages and disadvantages they offer.'], ['There are many studies on the characterization of volatile compound composition and the', 'antimicrobial and antioxidant activities of various selected groups of essential oils. Bozin et al.', 'characterized Lamiaceae species and the antimicrobial and antioxidant activities of the oils', 'of Ocimum basilicum L., Origanum vulgare L., and Thymus vulgaris L. [6], and the chemical', 'constituents of four populations of Piper aduncum L. from Distrito Federal, Brazil [7], were identiﬁed.', 'Many other essential oils have been characterized, namely, sweet lime (Citrus limetta Risso) [8],', 'Chenopodium ambrosioides, Philodendron bipinnatiﬁdum [9], cinnamon oil, eucalyptus oil, lemongrass', 'oil, peppermint oil, citronella oil, turpentine oil [10], citronella oil [11], Tithonia diversifolia (Hemsl.)', 'A. Gray [12], O. basilicum L. from Italy [13], and Iranian geranium oil [14]. The composition of two', 'species of mint (Mentha suaveolens Ehrh. and Mentha rotundifolia) grown in Or˘a¸stie-Romania has', 'been comparatively examinated [15]. From the 21 plant essential oils (cinnamon, clove, geranium,', 'lemon, lime, orange and rosemary, aniseed, eucalyptus, and camphor) tested against six bacterial', 'species four Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and', 'Proteus vulgaris) and two Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus [16], 19 oils', 'showed antibacterial activity.'], ['Consumers are worried about the presence of chemical preservatives, which can lead to benzoic', 'acid by the decarboxylating action of some spoilage microorganisms, and this is considered the cause', 'of many carcinogenic and teratogenic attributes and residual toxicity. Therefore, the studies to ﬁnd', 'natural and socially acceptable preservatives receive increasing attention by screening the composition', 'and the biological, antimicrobial, and antioxidant activities of plant extracts [17]. Essential oils can'], ['\x0cMaterials 2017, 10, 45'], ['3 of 24'], ['prevent fungal growth in food products, which may cause spoilage and result in a reduction in the', 'quality and quantity of food (shelf-life). Most EOs applied directly onto food or in the vapor phase', 'can reduce or stop the colony forming ability of molds. They are also regarded as safe (GRAS) and', 'are accepted by the FDA and by consumers. By their potential antimicrobial/antifungal/antioxidant', 'effects, EOs could be the answer to the current search for environmental solutions and to assuring the', 'microbial safety of food products in active packaging applications [18,19].'], ['In this study, four essential oils, namely, thyme (Thymus vulgaris L.), clove (Eugenia caryophyllus', 'from dried ﬂoral buds of Syzygium aromaticum), rosemary (Rosmarinus ofﬁcinalis L.), and tea tree', '(Melaleuca alternifolia aetheroleum) obtained from Romanian production (Fares Co., Or˘a¸stie, Romania)', 'are intent to be used as components in bioactive food packaging. Therefore, as a ﬁrst step, it should', 'be worthwhile to comparatively evaluate the composition and antimicrobial/antioxidant activity', 'of these four EOs. Antifungal and antibacterial activity against three target food spoilage fungi', '(Aspergillus brasiliensis, Fusarium graminearum, and Penicillium corylophilum) and three potential', 'pathogenic food bacteria (S. aureus, E. coli, and L. monocytogenes) have been evaluated. The minimum', 'inhibitory concentration (MIC) concentration was established in each case. The antioxidant activity', 'was determined, and the most efﬁcient oil for each type of activity was established. Preliminary tests', 'on the EO encapsulation into chitosan ﬁlms and their antimicrobial activity against the spoilage of beef', 'meat showed promising results, a detailed presentation for which will be provided in a future paper.'], ['2. Results and Discussion'], ['2.1. Chemical Composition'], ['2.1.1. Phenolic Content'], ['The phenolic content of extracts of many plants contributes signiﬁcantly to their total antioxidant', 'activity. The antioxidant feature of the investigated essential oils was determined by the phenolic', 'compouds presence in their composition by the Folin–Ciocâlteu method [20].'], ['The total concentration of phenolic compounds found in essential oil samples are presented', 'in Table 1, and a decrease in phenolic content concentration in the following order was found:', 'clove > thyme > tea tree > rosemary. Excepting the rosemary oil, all oils were found to have different', 'phenolic levels, ranging from 0.034 to 1.136 mg·GAE/g·DW, which can play a vital role in the increase', 'of food shelf-life. The clove oil has the highest content of phenolic compounds.'], ['Oil Type', 'Thyme', 'Clove'], ['Table 1. Total phenolic content in the investigated essential oils.', 'Total Phenolic Content (mg·GAE/g·DW) *'], ['Rosemary', 'Tea Tree', '* mg·GAE/g·DW (mg of gallic acid equivalent per g dry weight).'], ['0.349', '1.136', '0.000', '0.034'], ['2.1.2. GC-MSD and GC-FID Analysis'], ['The composition (including both main components and those in low amount but with signiﬁcant', 'biological/therapeutic effects) of the essential oils varies depending on the geographical position,', 'the plant’s origin and species, harvest time, distillation/extraction procedure, etc. [17]. Additionally,', 'the composition of the essential oils and consequently their biological/therapeutic activities depend', 'on the combination and ratio of their numerous different components. Gas chromatography coupled', 'with mass spectrometry (GC-MSD) and ﬂame ionization (GC-FID) detectors was used to determine', 'the quality and quantity of chemical compounds in the essential oils.'], ['The chromatograms of studied essential oils are shown in Figures 1–4.'], ['\x0cMaterials 2017, 10, 45'], ['4 of 24'], ['The essential thyme oil contains especially thymol, p-cymene, γ-terpinene, linalol, isothymol'], ['(carvacrol/biosol), and β-myrcene—Figure 1.'], ['Figure 1. The GC-MSD and GC-FID (insert) chromatograms of thyme oil.'], ['The clove essential oil had the simplest composition, based on eugenol/eugenol acetate and'], ['β-/α-caryophyllene, accompanied by several other sesquiterpenes—Figure 2.'], ['Figure 2. The GC-MSD and GC-FID (insert) chromatograms of clove oil.'], ['Materials 2017, 10, 45 4 of 23 The essential thyme oil contains especially thymol, p-cymene, γ-terpinene, linalol, isothymol (carvacrol/biosol), and β-myrcene—Figure 1.  Figure 1. The GC-MSD and GC-FID (insert) chromatograms of thyme oil. The clove essential oil had the simplest composition, based on eugenol/eugenol acetate and β-/α-caryophyllene, accompanied by several other sesquiterpenes—Figure 2.  Figure 2. The GC-MSD and GC-FID (insert) chromatograms of clove oil. Materials 2017, 10, 45 4 of 23 The essential thyme oil contains especially thymol, p-cymene, γ-terpinene, linalol, isothymol (carvacrol/biosol), and β-myrcene—Figure 1.  Figure 1. The GC-MSD and GC-FID (insert) chromatograms of thyme oil. The clove essential oil had the simplest composition, based on eugenol/eugenol acetate and β-/α-caryophyllene, accompanied by several other sesquiterpenes—Figure 2.  Figure 2. The GC-MSD and GC-FID (insert) chromatograms of clove oil. \x0cMaterials 2017, 10, 45'], ['5 of 24'], ['The rosemary oil is rich in light monoterpenes, containing mainly eucalyptol, camphor,'], ['α-/β-pinene, camphene, borneol, and limonene—Figure 3.'], ['Figure 3. The GC-MSD and GC-FID (insert) chromatograms of rosemary oil.'], ['The tea tree essential oil contained both light monoterpenes and numerous sesquiterpenes.', 'The main compound is 4-terpineol, followed by γ-terpinene, 2-carene, α-terpineol, α-terpinene,', 'α-pinene, o-cymene, limonene, β-caryophyllene, eucalyptol, and β-myrcene—Figure 4. Standards ask', 'for α-terpinene (5%–13%), which was not found in our tee tree sample, instead 2-carene was found in', 'high amounts of about 10%. Aromadendrene and δ-cadinene, mentioned by standards in amounts', 'varying from traces up to 7%–8% were conﬁrmed in the studied sample.'], ['Figure 4. The GC-MSD and GC-FID (insert) chromatograms of tea tree oil.'], ['Materials 2017, 10, 45 5 of 23 The rosemary oil is rich in light monoterpenes, containing mainly eucalyptol, camphor, α-/β-pinene, camphene, borneol, and limonene—Figure 3.  Figure 3. The GC-MSD and GC-FID (insert) chromatograms of rosemary oil. The tea tree essential oil contained both light monoterpenes and numerous sesquiterpenes. The main compound is 4-terpineol, followed by γ-terpinene, 2-carene, α-terpineol, α-terpinene, α-pinene, o-cymene, limonene, β-caryophyllene, eucalyptol, and β-myrcene—Figure 4. Standards ask for α-terpinene (5%–13%), which was not found in our tee tree sample, instead 2-carene was found in high amounts of about 10%. Aromadendrene and δ-cadinene, mentioned by standards in amounts varying from traces up to 7%–8% were confirmed in the studied sample.  Figure 4. The GC-MSD and GC-FID (insert) chromatograms of tea tree oil. Materials 2017, 10, 45 5 of 23 The rosemary oil is rich in light monoterpenes, containing mainly eucalyptol, camphor, α-/β-pinene, camphene, borneol, and limonene—Figure 3.  Figure 3. The GC-MSD and GC-FID (insert) chromatograms of rosemary oil. The tea tree essential oil contained both light monoterpenes and numerous sesquiterpenes. The main compound is 4-terpineol, followed by γ-terpinene, 2-carene, α-terpineol, α-terpinene, α-pinene, o-cymene, limonene, β-caryophyllene, eucalyptol, and β-myrcene—Figure 4. Standards ask for α-terpinene (5%–13%), which was not found in our tee tree sample, instead 2-carene was found in high amounts of about 10%. Aromadendrene and δ-cadinene, mentioned by standards in amounts varying from traces up to 7%–8% were confirmed in the studied sample.  Figure 4. The GC-MSD and GC-FID (insert) chromatograms of tea tree oil. \x0cMaterials 2017, 10, 45'], ['6 of 24'], ['The main classes of compounds identiﬁed in the studied essential oils are presented in Table 2.', 'Under the mentioned analysis parameters, only volatile compounds up to the level of sesquiterpenes', 'were detected.'], ['Table 2. The main classes of compounds found in the studied essential oils.'], ['Class of Compounds'], ['Monoterpenes'], ['(C10 with 2 isoprene units)'], ['Phenylpropanoides'], ['(C6 with C3 side chains)'], ['Sesquiterpenes'], ['(C15 with 3 isoprene units)'], ['Main Compounds'], ['Borneol, Camphene, Camphor, Carene, Carvol, Citral, p-Cymene,', 'Eucalyptol, Fenchone, Geraniol, Limonene, Linalol, Menthone, Myrcene,', 'Ocimene, Phellandrene, Piperitol, Terpinene, Thujene, Thymol'], ['Apiole, Chavicol, Cinnamaldehyde, Estragole, Eugenol, Myristicin'], ['Amorphene, Aromoadendrene, Cadinene, Caryophyllene, Cubebene,', 'Elemene, Farnesene, Globulol, Gurgujene, Isolendene, Maaliene,', 'Panasinsen, Selinene, Spathulenol'], ['Table 3 shows the quantitative composition (based on Gas Chromatographz with Flame Ioniyation', 'Detector (GC-FID) of the analyzed essential oils. The GC-FID analysis was performed on several', 'different samples obtained from the same company, i.e., Fares, Or˘a¸stie-Romania, and no relevant', 'difference was found between them. The results are reproducible.'], ['The composition of the four essential oils is within the limits of AFNOR (Association French', 'Normalization Organization Regulation)/ISO standards [21–25], except for a few differences, which', 'could be considered within the error limits of the analysis. Deviation in the composition from standard', 'values may not interfere with the therapeutic properties of the essential oils; however, if oils respect', 'the standard limits, it is safer to consider them usable according to the general practice.'], ['Table 3. The composition of the selected commercial essential oils from the Fares Company, Romania,', 'compared with AFNOR/ISO standards *.'], ['Chemical Compound', 'ORAC (µTE/100 g) **'], ['Thyme', '15,960'], ['Clove'], ['1,078,700'], ['α-Thujene', 'α-Pinene', 'Camphene', 'β-Pinene', 'β-Myrcene', '2-Carene', 'p-Cymene', 'Limonene', 'Eucalyptol', 'γ-Terpinene', 'α-Terpinene'], ['Linalol', 'Camphor', 'Borneol'], ['4-Terpineol', 'α-Terpineol'], ['Thymol', 'Carvacrol', 'Eugenol'], ['β-Caryophyllene', 'α-Caryophyllene', 'Aromadendrene'], ['Ledene'], ['Eugenol acetate'], ['δ-Cadinene'], ['Caryophyllene oxide'], ['0.4', '1.6', '1.0'], ['2.5', '2.3', '22.5'], ['7.9'], ['5.6'], ['1.1'], ['43.1', '2.7'], ['2.3'], ['85.7', '4.5', '0.9'], ['7.9 (>8)'], ['0.4'], ['Rosemary'], ['Tea Tree'], ['330'], ['11.4', '5.0'], ['9.4 (<9)'], ['1.3', '2.6', '43.1'], ['11.3', '3.0'], ['1.8'], ['3.2'], ['3.9'], ['2.5', '10.3', '2.8', '3.6', '2.3', '16.3'], ['4.1 (>5)'], ['38.7', '4.6'], ['2.2'], ['1.1', '1.1'], ['0.9'], ['* The bold numbers in Table 3 indicate values within the limits of the standards; italic numbers are below', 'the limits, while the underlined are over the limits, the bracket showing the closest limits in the standard.', '** ORAC—oxygen radical absorption capacity; expressed as µmol Trolox equivalent (TE) at 100 g, accuracy of', '±5%, as presented in the literature [26,27].'], ['\x0cMaterials 2017, 10, 45'], ['7 of 24'], ['The ORAC index shows the antioxidant capacity of the oils. According to databases, the antioxidant', 'capacity is highest for clove essential oil, followed at a high distance by thyme oil, while the antioxidant', 'capacity for tea tree is smaller than those of other essential oils. These statements are in accordance', 'with results obtained by the Folin–Ciocâlteu method.'], ['Based on the gas chromatography analysis coupled with mass spectrometry and ﬂame ionisation', 'detectors, the commercial essential oils of thyme (Thymus vulgaris L.), clove (Eugenia caryophyllus),', 'rosemary (Rosmarinus ofﬁcinalis L.), and tea tree (Melaleuca alternifolia aetheroleum) were found to have', 'compositions within the limits of the AFNOR/ISO standards.'], ['Most of the obtained results related to the composition of the studied essential oils are in', 'accordance with those found by other authors and constitute support for the explanation of', 'their potential biological activity spectrum [28–44]. Thyme essential oil (TEO) is obtained from', 'Thymus vulgaris L. and exhibits antimicrobial effects due to its constituents. Omidbeygi et al. [28] found', 'that the major compounds of TEO are thymol, carvacrol with similar chemical structures, linalool, and', 'ρ-cymene [28,29]. The presence of thymol (2-Isopropyl-5-methylphenol) and carvacrol enhanced the', 'TEO antimicrobial activity [30–32]. Clove essential oil (Caryophylli aetheroleum) (CEO) isolated from', 'the dry ﬂoral buds of Syzygium aromaticum, belonging to the Myrtaceae family has been used for its', 'antimicrobial activity. Goni [33], Shao [34], and Sebaaly [35] established that the CEO is composed', 'mainly of phenylpropanoides such as eugenol, β-caryophyllene, and the eugenyl acetate [33–36].'], ['Jiang et al. [37] found that the rosemary essential oil (Rosmarinus ofﬁcinalis L.) (REO) can be used', 'in the food industry as a ﬂavoring agent and preservative because of its antimicrobial and antioxidant', 'properties [37]. Among the terpenes found in the composition of REO, the main components are the', 'following: cineole, camphor, α-pinene, camphene, and α-terpineol [38,39].'], ['Sánchez-González et al. [33,40] established that the essential oil of Melaleuca alternifolia, also', 'known as tea tree essential oil (TTO) is composed of terpene, mainly monoterpenes, sesquiterpenes,', 'and tertiary alcohols [33,40–43]. The main components of TTO are terpineol, cineol, pinen, and terpinen', 'and demonstrated antimicrobial activity [40,44].'], ['2.2. Antifungal Activity of the Tested Essential Oils'], ['The antifungal activity of the essential oils against the three fungi strains examined was assessed', 'by the percentage of the inhibition rate (IR%) and minimum inhibitory concentration (MIC). In order', 'to evaluate the quantity of essential oils needed to be used, a preliminary investigation was carried out', 'with a volume of 10 µL and 20 µL of each of them. After this screening, the maximum concentration', 'used was 60 µL, while the minimum volume used was 2.5 µL. The results presented in Figures 5–7', 'for the antifungal activity of the essential oils tested are the ones from the 7th day of analyses. From', 'all tested oils, the most effective were the essential oils of clove, thyme, and tea tree. The efﬁcacy', 'of these essential oils can be attributed to the terpenes and phenylpropanoides present in their', 'composition. The research studies demonstrated that phenolic compounds are responsible for the', 'antifungal potential of these oils.'], ['2.2.1. Fusarium Graminearum G87'], ['Fusarium graminearum, being a plant pathogen, is a fungus commonly found in cereal grains', '(mostly wheat and barley). This pathogen has the potential to produce mycotoxines (deoxynivalenol', 'and zearalonene) that have a negative impact on human and animal health [45]. The contamination of', 'cereals by this toxigenic mold produces major economic impacts in agriculture.'], ['The essential oils selected were tested against Fusarium graminearum, and the results are presented'], ['in Figure 5.'], ['\x0cMaterials 2017, 10, 45'], ['8 of 24'], ['Figure 5. Inhibition of the Fusarium graminearum G87 growth by the thyme, clove, rosemary, and tea', 'tree essential oils.'], ['The fungal growth inhibition induced by essential oils, as determined by the disc diffusion assay,', 'was dependent mostly on the volume (varying from 5 µL to 60 µL) and nature of the essential oils.', 'According to the results presented in Figure 5, it can be noticed that the essential oils tested had', 'antifungal activity against Fusarium graminearum G87, but their efﬁcacy is different. Thyme essential', 'oil was the most effective, with an inhibition rate of over 80% at the lowest volume of essential oil used', 'for the growth inhibition of this fungus, namely, 87.04% for 7 µL. Clove oil was also effective against', 'the tested fungus, with an inhibition rate of 81.64% when using 40 µL. It is assumed that the high', 'antifungal potential of clove essential oil is due to its active principle, eugenol (85.7%). The results are', 'in accordance with the study of Abbaszadeh et al. [46] regarding the effectiveness of eugenol against', 'pathogenic fungi, which demonstrated that this phenylpropanoide had antifungal activity against all', 'tested fungi. The antifungal activity was in direct ratio with the concentration of eugenol added to', 'the media. In a study made by Marin et al. [47] who tested the efﬁcacy of cinnamon, clove, oregano,', 'palmarosa, and lemongrass oils against the mycotoxines produced by Fusarium graminearum, it was', 'shown that clove essential oils was the most efﬁcient against zearalenone and deoxynivalenol release.', 'Rosemary essential oil exhibits antifungal activity, but this was very low. For a volume of 60 µL', 'of this oil, the inhibition was below 10%. Tea tree essential oil had an inhibition rate higher than 80%', 'against F. graminearum G87 only for the highest volume used in this study (60 µL).'], ['2.2.2. Penicillium Corylophilum CBMF1'], ['Penicillium corylophilum is a fungus that may cause the spoilage of bakery products; for this', 'judgment, the essential oils selected were used to determine their antifungal activity against this mold.', 'tested essential oils against'], ['inhibition manifested by the'], ['Figure 6 shows'], ['the'], ['Penicillium corylophilum growth.'], ['Materials 2017, 10, 45 8 of 23 The essential oils selected were tested against Fusarium graminearum, and the results are presented in Figure 5.  Figure 5. Inhibition of the Fusarium graminearum G87 growth by the thyme, clove, rosemary, and tea tree essential oils. The fungal growth inhibition induced by essential oils, as determined by the disc diffusion assay, was dependent mostly on the volume (varying from 5 μL to 60 μL) and nature of the essential oils. According to the results presented in Figure 5, it can be noticed that the essential oils tested had antifungal activity against Fusarium graminearum G87, but their efficacy is different. Thyme essential oil was the most effective, with an inhibition rate of over 80% at the lowest volume of essential oil used for the growth inhibition of this fungus, namely, 87.04% for 7 μL. Clove oil was also effective against the tested fungus, with an inhibition rate of 81.64% when using 40 μL. It is assumed that the high antifungal potential of clove essential oil is due to its active principle, eugenol (85.7%). The results are in accordance with the study of Abbaszadeh et al. [46] regarding the effectiveness of eugenol against pathogenic fungi, which demonstrated that this phenylpropanoide had antifungal activity against all tested fungi. The antifungal activity was in direct ratio with the concentration of eugenol added to the media. In a study made by Marin et al. [47] who tested the efficacy of cinnamon, clove, oregano, palmarosa, and lemongrass oils against the mycotoxines produced by Fusarium graminearum, it was shown that clove essential oils was the most efficient against zearalenone and deoxynivalenol release. Rosemary essential oil exhibits antifungal activity, but this was very low. For a volume of 60 μL of this oil, the inhibition was below 10%. Tea tree essential oil had an inhibition rate higher than 80% against F. graminearum G87 only for the highest volume used in this study (60 μL). 2.2.2. Penicillium Corylophilum CBMF1 Penicillium corylophilum is a fungus that may cause the spoilage of bakery products; for this judgment, the essential oils selected were used to determine their antifungal activity against this mold. Figure 6 shows the inhibition manifested by the tested essential oils against  Penicillium corylophilum growth. \x0cMaterials 2017, 10, 45'], ['9 of 24'], ['Figure 6. Inhibition of the Penicillium corylophilum CBMF1 growth by the thyme, clove, rosemary, and', 'tea tree essential oils.'], ['The effectiveness of thyme oil as an antifungal agent can be noticed from the data presented', 'in Figure 6. As can be seen, thyme essential oil is more active than clove essential oil, showing', 'an inhibition rate of 100% at 10 µL, while clove oil was less effective (IR = 47.6%) at a volume of', '10 µL. As well, tea tree essential oil has antifungal properties, but when a higher oil volume is used.', 'It produced an inhibition of 89.04% for a quantity of 40 µL. It is considered that the antifungal activity of', 'tea tree oil was signiﬁcantly affected by the presence of high content of γ-terpinene (16.3%), 4-terpineol', '(38.7%), and α-terpineol (4.6%). Previous studies showed that tea tree oil has antifungal activity against', 'Botrytis cinerea and Rhizopus stolonifer under in vitro conditions, inhibiting the spore germination and', 'mycelial growth [43].'], ['For the highest volume used in this study (60 µL), rosemary oil did not inhibit the fungal growth'], ['of Penicillium corylophilum CBMF1, the IR being below 10%.'], ['2.2.3. Aspergillus Brasiliensis ATCC 16404'], ['Aspergillus brasiliensis is a black mold that can spoil food products, especially fruits and'], ['vegetables [48].'], ['The inhibition rate of the tested essential oils (thyme oil, clove oil, rosemary oil, tea tree oil) against', 'Aspergillus brasiliensis ATCC 16404 is presented in Figure 7. The results presented evidenced that thyme', 'oil was effective against A. brasiliensis ATCC 16404 at a volume of 5 µL (IR = 97.43%), while clove oil', 'shows antifungal activity at a volume of 30 µL (IR = 82.64%). The strong antifungal activity of thyme', 'oil is attributed to phenolic compounds carvacrol (2.75%) and thymol (43.1%), while the antimicrobial', 'effectiveness of clove oil is associated with the activity of eugenol (85.7%), β-caryophyllene (4.5%),', 'and eugenol acetate (7.9%). Similar studies were conducted by Abbaszadeh et al. [46], who', 'tested the antifungal efﬁciency of thymol, carvacrol, eugenol, and menthol on growth inhibition', 'of some important food-borne pathogens. The results showed that thymol and carvacrol inhibited', 'the fungal growth of Cladosporium spp., Aspergillus spp., Fusarium oxysporum, Botrytis cinerea,', 'Penicillium spp., Alternaria alternata, and Rhizopus oryzae, and the inhibition growth was dependent on', 'the concentration used.'], ['Materials 2017, 10, 45 9 of 23  Figure 6. Inhibition of the Penicillium corylophilum CBMF1 growth by the thyme, clove, rosemary, and tea tree essential oils. The effectiveness of thyme oil as an antifungal agent can be noticed from the data presented in Figure 6. As can be seen, thyme essential oil is more active than clove essential oil, showing an inhibition rate of 100% at 10 μL, while clove oil was less effective (IR = 47.6%) at a volume of 10 μL. As well, tea tree essential oil has antifungal properties, but when a higher oil volume is used. It produced an inhibition of 89.04% for a quantity of 40 μL. It is considered that the antifungal activity of tea tree oil was significantly affected by the presence of high content of γ-terpinene (16.3%), 4-terpineol (38.7%), and α-terpineol (4.6%). Previous studies showed that tea tree oil has antifungal activity against Botrytis cinerea and Rhizopus stolonifer under in vitro conditions, inhibiting the spore germination and mycelial growth [43]. For the highest volume used in this study (60 μL), rosemary oil did not inhibit the fungal growth of Penicillium corylophilum CBMF1, the IR being below 10%. 2.2.3. Aspergillus Brasiliensis ATCC 16404 Aspergillus brasiliensis is a black mold that can spoil food products, especially fruits and vegetables [48]. The inhibition rate of the tested essential oils (thyme oil, clove oil, rosemary oil, tea tree oil) against Aspergillus brasiliensis ATCC 16404 is presented in Figure 7. The results presented evidenced that thyme oil was effective against A. brasiliensis ATCC 16404 at a volume of 5 μL (IR = 97.43%), while clove oil shows antifungal activity at a volume of 30 μL (IR = 82.64%). The strong antifungal activity of thyme oil is attributed to phenolic compounds carvacrol (2.75%) and thymol (43.1%), while the antimicrobial effectiveness of clove oil is associated with the activity of eugenol (85.7%), β-caryophyllene (4.5%), and eugenol acetate (7.9%). Similar studies were conducted by Abbaszadeh et al. [46], who tested the antifungal efficiency of thymol, carvacrol, eugenol, and menthol on growth inhibition of some important food-borne pathogens. The results showed that thymol and carvacrol inhibited the fungal growth of Cladosporium spp., Aspergillus spp., Fusarium oxysporum,  Botrytis cinerea, Penicillium spp., Alternaria alternata, and Rhizopus oryzae, and the inhibition growth was dependent on the concentration used. \x0cMaterials 2017, 10, 45'], ['10 of 24'], ['Figure 7. Inhibition of the Aspergillus brasiliensis ATCC 16404 growth by the thyme, clove, rosemary,', 'and tea tree essential oils.'], ['The essential oil of rosemary showed an inhibition rate of 14.22% for the volume of', '60 µL. Jiang et al. [37] tested the antimicrobial activity of rosemary essential oil and its main', 'components, α-pinene and 1.8-cineole, against Gram-positive and Gram-negative bacteria and fungi', '(Candida albicans, Aspergillus niger). This study showed that the antimicrobial activity of rosemary', 'essential oil was superior to its active compounds, and the essential oil is more active against all the', 'bacteria used in this research compared to the tested fungi, concluding that the synergism between its', 'compounds determine the antifungal activity.'], ['Tea tree essential oil inhibited the growth of the tested fungus with an inhibition rate of 88.90%'], ['for a volume of 35 µL.'], ['Comparing antifungal activity results of the studied essential oils, the following decreasing', 'order of activity is evident: thyme oil > clove oil > tea tree oil >> rosemary oil. Concerning the', 'sensitivity to various fungi, the thyme oil and tea tree oil are very effective in the inhibition of the', 'Aspergillus brasiliensis, as is clove oil in the inhibition of the Penicillium corylophilum. The rosemary oil is', 'less effective as an antifungal agent but shows some activity against Aspergillus brasiliensis. However,', 'other studies have evidenced that high-quality rosemary oil has antitumor, antifungal, and antiparasitic', 'effects [49]. It has also analgesic, anticancer, anticatarrhal, anti-infection, anti-inﬂammatory, and', 'expectorant properties and stimulates the circulatory system [50].'], ['2.3. Minimum Inhibitory Concentration'], ['The minimum inhibitory concentrations of the tested essential oils (EOs of thyme, clove, rosemary,'], ['and tea tree oils) are presented in Table 4.'], ['Table 4. Minimum inhibitory concentration (MIC) of essential oils tested against Fusarium graminearum', 'G87, Penicillium corylophilum CBMF1, and Aspergillus brasiliensis ATCC 16404.'], ['Essential Oil Fungal Strain'], ['Penicillium corylophilum CBMF1'], ['Fusarium graminearum G87'], ['Aspergillus brasiliensis ATCC 16404'], ['Thyme', '(ppm)', '174.41', '162.79', '116.27'], ['Clove', '(ppm)', '465.11', '930.23', '697.67'], ['Rosemary'], ['(ppm)', 'NA', 'NA', 'NA'], ['Tea Tree', '(ppm)', '930.23', '1395.34', '813.95'], ['NA—not applicable.'], ['Materials 2017, 10, 45 10 of 23  Figure 7. Inhibition of the Aspergillus brasiliensis ATCC 16404 growth by the thyme, clove, rosemary, and tea tree essential oils. The essential oil of rosemary showed an inhibition rate of 14.22% for the volume of 60 μL.  Jiang et al. [37] tested the antimicrobial activity of rosemary essential oil and its main components, α-pinene and 1.8-cineole, against Gram-positive and Gram-negative bacteria and fungi  (Candida albicans, Aspergillus niger). This study showed that the antimicrobial activity of rosemary essential oil was superior to its active compounds, and the essential oil is more active against all the bacteria used in this research compared to the tested fungi, concluding that the synergism between its compounds determine the antifungal activity. Tea tree essential oil inhibited the growth of the tested fungus with an inhibition rate of 88.90% for a volume of 35 μL. Comparing antifungal activity results of the studied essential oils, the following decreasing order of activity is evident: thyme oil > clove oil > tea tree oil >> rosemary oil. Concerning the sensitivity to various fungi, the thyme oil and tea tree oil are very effective in the inhibition of the Aspergillus brasiliensis, as is clove oil in the inhibition of the Penicillium corylophilum. The rosemary oil is less effective as an antifungal agent but shows some activity against Aspergillus brasiliensis. However, other studies have evidenced that high-quality rosemary oil has antitumor, antifungal, and antiparasitic effects [49]. It has also analgesic, anticancer, anticatarrhal, anti-infection, anti-inflammatory, and expectorant properties and stimulates the circulatory system [50]. 2.3. Minimum Inhibitory Concentration The minimum inhibitory concentrations of the tested essential oils (EOs of thyme, clove, rosemary, and tea tree oils) are presented in Table 4. Table 4. Minimum inhibitory concentration (MIC) of essential oils tested against Fusarium graminearum G87, Penicillium corylophilum CBMF1, and Aspergillus brasiliensis ATCC 16404. Essential Oil  Fungal Strain Thyme(ppm) Clove(ppm) Rosemary (ppm)  Tea Tree  (ppm)  Penicillium corylophilum CBMF1 174.41 465.11 NA 930.23 Fusarium graminearum G87 162.79 930.23 NA 1395.34 Aspergillus brasiliensis ATCC 16404 116.27 697.67 NA 813.95 NA—not applicable. The data presented in Table 4 shows that, from all the essential oils tested in this study, rosemary oil did not show desirable results against the selected fungi. The results evidenced that the most effective essential oil from the tested oils was thyme essential oil, which displayed strong antifungal activity with MIC values between 116.27 and 174.41 ppm. MIC values of clove, rosemary, and tea tree essential oils were found within the range of 465.11–1395.34 ppm. \x0cMaterials 2017, 10, 45'], ['11 of 24'], ['The data presented in Table 4 shows that, from all the essential oils tested in this study, rosemary'], ['oil did not show desirable results against the selected fungi.'], ['The results evidenced that the most effective essential oil from the tested oils was thyme essential', 'oil, which displayed strong antifungal activity with MIC values between 116.27 and 174.41 ppm. MIC', 'values of clove, rosemary, and tea tree essential oils were found within the range of 465.11–1395.34 ppm.'], ['2.4. Antibacterial Inhibition'], ['The antibacterial effect of EOs against the growth of S. aureus, E. coli, and L. monocytogenes', 'was established by the agar disc diffusion method, and the results are presented in Figure 8.', 'On Mueller–Hinten (MH) agar plates, the inhibition zones of S. aureus are 64.7 ± 1.2 mm for thyme oil,', '27.8 ± 3.4 mm for tea tree oil, 27.8 ± 4.0 mm for clove oil, and 12.8 ± 4.3 mm for rosemary oil. For', 'E. coli, the inhibition zones are 35.5 ± 4.6 mm for thyme oil, 27.0 ± 3.4 mm for tea tree oil, 19.5 ± 0.5 mm', 'for clove oil, and 15.1 ± 0.5 mm for rosemary oil. The diameters of the inhibition zones in the case', 'of L. monocytogenes growth are of 69.5 ± 6.4 mm for thyme oil, 22.0 ± 2.8 mm for tea tree oil, and', '28.5 ± 2.1 mm for clove oil. There is no inhibition zone for rosemary.'], ['Figure 8. Antimicrobial activity of the four essential oils (EOs) (10 µL on each disc) against S. aureus,', 'E. coli, and L. monocytogenes using the agar disc diffusion method on an Mueller–Hinten (MH) Plate.'], ['Thyme oil had the highest inhibition zones for the three test strains, followed by tea tree oil, and'], ['then by clove oil. Rosemary oil was the least active.'], ['Thyme essential oil is strongly antimicrobial, antifungal, antiviral, antiparasitic, mainly due to the', 'high content of thymol [49]. This is in accordance with other studies that reported that thyme essential', 'oil is efﬁcient against Gram-positive and Gram-negative bacteria [29,30] and fungi [28,51,52].'], ['Due to its antimicrobial, antifungal, antivermin, and antiviral activity [34,35], clove essential', 'oil is used as a preservative but also exhibits other effects with positive impacts on consumer', 'health, including antiseptic, antihelmintic, anti-inﬂammatory, antispastic, carminative, anti-neuralgic,', 'antiulcer, anti-thrombotic, anticancerinogenic, and anticoagulant activities. Moreover, it acts as a local', 'analgesic [36].'], ['The inhibition zones observed after 24 h were stable and identical after 48 and 72 h of incubation.', 'EOs are less effective in the vapor disc diffusion method compared to the disc diffusion method.', 'Thyme and clove oils had volatile components that had inhibition zones even when the disc with EO', 'had no contact with the bacteria. Thyme had the best effect for all test microorganisms, while clove', 'only had an effect on S. aureus (Table 5).'], ['Materials 2017, 10, 45 11 of 23 2.4. Antibacterial Inhibition The antibacterial effect of EOs against the growth of S. aureus, E. coli, and L. monocytogenes was established by the agar disc diffusion method, and the results are presented in Figure 8. On Mueller–Hinten (MH) agar plates, the inhibition zones of S. aureus are 64.7 ± 1.2 mm for thyme oil, 27.8 ± 3.4 mm for tea tree oil, 27.8 ± 4.0 mm for clove oil, and 12.8 ± 4.3 mm for rosemary oil. For E. coli, the inhibition zones are 35.5 ± 4.6 mm for thyme oil, 27.0 ± 3.4 mm for tea tree oil, 19.5 ± 0.5 mm for clove oil, and 15.1 ± 0.5 mm for rosemary oil. The diameters of the inhibition zones in the case of  L. monocytogenes growth are of 69.5 ± 6.4 mm for thyme oil, 22.0 ± 2.8 mm for tea tree oil, and 28.5 ± 2.1 mm for clove oil. There is no inhibition zone for rosemary. Thyme oil had the highest inhibition zones for the three test strains, followed by tea tree oil, and then by clove oil. Rosemary oil was the least active. Thyme essential oil is strongly antimicrobial, antifungal, antiviral, antiparasitic, mainly due to the high content of thymol [49]. This is in accordance with other studies that reported that thyme essential oil is efficient against Gram-positive and Gram-negative bacteria [29,30] and fungi [28,51,52]. Due to its antimicrobial, antifungal, antivermin, and antiviral activity [34,35], clove essential oil is used as a preservative but also exhibits other effects with positive impacts on consumer health, including antiseptic, antihelmintic, anti-inflammatory, antispastic, carminative, anti-neuralgic, antiulcer, anti-thrombotic, anticancerinogenic, and anticoagulant activities. Moreover, it acts as a local analgesic [36].  Figure 8. Antimicrobial activity of the four essential oils (EOs) (10 μL on each disc) against S. aureus, E. coli, and L. monocytogenes using the agar disc diffusion method on an Mueller–Hinten (MH) Plate. The inhibition zones observed after 24 h were stable and identical after 48 and 72 h of incubation. EOs are less effective in the vapor disc diffusion method compared to the disc diffusion method. Thyme and clove oils had volatile components that had inhibition zones even when the disc with EO had no contact with the bacteria. Thyme had the best effect for all test microorganisms, while clove only had an effect on S. aureus (Table 5). Table 5. Mean inhibition zone diameter (mm) by the vapor disc diffusion method. Essential Oil VaporPhase MethodS. aureus E. coli L. monocytogenes Thyme 56.8 ± 6.9 28.3 ± 1.3 48.5 ± 4.9 Clove 14.2 ± 2.6 0 ± 0 0 ± 0 Rosemary 0 ± 0 0 ± 0 NA Tea tree 0 ± 0 0 ± 0 0 ± 0 \x0cMaterials 2017, 10, 45'], ['12 of 24'], ['Table 5. Mean inhibition zone diameter (mm) by the vapor disc diffusion method.'], ['Essential Oil'], ['Thyme', 'Clove'], ['Rosemary', 'Tea tree'], ['Vapor Phase Method'], ['S. aureus', '56.8 ± 6.9', '14.2 ± 2.6'], ['0 ± 0', '0 ± 0'], ['E. coli', '28.3 ± 1.3'], ['0 ± 0', '0 ± 0', '0 ± 0'], ['L. monocytogenes'], ['48.5 ± 4.9'], ['0 ± 0', 'NA', '0 ± 0'], ['The minimal inhibitory concentration of an antimicrobial agent is the lowest (i.e., minimal)', 'concentration of the antimicrobial agent that inhibits a given bacterial isolate from multiplying and', 'producing visible growth in the test system. This concentration was determined by incubating a', 'known quantity of bacteria with speciﬁed dilutions of the antimicrobial agent. Using a similar broth', 'dilution method, Zhang et al. [53] found that MIC was 1.0 mg/mL of cinnamon for both S. aureus', 'and E. coli. Broth microdilution is the most widely used method in clinical laboratories, but agar', 'diffusion is also used. Due to the oily content of EOs, using solvents is needed to obtain homogeneous', 'dilutions. The results obtained using three solvents namely Tween-20, DMSO, and ethanol by the two', 'test methods (agar MIC testing and the broth microdilution test) are shown in Table 6. There are some', 'differences between the test results, but they all showed that thyme is the most effective EO, with a', 'very low concentration needed to inhibit the growth of S. aureus and E. coli, followed by clove and tea', 'tree oils. The thyme oil MIC values with the broth dilution method was approximately 0.39%–3.13%', 'for S. aureus and 1.56%–3.13% for E. coli. Similarly, clove MIC value range were approximately', '3.13%–6.26% for S. aureus and 6.25% for E. coli. A strong antimicrobial activity of thyme was also', 'found by Mith et al. [54] when they tested different EOs and determined MIC against both food-borne', 'pathogens and spoilage bacteria with a broth dilution method. The MIC value ranges of tea tree oil', 'were approximately 0.80%–25.00% for S. aureus and 12.5%–25.00% for E. coli.'], ['Table 6. The minimal inhibitory concentration (MIC) (%) of essential oils in three different', 'solvents (v/v).'], ['EO'], ['Method'], ['MICs Value for S. aureus', 'Concentration of Oil (v/v)', '0.5%'], ['5%'], ['100%', 'Ethanol'], ['MICs Value for E. coli'], ['Concentration of Oil (v/v)', '0.5%'], ['5%'], ['Tween-20'], ['DMSO'], ['100%', 'Ethanol'], ['Thyme'], ['Clove'], ['Tea Tree'], ['Agar', 'Broth', 'Agar', 'Broth', 'Agar', 'Broth'], ['Tween-20'], ['1.56', '0.39', '3.13', '6.25', '6.25', '0.80'], ['DMSO', '12.50', '0.78', '12.50', '3.13', '25.00', '25.00'], ['1.56', '3.13', '12.50', '3.13', '25.00', '25.00'], ['3.13', '3.13', '12.50', '6.25', '12.50', '12.50'], ['6.25', '3.13', '12.50', '6.25', '12.50', '25.00'], ['1.56', '3.13', '3.13', '6.25', '12.50', '25.00'], ['TTO has gained attention in the food industry for its antimicrobial [41,42] and antifungal'], ['activity [43], and it was successfully used in respiratory or genito-urinary tract infections [44].'], ['2.5. Antioxidant Activity Evaluation'], ['The results obtained by the ABTS method—Figure 9—are well correlated with those obtained by'], ['2,2-diphenyl-1-picrylhydrazyl (DPPH)—Figure 10.'], ['\x0cMaterials 2017, 10, 45'], ['13 of 24'], ['Figure 9. ABTS radical discoloration of studied essential oils (a) and the values of sample concentration', 'required to scavenge 50% of the ABTS free radicals (IC50); (b) of selected essential oils.'], ['Figure 10. The values of the sample concentration required to scavenge 50% of the DPPH free radicals', '(IC50) of selected essential oils.'], ['For the analyzed essential oils, IC50 is in the µg/mL range assigned to a very good antioxidant', 'activity [55]. From the selected essential oils, the clove oil is by far the most radical scavenging active,', 'with an IC50 of 8 µg/mL. The antioxidant activity was associated with the presence of phenolic', 'compounds in the composition of essential oils, as determined by the Folin–Ciocâlteu method and', 'the GC-FID technique and ORAC values. The antioxidant activity varies in the following order:', 'clove oil > thyme oil > rosemary oil >> tea tree oil. Based on the radical scavenging tests, the clove oil', 'is the most effective antioxidant is recommended for further such applications.'], ['2.6. Preliminary Results on the Use of Encapsulated Essential Oils into Chitosan Films as Food', 'Packaging Material'], ['The stability of the chitosan/essential oil emulsions was sustained by dynamic light scattering', '(DLS) analysis, which showed a main particle population with low dimensions (Figure 11), with', 'the distribution showing, in the low dimension range, a single peak and a polydispersity index', '(PDI)-approached value of 1.0 (Table 7). The chitosan/essential oil emulsions contain two kinds', 'of particle populations: the ﬁrst one is very small, where d = 5 nm for the chitosan solution and', '11–70 (350) nm for the emulsion containing EOs, and the second contains larger particles of 850 nm', 'for CS and 2500–8800 nm for CS/EOs emulsions. Oils are probably concentrated around CS particles', 'because of aggregation.'], ['Materials 2017, 10, 45 12 of 23 The minimal inhibitory concentration of an antimicrobial agent is the lowest (i.e., minimal) concentration of the antimicrobial agent that inhibits a given bacterial isolate from multiplying and producing visible growth in the test system. This concentration was determined by incubating a known quantity of bacteria with specified dilutions of the antimicrobial agent. Using a similar broth dilution method, Zhang et al. [53] found that MIC was 1.0 mg/mL of cinnamon for both S. aureus and E. coli. Broth microdilution is the most widely used method in clinical laboratories, but agar diffusion is also used. Due to the oily content of EOs, using solvents is needed to obtain homogeneous dilutions. The results obtained using three solvents namely Tween-20, DMSO, and ethanol by the two test methods (agar MIC testing and the broth microdilution test) are shown in Table 6. There are some differences between the test results, but they all showed that thyme is the most effective EO, with a very low concentration needed to inhibit the growth of S. aureus and E. coli, followed by clove and tea tree oils. The thyme oil MIC values with the broth dilution method was approximately 0.39%–3.13% for S. aureus and 1.56%–3.13% for E. coli. Similarly, clove MIC value range were approximately 3.13%–6.26% for S. aureus and 6.25% for E. coli. A strong antimicrobial activity of thyme was also found by Mith et al. [54] when they tested different EOs and determined MIC against both food-borne pathogens and spoilage bacteria with a broth dilution method. The MIC value ranges of tea tree oil were approximately 0.80%–25.00% for S. aureus and 12.5%–25.00% for E. coli. Table 6. The minimal inhibitory concentration (MIC) (%) of essential oils in three different solvents (v/v). EO Method MICs Value for S. aureusMICs Value for E. coli Concentration ofOil (v/v)Concentration of Oil (v/v) 0.5% Tween-20 5% DMSO 100% Ethanol 0.5% Tween-20 5% DMSO 100% Ethanol   Thyme Agar 1.56 12.50 1.56 3.13 6.25 1.56 Broth 0.39 0.78 3.13 3.13 3.13 3.13 Clove Agar 3.13 12.50 12.50 12.50 12.50 3.13 Broth 6.25 3.13 3.13 6.25 6.25 6.25 Tea Tree Agar 6.25 25.00 25.00 12.50 12.50 12.50 Broth 0.80 25.00 25.00 12.50 25.00 25.00 TTO has gained attention in the food industry for its antimicrobial [41,42] and antifungal activity [43], and it was successfully used in respiratory or genito-urinary tract infections [44]. 2.5. Antioxidant Activity Evaluation The results obtained by the ABTS method—Figure 9—are well correlated with those obtained by 2,2-diphenyl-1-picrylhydrazyl (DPPH)—Figure 10. 050100150020406080100020004000600080001000012000020406080100ABTS Inhibition [%]Oil concentration [ppm] Tea tree oil Rosemary oilABTS Inhibition [%]Oil Concentration [ppm] Clove oil Thyme oilCloveThymeRosemaryTea tree050300040005000IC50 [\uf06dg/mL](a)(b)Figure 9. ABTS radical discoloration of studied essential oils (a) and the values of sample concentration required to scavenge 50% of the ABTS free radicals (IC50); (b) of selected essential oils. Materials 2017, 10, 45 13 of 23  Figure 10. The values of the sample concentration required to scavenge 50% of the DPPH free radicals (IC50) of selected essential oils. For the analyzed essential oils, IC50 is in the μg/mL range assigned to a very good antioxidant activity [55]. From the selected essential oils, the clove oil is by far the most radical scavenging active, with an IC50 of 8 μg/mL. The antioxidant activity was associated with the presence of phenolic compounds in the composition of essential oils, as determined by the Folin–Ciocâlteu method and the GC-FID technique and ORAC values. The antioxidant activity varies in the following order: clove oil > thyme oil > rosemary oil >> tea tree oil. Based on the radical scavenging tests, the clove oil is the most effective antioxidant is recommended for further such applications. 2.6. Preliminary Results on the Use of Encapsulated Essential Oils into Chitosan Films as Food  Packaging Material The stability of the chitosan/essential oil emulsions was sustained by dynamic light scattering (DLS) analysis, which showed a main particle population with low dimensions (Figure 11), with the distribution showing, in the low dimension range, a single peak and a polydispersity index (PDI)-approached value of 1.0 (Table 7). The chitosan/essential oil emulsions contain two kinds of particle populations: the first one is very small, where d = 5 nm for the chitosan solution and 11–70 (350) nm for the emulsion containing EOs, and the second contains larger particles of 850 nm for CS and 2500–8800 nm for CS/EOs emulsions. Oils are probably concentrated around CS particles because of aggregation.  (a) (b)Figure 11. Particle size distribution of the chitosan (a) and chitosan/tea tree oil (b) emulsions in diluted acetic acid containing surfactant Tween 80. \x0cMaterials 2017, 10, 45'], ['14 of 24'], ['Figure 11. Particle size distribution of the chitosan (a) and chitosan/tea tree oil (b) emulsions in diluted', 'acetic acid containing surfactant Tween 80.'], ['Table 7. DLS results for essential oil/chitosan emulsions used to obtain ﬁlms for food packaging.'], ['Sample', 'Chitosan'], ['Thyme (Thymus Vulgaris L.)'], ['Cloves (Eugenia caryophyllus from dried'], ['ﬂoral buds of Syzygium aromaticum)', 'Rosemary (Rosmarinus ofﬁcinalis L.)'], ['Tea tree (Melaleuca alternifolia aetheroleum)'], ['Z First Peak, d (nm)'], ['Z Average (nm)'], ['5'], ['11.5 and 350'], ['10.5', '60'], ['15 and 70'], ['850', '6190', '8790', '2550', '6360'], ['PDI', '0.997', '0.735', '1.0', '1.0', '1.0'], ['Scanning electron microscopy examination reveals the morphology of the ﬁlms obtained by the', 'incorporation of essential oils into chitosan ﬁlms—Figure 12. The ﬁlms are relatively homogeneous,', 'and dispersion of the essential oil in the ﬁlm obtained by solvent casting is homogeneous, as is evident', 'from the SEM images taken at high magniﬁcation (2000×).'], ['Figure 12. SEM images of (a) CS and (b) CS/rosemary or clove oil/T80 ﬁlms at a 2000× magniﬁcation.'], ['Materials 2017, 10, 45 13 of 23  Figure 10. The values of the sample concentration required to scavenge 50% of the DPPH free radicals (IC50) of selected essential oils. For the analyzed essential oils, IC50 is in the μg/mL range assigned to a very good antioxidant activity [55]. From the selected essential oils, the clove oil is by far the most radical scavenging active, with an IC50 of 8 μg/mL. The antioxidant activity was associated with the presence of phenolic compounds in the composition of essential oils, as determined by the Folin–Ciocâlteu method and the GC-FID technique and ORAC values. The antioxidant activity varies in the following order: clove oil > thyme oil > rosemary oil >> tea tree oil. Based on the radical scavenging tests, the clove oil is the most effective antioxidant is recommended for further such applications. 2.6. Preliminary Results on the Use of Encapsulated Essential Oils into Chitosan Films as Food  Packaging Material The stability of the chitosan/essential oil emulsions was sustained by dynamic light scattering (DLS) analysis, which showed a main particle population with low dimensions (Figure 11), with the distribution showing, in the low dimension range, a single peak and a polydispersity index (PDI)-approached value of 1.0 (Table 7). The chitosan/essential oil emulsions contain two kinds of particle populations: the first one is very small, where d = 5 nm for the chitosan solution and 11–70 (350) nm for the emulsion containing EOs, and the second contains larger particles of 850 nm for CS and 2500–8800 nm for CS/EOs emulsions. Oils are probably concentrated around CS particles because of aggregation.  (a) (b)Figure 11. Particle size distribution of the chitosan (a) and chitosan/tea tree oil (b) emulsions in diluted acetic acid containing surfactant Tween 80. Materials 2017, 10, 45 14 of 23 Table 7. DLS results for essential oil/chitosan emulsions used to obtain films for food packaging. Sample Z First Peak, d (nm)ZAverage (nm) PDIChitosan 5 850 0.997 Thyme (Thymus Vulgaris L.) 11.5 and 350 6190 0.735 Cloves (Eugenia caryophyllus from dried floral buds of Syzygium aromaticum) 10.5 8790 1.0 Rosemary (Rosmarinus officinalis L.) 60 2550 1.0 Tea tree (Melaleuca alternifolia aetheroleum) 15 and 70 6360 1.0 Scanning electron microscopy examination reveals the morphology of the films obtained by the incorporation of essential oils into chitosan films—Figure 12. The films are relatively homogeneous, and dispersion of the essential oil in the film obtained by solvent casting is homogeneous, as is evident from the SEM images taken at high magnification (2000×).  (a) (b)Figure 12. SEM images of (a) CS and (b) CS/rosemary or clove oil/T80 films at a 2000× magnification It can be observed that the surfaces are very smooth, and, by essential oil incorporation, very small particles are evidenced at high magnification. The good homogeneity of the films and the encapsulation of the oils is assured by the presence of a nonionic surfactant and emulsifier T80. The encapsulation of active (antimicrobial and antioxidant) essential oils into the chitosan matrix leads to a significant decrease in the total number of germs for beef meat packed in such films—from 2400–1020 CFU/cm2 for films containing only CS to 700 CFU/cm2 for films containing both CS and clove oil. These films proved to have a good antimicrobial activity for delaying the spoilage of beef meat, with both antimicrobial agents acting synergistically [56]. This research will be the topic of the subsequent paper. 3. Experimental Section 3.1. Materials Essential Oils Four commercial essential oils used in this study were purchased from the Fares company (Orăştie, Romania): thyme (Thymus Vulgaris L.), clove (Eugenia caryophyllus—from dried floral buds of Syzygium aromaticum), rosemary (Rosmarinus officinalis L.), and tea tree  (Melaleuca alternifolia aetheroleum) obtained using a distillation apparatus (according to European Pharmacopoeia 7th edition, method 8.2.12). The Folin–Ciocâlteu phenol reagent 2N was purchased from Sigma-Aldrich (Buchs, Switzerland), and sodium carbonate decahydrate (Na2CO3·10H2O, M = 286.14) was from Chimopar S.A., Bucuresti, Romania. Methanol and gallic acid (97%, M = 170.12) were purchased from Sigma-Aldrich (Buchs, Switzerland). \x0cMaterials 2017, 10, 45'], ['15 of 24'], ['It can be observed that the surfaces are very smooth, and, by essential oil incorporation, very', 'small particles are evidenced at high magniﬁcation. The good homogeneity of the ﬁlms and the', 'encapsulation of the oils is assured by the presence of a nonionic surfactant and emulsiﬁer T80.'], ['The encapsulation of active (antimicrobial and antioxidant) essential oils into the chitosan matrix', 'leads to a signiﬁcant decrease in the total number of germs for beef meat packed in such ﬁlms—from', '2400–1020 CFU/cm2 for ﬁlms containing only CS to 700 CFU/cm2 for ﬁlms containing both CS and', 'clove oil. These ﬁlms proved to have a good antimicrobial activity for delaying the spoilage of beef', 'meat, with both antimicrobial agents acting synergistically [56]. This research will be the topic of the', 'subsequent paper.'], ['3. Experimental Section'], ['3.1. Materials'], ['Essential Oils'], ['Four commercial essential oils used in this study were purchased from the Fares company', '(Or˘a¸stie, Romania): thyme (Thymus Vulgaris L.), clove (Eugenia caryophyllus—from dried ﬂoral buds of', 'Syzygium aromaticum), rosemary (Rosmarinus ofﬁcinalis L.), and tea tree (Melaleuca alternifolia aetheroleum)', 'obtained using a distillation apparatus (according to European Pharmacopoeia 7th edition,', 'method 8.2.12).', 'The Folin–Ciocâlteu phenol reagent 2N was purchased from Sigma-Aldrich (Buchs, Switzerland),', 'and sodium carbonate decahydrate (Na2CO3·10H2O, M = 286.14) was from Chimopar S.A., Bucuresti,', 'Romania. Methanol and gallic acid (97%, M = 170.12) were purchased from Sigma-Aldrich', '(Buchs, Switzerland).', 'For the determination of the antioxidant activity 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich,', 'Darmstadt, Germany), 2,2(cid:48)-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS,', 'purity ≥ 98% by HPLC, Sigma-Aldrich, Darmsadt, Germany) and potassium persulfate (KPS, ACS', 'reagent, purity ≥ 99.0%, Fluka, Buchs, Switzerland) were used. High purity solvents such as Tween-20', 'from Sigma-Aldrich (Buchs, Switzerland) containing lauric acid, ≥40% (balance primarily myristic,', 'palmoyic, and stearic acids; dimethyl sulfoxide (DSMO, Sigma-Aldrich, Buchs, Switzerland), or ethanol', '(Sigma-Aldrich, Buchs, Switzerland) were used in the antibacterial activity test.', 'Chitosan (CS) from crab shells, with a dynamic viscosity of >400 mPa·s in 1% acetic acid (20 ◦C)'], ['where MW = 310,000–375,000 g/mol, was purchased from Sigma-Aldrich (Buchs, Switzerland).'], ['Polysorbate 80 or Tween® 80 (T80) is a viscous, water-soluble viscous yellow liquid, a nonionic', 'surfactant and emulsiﬁer often used in foods and cosmetics. It was offered by Sigma-Aldrich (Buchs,', 'Switzerland) for research purposes.'], ['3.2. Microorganisms'], ['3.2.1. Fungi'], ['Three food-related fungi were used as target microorganisms in this study: Aspergillus brasilliensis', 'ATCC 16404, Penicillium corylophilum CBMF1, and Fusarium graminearum G87, and they were provided', 'from the collection of Faculty of Biotechnology of University of Agronomic Sciences and Veterinary', 'Medicine Bucharest (Bucharest, Romania).'], ['3.2.2. Bacteria'], ['The three tested bacterial strains were obtained from the Culture Collection at the University of', 'Gothenburg: Staphylococcus aureus CCUG 1828, Escherichia coli CCUG 10979, and Listeria monocytogenes', 'CCUG 15527. A test culture was prepared by growth on MH agar overnight, and colonies were diluted', 'to a 0.5 McFarland in MH broth.'], ['\x0cMaterials 2017, 10, 45'], ['3.3. Methods of Investigation'], ['16 of 24'], ['3.3.1. Determination of Total Phenolic Content in Vegetable Oils by the Folin–Ciocâlteu', 'Reagent Method'], ['The amount of total phenols was determined by the Folin–Ciocâlteu reagent method as described', 'by Scalbert et al. [18]. A 10 µL volume of essential oil was dissolved in 10 mL of methanol, and', '0.1 mL of methanol solution was then transferred into a volumetric ﬂask and diluted with 0.4 mL of', 'double distilled water and used for analysis. A Folin–Ciocâlteu reagent solution 1:10 v/v in double', 'distilled water (1 mL) was added to the diluted methanol/oil mixture (0.5 mL), which was then mixed', 'thoroughly and leaved for 10 minutes at room temperature. A 2 mL solution based on 15% sodium', 'carbonate (Na2CO3·10H2O) was added; after 1 h of incubation at room temperature, the absorbance', 'was measured at 740 nm with a UV-Vis 60 Cary spectrophotometer against a blank sample that was', 'concomitantly prepared. The blank sample contained 0.1 mL of methanol without oil, 0.4 mL of water,', '1 mL of a Folin–Ciocâlteu reagent solution, and 2 mL of a Na2CO3·10H2O solution. Gallic acid was', 'used as the standard for the calibration curve, which was drawn using solutions of the gallic acid in', 'methanol of different concentrations ranging between 0.01 and 1 mg/mL. Based on the measured', 'absorbance at 740 nm, the concentration of phenolics was read (mg/mL) from the calibration line, and', 'the total phenolics values were then expressed as mg·GAE/g·DW. Each determination was made in', 'triplicate, and average values were reported.'], ['3.3.2. Gas Chromatography-Mass Spectrometry Detector and Flame-Ionized Detector', 'Analysis (GC-MSD/FID)'], ['The essential oils were characterized by gas chromatography coupled with mass spectrometry', 'detector (GC-MSD) for qualitative analysis and, with a ﬂame ionized detector (GC-FID) (Agilent,', 'Santa Clara, CA, USA), quantitative analysis.'], ['The GC analysis was performed on a 6890 Agilent Technologies chromatograph using the', 'following parameters: a Teknokroma TR-520232 (95% dimethyl- and 5% diphenyl-polysiloxane,', 'crosslinked; 30 m × 250 µm × 0.25 µm) capillary column, a 1 mL/min helium ﬂow, an inlet at 175 ◦C,', 'a 100:1 split ratio, and a temperature program starting at 60 ◦C, with a heating rate of 7 ◦C/min up to', '200 ◦C and then 15 ◦C/min up to 300 ◦C, followed by isothermal heating at 300 ◦C for 3.33 min.'], ['The MS detection was performed on a 5975 Inert XL Agilent Technologies detector (Agilent,', 'Santa Clara, CA, USA), at 70 eV. Identiﬁcation of chromatographic peaks was made by reference to', 'Institute of Standards and Technology (NIST) database, with a quality of recognition above 90%.'], ['3.4. Fungi Spore Suspension Preparation'], ['To evaluate the antifungal activity of the tested essential oils, the selected fungi were grown', 'on a Potato Dextrose Agar (PDA, Scharlau Microbiologi, Barcelona, Spain) medium in 90 mm Petri', 'dishes for 7–9 days and stored at 25 ◦C. Fungal spore suspension was obtained in aseptic conditions', 'in a laminar ﬂow cabinet after ultraviolet sterilization for 20 min. Fungal spore suspensions were', 'collected from the surface of fungal colonies by gentle scraping with a sterile inoculation loop and', 'suspended in 10 mL of sterile water. Spore population was counted using a hemocytometer (Thoma', 'camera, 0, 100 mm depth, from Hirschmann Techcolor, Eberstadt, Germany). The concentration of', 'spore suspensions was 106 spores/mL.'], ['3.5. Antifungal Assay'], ['The antifungal activity of the tested oils was determined with the disc diffusion method. Potato', 'dextrose agar medium was poured into 90 mm Petri dishes, and, after its solidifying, in each Petri', 'dish, 100 µL of spore suspensions (containing 106 spores/mL) were spread onto the surface of the', 'media. The dishes were left to facilitate the incorporation of fungi in the PDA medium for 30 min.', 'Subsequently, ﬁlter paper discs (6 mm Ø; Whatman) were placed on the surface of the Petri dishes and'], ['\x0cMaterials 2017, 10, 45'], ['17 of 24'], ['impregnated with different amounts (volume from 2.5 to 60 µL) of essential oil. All determinations', 'were performed in ﬁve replicates. Control plates (without essential oils) were inoculated using the', 'same procedure. Each sample was evaluated by three analysts, and three averages of the ﬁve replicates', 'were obtained. The ﬁnal average is the arithmetic average of the three other averages. Samples were', 'evaluated through estimation of the degree to which the Petri dish surface was covered by fungal', 'mycelium and through an inhibition rate (IR) calculation.'], ['The inhibition rate was calculated as follows:'], ['IR(%) ='], ['C − S', 'C'], ['× 100'], ['(1)'], ['where C is the rate of the fungal mycelium growth of the control, and S is the rate of the fungal', 'mycelium growth of the samples.', 'The dishes were sealed with paraﬁlm tape to prevent the volatilization of essential oils and', 'incubated for 7 days at 25 ◦C. The efﬁcacy of the treatment was evaluated after 7 days by the inhibition', 'rate IR (%).'], ['3.6. Determination of the Minimum Inhibitory Concentration (MIC) of Fungi'], ['Essential oils were screened to determine their minimum inhibitory concentration against the', 'food spoilage microorganisms selected. The same procedure as the one described for the antifungal', 'assay was used to determine the minimum inhibitory concentration (MIC). The deﬁnition of the MIC', 'differs between various publications [57–59], so it was decided that the MIC would be deﬁned as', 'the lowest volume of essential oil that inhibited the growth of the tested fungi with an inhibition', 'rate higher than 80%. The MIC is expressed in different units including mg/mL [60], µg/mL [61,62],', 'µL/L [63,64], µL/mL [65], and ppm [62,66]. In this study, the MIC of fungi was expressed as ppm.'], ['3.7. The Agar Disc Diffusion Method'], ['The protocol of the Clinical and Laboratory Standards Institute (CLSI) [67,68] was followed', 'with some modiﬁcations. The bacterial suspensions were diluted to a McFarland standard of 0.5', '(0.5 McFarland standard is prepared by mixing 0.05 mL of 1.175% barium chloride dihydrate', '(BaCl2·2H2O), with 9.95 mL of 1% sulfuric acid (H2SO4), and adjusted to about 104–105 bacteria/mL', 'before use. A 0.1 mL portion from this bacterial dilution was spread on Mueller–Hinten (MH) agar', '(MHA, Sigma-Aldrich, Darmstadt, Germany). Subsequently, sterile paper discs (Macherey-Nagel,', 'Düren, Germany, LOT: 141112) 6 mm in diameter were placed within 15 min onto the inoculated agar', 'surface containing 10 µL of the essential oil or 10 µL of sterile water as control. Petri dishes were', 'incubated at 37 ◦C for 24 ± 1 h. After incubation, the inhibition zones were measured as the complete', 'inhibition in mm, as a diameter including the disc. The inhibition zones were measured in triplicate', 'with a vernier caliper. The distance from the colonies closest to the disc to the center of the disc was', 'doubled to obtain a diameter.'], ['3.8. The Vapor Disc Diffusion Method'], ['MH-agar plate surfaces inoculated with E. coli and S. aureus and Tryptone Soya Agar (TSA,', 'CM 0131, Oxoid Ltd., Thermo Fisher Scientiﬁc, Basingstoke, Hampshire, UK) with L. monocytogenes', 'were prepared as described in Section 3.7. A volume of 10 µL of each pure EO was added to sterile', 'ﬁlter discs with a diameter of 6 mm, and sterile water was used as control. The discs were placed on', 'the inside center of a Petri dish lid in a sterile working hood. A control was also prepared by adding', '10 µL of sterile water to the ﬁlter discs.'], ['The inoculated agar plate was then placed to ﬁt the lid but with the agar upwards. This created', 'a closed Petri dish with a distance of about 1 cm between the disc and the bacteria inoculated agar', 'surface in accordance with Goni et al. [69]. The Petri dishes were sealed with adhesive tape to prevent', 'volatile components of the essential oils to escape.'], ['\x0cMaterials 2017, 10, 45'], ['18 of 24'], ['3.9. Determination of the Minimum Inhibitory Concentration (MIC) of Bacteria'], ['3.9.1. Agar MIC Testing'], ['The preparation of agar, broth, and bacteria were carried out as described in Section 3.7. MICs', 'was determined for thyme, clove, and tea tree against S. aureus and E. coli according to the method', 'used by Zhang and Mith [53,54] with some minor modiﬁcations. The original solution of each EO was', 'dissolved in Tween-20 (0.5%), dimethyl sulfoxide (DSMO, 5%), or ethanol (100%) in a 1:1 ratio to make', 'serial 2-fold dilutions, yielding solutions with a gradient from 100% oil to 0.2% oil (v/v). A volume of', '10 µL of each oil dilution was added to the 6 mm paper disc and then placed on MH agar plates with', 'a 100 µL bacterial concentration containing a 104–105 CFU/mL suspension of each tested bacterium.', 'DSMO without bacteria was used as a control.'], ['3.9.2. Broth Microdilution MIC Testing'], ['Similar dilutions in either Tween-20 (0.5%), dimethyl sulfoxide (DSMO, 5%), or ethanol (100%)', 'were transferred to sterile microtiter plates (honeycomb plates with 100 wells). Three parallel dilution', 'series were used with 100 µL in each well. The microtiter plates were mounted in a Bioscreen', 'C (Oy Growth Curves Ab Ltd., Helsinki, Finland), an automated growth curve analysis system,', 'programmed to measure absorbance at 600 nm (abs 600 nm) at regular time intervals at 37 ± 0.1 ◦C.', 'Prior to each measurement, the plates were shaken for 10 s (default setting). Growth curves of both', 'strains were recorded. Time-to-detection (TTD) were deﬁned as ABS600nm = 0.2. Lack of absorbance', 'above 0.2 were deﬁned as growth inhibition.'], ['3.10. Antioxidant Activity Evaluation'], ['The inhibition concentration of four essential oils (thyme, clove, rosemary, and tea tree) and', 'the stability of natural antioxidant at different concentrations were evaluated using 2,2’-azino-bis', '3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical methods.', '3.10.1. ABTS•+ (2,2’-Azino-bis 3-ethylbenzthiazoline-6-sulfonic Acid) Radical Cation', 'Scavenging Assay'], ['The ABTS•+ scavenging test is used to determine the antioxidant activity of both hydrophilic', 'and hydrophobic compounds. ABTS•+ is generated by mixing 2.5 mL of 7 mM ABTS with 2.5 mL of', '14.7 mM potassium persulfate (KPS) water solutions and stored in the dark at room temperature for', '16 h. The reaction between ABTS•+ and potassium persulfate directly generates the blue green ABTS•+', 'chromophore, which can be reduced by an antioxidant, thereby resulting in a loss of absorbance at', '750 nm.'], ['The antioxidant capacity is expressed as percentage inhibition, calculated using the following'], ['formula [70]:'], ['Inhibition(%) ='], ['(cid:20) Acontrol − Asample'], ['Acontrol'], ['(cid:21)'], ['× 100'], ['(2)'], ['where Acontrol is the absorbance of the ABTS radical in methanol, and Asample is the absorbance of an', 'ABTS radical solution mixed with the sample. All determinations were performed in triplicate.'], ['3.10.2. DPPH Radical Scavenging Assay'], ['The DPPH radical'], ['scavenging assay for essential oils was measured using the', '2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The DPPH is a stable free radical with a violet color', 'that, under the action of proton-donating compounds, is reduced to a light yellow color, and this', 'change can be monitored at 517 nm [71]. Concentrations of the methanolic solution between 0.156 and', '20 mg/mL were tested.'], ['\x0cMaterials 2017, 10, 45'], ['The radical scavenging activity was calculated according to the following equation:'], ['%RSA = 100 ×'], ['(cid:18) Acontrol − Asample'], ['(cid:19)'], ['Acontrol'], ['19 of 24'], ['(3)'], ['where Asample represents the absorbance of the sample solution in the presence of DPPH, and Acontrol', 'is the absorbance of the standard DPPH solution. For more details, see ref. [72].'], ['3.11. The Encapsulation of Essential Oils into the Chitosan Matrix by the Emulsion/Solvent Casting Method'], ['The ﬁlm forming solutions were prepared using highly viscous chitosan from crab shells where', 'MW = 310,000–375,000 g/mol, with a dynamic viscosity of >400 mPa·s in 1% acetic acid (20 ◦C).', 'The chitosan ﬁlm-forming solution was prepared by dissolving 2 g of chitosan per 100 mL of', 'a 5% acetic acid aqueous solution. Food grade essential oils (clove, thyme, rosemary, and tea', 'tree oils) were incorporated in a proportion of 0.75 mL/g chitosan, and Tween 80 (Sigma-Aldrich,', 'Darmstadt, Germany) (0.125 g/g chitosan) was added as an emulsifying agent. The essential oil-added', 'ﬁlm-forming solution was homogenized with an ultrasonic processor UP50H (Hielscher—Ultrasound', 'Technology, Teltow, Germany) using a power of 50 W at 30 kHz). The ﬁlms were obtained by casting', '50 mL in glass Petri dishes (153 cm2) and drying ﬁrst at 25 ◦C in a forced-air oven for 24 h and then', 'at 40 ◦C in a vacuum oven to yield a uniform thickness in all cases. Prior to analyses, the ﬁlms', 'were conditioned in desiccators for 2 days at 22 ◦C over a saturated solution of NaBr (58% relative', 'humidity) [56].'], ['3.12. The Dynamic Light Scattering (DLS) Analysis'], ['The DLS analysis of the oils-in-chitosan emulsions was carried out using a Zetasizer NS (Malvern', 'Instruments Ltd., Malvern, UK), according with standard ISO 13321 (1996). This analysis only gives', 'a mean particle size (z-average) and an estimate of the width of the distribution (polydispersity', 'index (PDI)). It is very sensitive to the presence of aggregates due to the inherent intensity weighting.', 'The oil/chitosan ratio was kept at 0.75 mL/1 g chitosan. For DLS, the concentration of the initial', 'solution was 1.5 wt % chitosan and the corresponding concentration of the oils. The chitosan/essential', 'oil emulsions of 1.5 wt % were diluted three times prior to analysis with the 5 wt % acetic acid', 'aqueous solution.'], ['3.13. Scanning Electron Microscopy (SEM)'], ['SEM examination was carried out using QUANTA 200 scanning electronic microscope', '(FEI Company, Hillsboro, OR, USA), with an integrated EDX system, GENESIS XM 2i EDAX', '(FEI Company, Hillsboro, OR, USA) with a SUTW detector and without any further treatments', 'at different magniﬁcations given on the image micrographs.'], ['3.14. Statistical Analysis'], ['Each analysis was performed in ﬁve replicates. Data were analyzed statistically with the one-way', 'analysis of variance ANOVA test using the SPSS statistics. Signiﬁcant differences between tested', 'samples and the control were deﬁned at p < 0.05.'], ['4. Conclusions'], ['Based on GC-MSD/FID analysis, it has been established that the commercially available thyme,'], ['rosemary, clove, and tea tree essential oils correspond to AFNOR/ISO standards.'], ['The results of this study demonstrate the potential of thyme oil, clove oil, and tea tree oil for use', 'as antifungal agents against tested fungi strains (Fusarium graminearum G87, Penicillium corylophilum', 'CBMF1, and Aspergillus brasiliensis ATCC 16404) and three potential pathogenic food bacteria:', 'Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. The overall results of the MIC values'], ['\x0cMaterials 2017, 10, 45'], ['20 of 24'], ['show that thyme oil is the most effective with a very low concentration used against both on the three', 'fungi and bacteria, followed by clove and tea tree oils, while the rosemary oil is less active or inactive.', 'Concerning the antioxidant activity, the IC50 of the analyzed essential oils is in the µg/mL range', 'that is assigned to a very good antioxidant activity which varies in the following order: clove oil >', 'thyme oil > rosemary oil > tee tree oil, and the clove oil exhibits the highest radical scavenging activity.', 'Promising results were obtained by their incorporation into chitosan emulsions and ﬁlms, with', 'potential applications in food packaging that will be detailed in a future paper. Taking into account', 'their biological activity in addition, it is expected that their use can also beneﬁt consumers’ health.'], ['On the basis of the obtained results, it can be concluded that these essential oils can be suitable', 'alternatives to chemical additives, satisfying the consumer demand for naturally preserved food', 'products and ensuring their safety at the same time.'], ['Acknowledgments: The research leading to these results was funded by the Romanian EEA Research Programme', 'operated by MEN under the EEA Financial Mechanism 2009–2014, project contract No. 1SEE/2014. We also thank', 'the Norwegian Research council for its ﬁnancial support, and Bioing. A. Diaconu for the DLS measurements.', 'Author Contributions: C.V., M.S., A.C.M., M.A.B., and E.S. designed research; E.S., M.A.B., J.T.R., E.E.T., W.K.,', 'D.P., C.P.C., and A.I. performed research; C.V., M.S., A.C.M., M.A.B., E.S., and D.P. analyzed the data; C.V., M.S.,', 'and A.C.M. wrote the paper. 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