Constituents of the Essential Oils of Garlic and Citronella and Their Vapor-phase Inhibition Mechanism against S.aureus

Abstract

The purpose of this study was to evaluate the vapor-phase antibacterial efficacy of garlic essential oil (GEO) and citronella essential oil (CEO) against four foodborne bacteria and their vapor-phase antibacterial mechanism against Staphylococcus aureus (S.aureus). GC-MS analysis confirmed that diallyl disulfide (29.258 %) and citronellal (36.940 %) were the major constituents of GEO and CEO, respectively. GEO and CEO at concentrations of 80 µL/L could completely inhibit the growth of all tested bacteria and the combinations of GEO with CEO displayed additive effects against the four tested bacteria with fractional inhibitory concentration index (FICI) values of 0.75 or 0.50. Furthermore, the scanning electron microscopy observations, the change of proteins concentration and catalase activity of S.aureus confirmed that the EOs impacting the membrane integrity and catalase activity of S.aureus. Thus, our study will provide a theoretical foundation for the applicability of EOs on the non-contact preservative packaging for food.

Introduction

Food safety has received increasing attention in recent years. Food spoilage caused by foodborne bacteria are the major problem of food safety (Zhang et al, 2015). Literatures report that Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) and other pathogens contaminated food is a serious threat to human health (Li et al., 2014; Patrignani et al., 2015). Among the classical pathogens that are considered threats to the safety of foods, S.aureus has been particularly concerning (Almadiy et al., 2016). Inhibiting foodborne pathogens is one of the main measures to prolonging the shelf life of food.

In recent years, a lot of researches have shown that various essential oils (EOs) which extracted from foodborne plants, have excellent antibacterial effect (Ammer et al., 2016; Mohammadhosseini et al., 2017), EOs and plant extracts of cinnamon, fennel, onions and garlic have in vitro anti-biofilm activities against S.aureus and E.coli (Benkeblia, 2004; Diao et al., 2014; Zhang et al., 2015). The utilization of various EOs to combat foodborne microbes is a promising approach in antimicrobial research, and some EOs have played major roles in food preservation (Van Haute et al., 2016; Yang et al., 2011). However, these studies have all focused on the contact type antibacterial mechanism and fresh-keeping effects of EOs, for example, the EOs are coated onto the surface of the food or incorporated into the packaging material, which can be paper, plastic or a biofilm (Camo et al., 2011; dos Santos Rodrigues et al., 2017; Kwon et al., 2017; Muriel-Galet et al., 2015). Direct contact with EOs can affect the sensory qualities such as the color of food and the consumers' desire for purchase, so to study the non-contact type antibacterial mechanism of EOs such as vapor-phase fumigation, will significantly broaden the applicability of EOs.

Studies have shown that the combination of EOs may result in synergistic antimicrobial effects, which can reduce the total volume of EOs required (de Medeiros Barbosa et al., 2016; Duarte et al., 2012; Luís et al., 2016). For food preservation, the use of a small amount of EOs may provide a balance between sensory acceptability and antimicrobial efficacy (Govaris et al., 2010; Michalczyk et al., 2012). Therefore using combination of EOs at appropriately lowered doses may have a positive effect on the inhibition of microorganisms in food and minimize the potentially negative effects of large doses of individual oils on the sensory qualities of food.

In this study, garlic essential oil (GEO) and citronella essential oil (CEO) were used as natural sanitizers. Garlic and citronella are the traditional seasonings in many Asian countries (Ud-Daula et al., 2016), and have been certified safe by the Food and Drug Administration (FDA).

However, plant EOs as antibacterial agents are inadequate for major practical use due to the limitations associated with their color, odor, liquid state and other related restrictions. The urgent need now is the search for innovative techniques for developing and strengthening the use of plant EOs as natural antimicrobials with high safety margins. In this context, the technology of controlled release microcapsules has become a new field of interdisciplinary research, and it is necessary to develop a kind of core material with broad spectrum activity and a good odor.

The purpose of this investigation was first to determine the principal chemical components of GEO and CEO by gas chromatography–mass spectrometry (GC/MS), then the vapor-phase minimum inhibitory concentrations (V-MICs) of the individual EOs on four kinds of tested bacteria and synergistic antibacterial activity of EOs were evaluated. These results would allow the compound EO would provide good antibacterial activity, the lower required volume of EO and an acceptable odor, and the mixture of EOs could serve as a non-contact antibacterial fresh-keeping packaging material, such as the core material for microcapsules. Finally, the vapor-phase inhibition mechanisms of the combination of GEO and CEO on S.aureus were explored by evaluation of the growth curve assay, scanning electron microscopy (SEM), and monitoring changes in the cell membrane integrity and catalase activity, and these data can provide a theoretical and technical foundation for the application of EOs in the food preservative packaging industry.

Materials and Methods

Bacteria and chemicals    Four foodborne and food spoilage bacteria, including gram-positive S. aureus ATCC 6538 and B. subtilis ATCC 6633 and gram-negative E. coli ATCC 8739 and S. enteritidis ATCC 49214, were used to assess the antimicrobial properties of the essential oils of garlic and citronella. All strains were obtained from the China General Microbiological Culture Collection Center and maintained in slants of nutrient agar (NA; Abxing, Beijing, China) at 4 °C. Active cultures were prepared by transferring a loop of cells from the agar slant into a test tube containing 5 mL of nutrient broth (NB; Abxing, Beijing, China) for bacteria. Cells were then incubated overnight at 37 °C to the logarithmic phase of growth. Culture purity was examined by streaking each culture on plates of NA for bacteria. The turbidity of each cell suspension was measured at 600 nm, and the suspensions were adjusted to the required concentration (approximately 1 × 10⁸ CFU/mL) using the McFarland standard. DuPont Tyvek (1073D) was purchased from Changzhou Road trading company of Jiangsu Province, and 500-mL PP disposable plastic bowls (bottom diameter 88 mm, height 68 mm, and top diameter 118 mm) were purchased from Chengdu AnBao Paper Group of Sichuan Province;Catalase (CAT) test kit (Visible light, product number: A007-1-1) was purchased from Nanjing Jiancheng Bioengineering Institute of Jiangsu Province.

Essential oils    Citronella, purchased from Yunnan Yunyao Polytron Technologies Inc., was dried to 40 °C to a constant weight and cut into 2-cm long pieces with scissors. Approximately 40 g of the pieces of citronella was mixed with 600 mL of water, subjected to ultrasonication at 420 W for 65 min, and steam distilled for 3 h. Garlic was purchased from Bo Jiang food market in the Hexi District of Tianjin City. Approximately 150 g of fresh peeled garlic was homogenized with a small amount of distilled water, added to 600 mL of distilled water at 35 °C, subjected to enzymolysis for 3 h, and steam distilled for 1.5 h. The GEO and CEO were collected, dried over anhydrous sodium sulfate and stored in amber bottles at −4 °C until analysis. The concentrations of EOs in a liquid phase rather than in the gas phase were reported in this study, because we wanted to report the total and accurate constituents of the EOs and their corresponding concentration rather than the partial volatile gas constituents and their corresponding concentration at a certain temperature.

GC/MS analysis    The constituents of the EOs were identified by GC/MS (VARIAN 4000 GC/MS, USA) using the method described by Han et al., (2014) with just a minor modification. of the temperature program used: the initial temperature (60 °C) was maintained for 3 min, then increased to 280 °C at 8 °C/min, and then 280 °C was maintained for 6 min.

Determination of the vapor-phase minimum inhibitory concentrations (V-MICs) of the essential oils    The V-MIC was defined as the lowest concentration of the volatile gas of EO required to prevent visible bacterial growth (de Medeiros Barbosa et al., 2016). The V-MICs were determined according to the method used by Duarte et al., (2012) with minor modifications. Briefly, 20 mL of agar medium was transferred to a 500-mL PP disposable plastic bowl, and after the agar had solidified, 100 µL of 10⁸ CFU/mL activated bacterial suspension was evenly spread on the agar plates. Sterile filter paper with a diameter of 6 mm was placed on the inside of the plastic bowl cover. A pipette was used to transfer 10, 15, 20, 25, 30, 35, 40, 45, and 50 µL of EO to the filter paper, and sterile water was used as the blank control. After the plastic bowl and the lid had been sealed with sealing film, they were inverted and cultured at 37 °C for 24 h before evaluation.

Determination of the fractional inhibitory concentration index (FICI)    The checkerboard method described by Arabe Rima de Oliveira et al., (2015) with a slight modification was used to assess the synergistic inhibition in vitro. The checkerboard method used an agar pipette that was divided into four regions, and 2 µL of the different bacterial suspensions were individually added to the different areas. Then, the essential oil of each group was transferred to a piece of sterile filter paper, which was placed on the disposable bowl cover, and then the bowl and the cover were sealed, inverted and cultured at 37 °C for 24 h.

The antimicrobial effects of the essential oil combinations were evaluated based on their FICI values. FICI serves as a simple mathematical approach to quantitatively describe interactions (de Medeiros Barbosa et al., 2016; Han et al., 2013), and it was calculated as Eq. 1:   

The concentrations of GEO and CEO were assayed at 1 V-MIC, 1/2 V-MIC, 1/4 V-MIC and 1/8 V-MIC individually and in combinations with the other one. The results were interpreted as synergy (FICI ≤ 0.5), additive (0.5 ≤ FICI≤ 1), indifference (1 ≤ FICI ≤ 4) and antagonism (FICI > 4).

Methods of elucidating the antimicrobial mechanism

Vapor-phase bacteriostatic tests    A 0.25-mL aliquot of activated S.aureus suspension with a concentration of approximately 1 × 10⁸ CFU/mL was added to 24.75 mL of fresh broth medium. The samples were transferred to paper cups that were adhered together with Tyvek1073D, and the bottoms of the paper cups were arranged in a sawtooth pattern. The suspensions were incubated at 37 °C to the logarithmic growth phase in the air bath of a constant temperature oscillator (120 rpm). The EOs were then placed on the bottoms of the 500-mL PP plastic bowls; the cups were placed in the bowls, the bowls were sealed, and culturing of the bacterial suspensions was continued (Table 1). For the blank, an equivalent amount of sterile normal saline (0.85 % NaCl) was added instead of the oil. Samples were taken at 0, 1, 2, 3, 5, 9, and 11 h to evaluate the indicators.

Table 1. The proportions of the essential oils used to evaluate the antibacterial mechanism

Added amount of EOs µL/L
Groups	GEO	CEO
CK	-	-
GEO	160	-
CEO	-	160
GC	80	80

Note: “-” indicates that the group was not included in the essential oil mixture; GC represents a mixture of GEO and CEO.

The evaluation of the cell integrity of S.aureus    The cell integrity of the S.aureus strains was established by measuring the release of cell constituents into the supernatant according to the method described by Ma et al., (2016); and Moghimi et al., (2016) with some modifications. The release of protein being determined by measuring the OD₂₈₀ values of the cells treated with the test solutions. At 0, 2, 4, 8, and 10 h after the addition of the EOs, 2-mL aliquots of the bacterial suspensions were collected, combined with 4 mL of sterile normal saline (0.85 % NaCl), and centrifuged at 3000 rpm for 10 minutes at 27 °C. The optical density values of the supernatants of the bacterial suspensions were measured at 280 nm by a UV-visible spectrophotometer (UV-2700, Shimadzu Corporation, Japan). The bacterial suspension with sterile normal saline was used as the blank control.

The determination of the catalase activity (CAT) of S. aureus    At 0, 2, 4, 6, 8, and 10 h after the addition of the EOs to each bacterial suspension, 0.9-mL aliquots were removed and diluted 1/10 with sterile normal saline. The dilute samples were ultrasonicated (power 300 W) in an ice water bath for 5 min, centrifuged at 2500 rpm for 10 minutes at 27 °C, and then 0.1 mL of the supernatant was diluted with 0.9 mL of sterile normal saline. Then, 0.05 mL of the dilute solution was used to test the catalase activity of the bacterial solution using a UV–visible spectrophotometer at 405 nm (0.5 cm path length) with a catalase (CAT) test kit. The activity was calculated according to the instructions of the catalase (CAT) test kit.

Scanning electron microscopy (SEM) analysis    SEM was used to explore the effects of the EOs on the morphology of S.aureus according to the method described by Zhang et al., (2015) with some modifications. The bacteria were incubated in broth medium at 37 °C, and the suspensions were treated with combinations of the EOs in different proportions (Table 1). All samples were incubated at 37 °C for 10 h, and after incubation, the suspensions were centrifuged at 3000 rpm for 5 min and washed twice with 0.1 M phosphate buffer solution (PBS, pH 7.4). The bacterial cells were fixed in 2.5 % glutaraldehyde for 10 h at 4 °C. The samples were dehydrated in a graded ethanol series (30 %, 50 %, 70 %, 85 %, 90 %, and 100 %), then cooled to −50 °C for 4 h, and vacuum freeze dried for 24 h until the samples were completely dry. Finally, all samples were sputter-coated with gold in an ion coater for 2 minutes and analyzed using a scanning electron microscope (SU-1510, Hitachi Corporation, Japan).

Reproducibility and statistics    All experiments were performed in triplicate. In addition, the data are expressed as the means ± SD. Analysis of variance (ANOVA) using the least squares difference method of the general linear model procedure in SPSS17.0 was used to express the significance of the differences between means. Differences were considered significant at the P > 0.05 level.

Results and Discussion

Chemical composition of the EOs    The main components (>0.1 %) of GEO and CEO are listed in Tables 2–3 according to their elution order (the contents of constituents greater than 3 % are shown in bold). Twenty-eight compounds were tentatively identified in GEO, and they account for 98.312 % of the total peak area. Twenty-nine compounds accounting for 99.196 % of the total peak area were tentatively identified in CEO.

Table 2. Main chemical constituents of GEO

No	Compositions	Area percentage (%)
1	(Z)-methyl 1-propenyl sulfide	0.158
2	Dimethyl disulfide	0.148
3	Diallyl sulfide	1.369
4	1-Propene,1,1′-thiobis-	0.129
5	Thiophene,2,4-dimethyl-	0.189
6	1,3-Dithiane	3.670
7	Methyl prop-1-enyl disulphide	0.499
8	1,2-Bis(methylthio)ethene	1.047
9	Dimethyl trisulfide	22.150
10	Thiourea,N,N,N′-trimethyl-	0.406
11	Diallyl disulfide	29.258
12	2-Ethenyl-1,3-dithiane	4.164
13	2-Methyl-1,3-oxathiane	1.415
14	2-Methoxy-3-methyl-butyricacid	7.582
15	1,3,5-Trithiane	0.399
16	3-Ethenyl-3,6-dihydrodithiine	5.251
17	Disulfide,bis(1,1-dimethylpropyl) (9CI)	2.451
18	3-Vinyl-3,4-dihydro-1,2-dithiine	1.750
19	2-Ethylidene-1,3-dithiane	0.160
20	8-Thiabicyclo[3.2.1]octane	0.240
21	Carvacrol	0.966
22	Diallyl trisulfide	4.112
23	3-Methoxythiophene	3.004
24	1-Propene,1,1′-thiobis-	0.165
25	1,3-Dithiole-2-thione	0.669
26	Diallyltetra sulfide	1.447
27	2-Hydroxy-5-methyl-p-benzoquinone	3.076
28	Cyclic octaatomic sulfur	2.438

Table 3. Main chemical constituents of CEO

No	Compositions	Area percentage (%)
1	Beta-Pinene	0.805
2	Bicyclo[2.2.1]heptane,7,7-dimethyl-2-methylene-	4.627
3	(Z)-13,7-dimethyl-3,6-octatriene	0.120
4	Linalool	0.195
5	1,3-Cyclohexadiene,1,3,5,5-tetramethyl	0.120
6	Citronellal	36.940
7	Cyclohexanol,5-methyl-2-(1-methylethenyl)-	0.881
8	1-Methoxy-3,7-dimethyl-octa-2,6-diene	0.183
9	Citronellol	16.320
10	Geraniol	16.780
11	Carane	1.451
12	Eugenol	0.621
13	Geranyl butyrate	0.320
14	β-Elemene	3.511
15	β-Cubebene	0.511
16	(-)-Isoledene	0.111
17	Naphthalene,1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-	0.516
18	(3aS,3bR,4S,7R,7aR)-7-Methyl-3-methylidene-4-(propan-2-yl)octahydro-1H-cyclopenta[1,3]cyclopropa[1,2]benzene	1.983
19	a-Muurolene	0.828
20	β-Cadinene	4.054
21	a-Muurolene	0.116
22	Cyclohexanemethanol,4-ethenyl-a,a,4-trimethyl-3-(1-methylethenyl)-, (1R,3S,4S)-	4.030
23	1-Hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene	0.195
24	Selinenol	0.263
25	t-Cadinol	0.716
26	a-Eudesmol	1.981
27	Hexadecanoic acid, methyl este	0.252
28	Thunbergol	0.164
29	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	0.602

Diallyl disulfide (29.258 %), dimethyl trisulfide (22.150 %), 2-methoxy-3-methyl-butyricacid (7.582 %), 3-ethenyl-3,6-dihydrodithiine (5.251 %), 2-ethyl-1,3-dithiane (4.164 %), diallyl trisulfide (4.112 %), 1,3-dithiane (3.670 %), 2-Hydroxy-5-methyl-p-benzoquinone (3.076 %), and 3-methoxythiophene (3.004 %) were the major components of GEO.

Citronellal (36.940 %), geraniol (16.780 %), citronellol (16.320 %), bicyclo[2.2.1]heptane-7,7-dimethyl-2-methylene (4.727 %), β-cadinene (4.054 %), and cyclohexylmethanol (4.030 %) were the major components of CEO and accounted for 82.85 % of the total peak area. In addition, there are small amounts of a variety of alkenes, alcohols, lipids and other compounds.

Previous reports have also determined the components of GEO and CEO. For example, Timung et al., (2016) used GC/MS analysis to evaluate the composition of hydro-distilled citronella oils extracted from different parts of the plant (leaves, stems and whole aerial) and found 50 compounds in the CEO. Out of those 50 compounds, 13 accounted for 99.210 % of the total peak area, and those 13 compounds included citronellal, citronellol, and geraniol. Similarly, other researchers have reported these three constituents are the major constituents of citronella (Kakaraparthi et al., 2014; Teixeira et al., 2013).

According to the results reported by El-Sayed et al., (2017), the major components of white-skin garlic were diallyl trisulfide followed by diallyl disulfide. These two components together account for 61.39 % of the total composition, which is consistent with our results.

Antibacterial activity of EOs    The antibacterial activity of EOs was qualitatively and quantitatively assessed by the values of vapor-phase minimum inhibition concentration (V-MIC).

As shown in table 4, two EOs exhibited antimicrobial activity against all tested bacteria. Untreated group (sterile normal saline) did not show inhibitory effects on any of the tested bacteria. Analysis of V-MICs showed that two EOs showed potent, broad inhibitory activity (V-MIC<100µL/L). V-MICs for EOs in the tested bacteria were measured as 60 °C 80 µL/L, we can see that the concentration of the volatile oil gas volatilized from 80 µL liquid GEO or CEO in 1 L sealed container could completely inhibit the growth of all tested bacteria, which indicated that the two EOs had good vapor-phase antibacterial effect on common bacteria, that is to say, when EOs are not in contact with food, it is feasible to use EO volatile gas for food preservation.

Table 4. V-MICs of the EOs on the four tested bacteria

V-MIC(µL/L)
EOs	E. coli	S.enteritidis	S. aureus	B.subtilis
CEO	70	70	80	60
GEO	70	80	60	80

Note: “60”, “70”, “80“ represent the concentration of the volatile oil gas which volatilized from 60 µL, 70 µL, 80 µL liquid GEO or CEO in 1 L sealed container, respectively.

The fractional inhibitory concentration indices (FICIs) of mixtures of two EOs    To minimize the undesirable effects of EOs on the organoleptic properties of food, we investigated the synergistic activities of the two EOs.

The FICIs of the mixed EOs based on the checkerboard test are shown in Table 5. Based on the FICI scale, the combination of GEO and CEO displayed a useful additive effect with FICIs of 0.75 against E. coli, S. aureus and S. enteritidis and 0.50 against B. subtilis, which both additive effect.

Table 5. FIC indices (FICIs) of mixtures of EOs against the tested bacteria^a

EOs	E.coli		S.enteritidis		S. aureus		B.subtilis
	FIC	FICI	FIC	FICI	FIC	FICI	FIC	FICI
GEO	0.25	0.75(A)b	0.25	0.75(A)	0.25	0.75(A)	0.25	0.5(A)
CEO	0.5		0.5		0.5		0.25

a  The results are the means of triplicate experiments.

b  The results were interpreted as synergy (S,FICI≤0.5), addition(A,0.5≤FICI≤1), indifference(I,1≤FICI≤4) and antagonism (AN, FICI>4)

de Medeiros Barbosa et al., (2016) observed that the combined application of OEO and rosemary EO shows synergistic effects in the inhibition of L. monocytogenes, E. coli and S. enteritidis. Kirkpinar et al., (2011) determined the individual and combined effects of OEO and GEO on the intestinal microflora of broilers, and their results showed that the clostridium counts using a combination of the oils were significantly lower than the counts when the oils were used individually.

Additive effect was observed in the present study, indicating that the combination of GEO and CEO can efficiently inhibit the growth of the tested strains at lower concentrations than those required when the EOs are used individually; in addition, the GEO has a special odor while CEO aromatic flavor and has strong antibacterial effect of vapor-phase. Adding a certain amount of CEO to GEO can further reduce the amount of mixed EOs and make it smell pleasant. Therefore, the combination of the two EOs can get a pleasant smell and good antibacterial effect on common bacteria,we only need to utilize volatile gas of mixed EOs in food preservation so that we can completely change the physical state of the EOs, such as solidify EOs in the form of microcapsules, and placing it in the non-woven bag used as non-contact fresh-keeping material. When it be used in the field of fresh-keeping of fresh agricultural products and fresh meat, can avoid direct contact between food and antibacterial agent, reduce the impact of EOs on the sensory qualities such as the color of food and the consumers' desire for purchase, prolong the shelf life of fresh agricultural products and fresh meat, achieve the purpose of antiseptic preservation. If we control the release rate of the volatile gas of the essential oil and release it at a certain rate of release in a specific environment, we can save the dosage of the preservative and achieve a better effect of long-term preservation.

Antibacterial mechanism

Effect of the EOs on the cell integrity of S.aureus    The cell membrane can maintain the stability of the intracellular solution and the normal life activity of the cell. When bacteria are exposed to an antiseptic, such as the essential oil of a plant, the function of the cell membrane is damaged, and intracellular substances such as proteins can be exuded. It is easy to determine the protein contents with an UV-visible spectrophotometer at 280 nm. Therefore, the optical densities of the bacterial suspension at 280 nm (OD₂₈₀) can be used as indicators of the effect of the essential oil or oils on the integrity of the S.aureus membrane (Ahmad et al., 2017; Li et al., 2015).

The influence of different combinations of EOs on the protein contents in the S.aureus suspensions are shown in Figure 1.

Fig. 1.

The curve of the OD₂₈₀ value of the S.aureus

Figure 1 shows that the OD₂₈₀ values of the S.aureus in the CK group fluctuated but remained at approximately 0.225 throughout the experiment, indicating that the S.aureus grew normally. The OD₂₈₀ values of the treated groups increased with time, indicating that the volatile gases of the individual or combined EOs destroyed the S.aureus cell membranes, leading to leakage of the intracellular substances, such as proteins, from the cells (Bajpai et al., 2013). The OD₂₈₀ values of the S.aureus in the GEO and CEO groups increased from 0.225 to 0.238 and 0.250, respectively, and were less than the final value of the GC group (0.260). The results were clearly indicated that the inhibitory effects of the volatile gases from the combinations of EOs on S.aureus were stronger than those of the individual EOs.

The changes in the contents of the intracellular substances of the S.aureus further confirmed the destructive effect of volatile gas fumigation on the cell membrane of S.aureus. Based on the release of proteins into the media, the gases can increase the permeability of the S.aureus cell membranes, and the normal living environment and metabolism of S.aureus were severely damaged, which led to S.aureus cell death.

Other researchers have reported similar modes of action. EOs can destroy the integrity of cell membranes and change the membrane permeability, which leads to the leakage of important cell contents, such as proteins (Cui et al., 2015; Gao et al., 2011; Knezevic et al., 2016; Skočibušić et al., 2006).

There are several reports that support that the bioactive components in the EOs might penetrate the phospholipid bilayer of the cell membrane and disturb the structural integrity of the cell membrane, which is detrimental to cell metabolism and can lead to cell death (Bajpai et al., 2013; Lv et al., 2011).

Based on their tests of the membrane permeability and protein leakage and scanning electron microscopy analysis of S.aureus, C. Chen et al., (2018) reported that the antimicrobial activity of garlic extracts may be due to the destruction of the structural integrity of cell membranes, leading to cell death. Diao et al., (2014) believed that the hydrophobicity of aromatic essential oils and their components enables them to bind to cell surfaces and penetrate into the cell plasma membrane. Z. Chen et al., (2016) reported that essential oils might increase bacterial membrane permeability and directly cause destruction of the cellular structure.

Effects of the EOs on the catalase (CAT) activity of the S.aureus    The changes in catalase activity reflect the physiological condition of the S.aureus and can be used as an important physiological indicator to determine if the S.aureus has been damaged external factors.

Figure 2 shows the changes in the CAT activity in the S.aureus after gas fumigation with various EOs or combinations of EOs. The CAT activity of the S.aureus in the CK group was stable at approximately 49.80 U/mg-prot throughout the experiment, while the CAT activities of the treated groups initially increased and then decreased. Of the treated groups, the activity of the GC group increased most rapidly, followed by those of the GEO and CEO groups. The CAT activities of the GC, GEO and CEO groups reached 75.88 U/mg-prot, 55.56 U/mg-prot and 64.46 U/mg-prot, respectively, at 2 h and 4 h. The results showed that fumigation with the vapor of individual or combinations of EOs caused rapid oxidative damage to S.aureus by increased the intracellular hydrogen peroxide content in the S.aureus.

Fig. 2.

The effects of different combinations of essential oils on the CAT activities in S. aureus

The higher the CAT value, the more damage was caused to the bacteria, so the combined effect seen in the GC group after 2 h was greater than the effects seen in the GEO and CEO groups. After 2 h, the CAT activities of the S.aureus decreased, indicating the S.aureus had been destroyed. The intrinsic antioxidant system of the S.aureus was unable to mitigate the threat of the EOs, which lead to the apoptosis of the S.aureus, causing the CAT activities in the S.aureus to decrease significantly. The activity in the GC group drops fastest, and the value is 24.39 U/mg-prot after 10 h, which is the lowest activity seen in any group, all the values of the treatment groups were lower than that of the CK group (46.97 U/mg-prot). This result indicated that fumigation with these gases can destroy the catalase in the S.aureus, and the GC group, which was treated with two EOs, showed the most damage.

A few studies have reported that some EOs possess strong antioxidant activities (Xiang et al., 2018, 2017). Bajalan et al., (2017) proved that the antioxidant activity of rosemary EOs increased steadily with increasing concentration. Nakkala et al., (2017) observed that cells in which apoptosis had been induced were associated with alterations to the cellular oxidant/antioxidant balance. Zardi-Bergaoui et al., (2018) argued that loss of catalase activity can cause a number of adverse effects such as cell death. Therefore, fumigation with the gases of the selected EOs can destroy the catalase of the S.aureus and lead cell death.

The effects of the EOs on the morphology of S.aureus    SEM analysis of bacteria treated with EOs was carried out to observe any morphological changes. As shown in Figure 3, the S.aureus cells of CK group were plump, round and smooth, with typical S.aureus globular cell structure and intact structure (column. 3A) , In contrast, some cells treated with GEO and CEO atrophied and lost their luster, and the cell structures of which had been deformed and shriveled. (column. 3B-3C). Scanning electron microscopy showed that vapor fumigation with single EO could result in severe membrane damage to S.aureus cells.

Fig. 3.

SEM pictures of S.aureus cells

Although two EOs are used only half the amount of their individual, however, it can be seen clearly from figure 3 (column. 3D) that the ultrastructural damage of S.aureus under low concentration of compound EOs is more obvious than that of a single high concentration of EO. Almost all S.aureus cells were obviously deformed, the cells were shrivelled and depressed, and the cell structure deformation is more severe, cell depression is more obvious. This phenomenon also demonstrates, intuitively, that compound of two EOs has better antibacterial properties than single EO.

The antimicrobial effect of OEs seems to be enhanced when studied in synergy that can be resulted from the mixture of two or more different EOs, Moon et al., (2011) hypothesized this may be the result of interactions of different active components of both EOs.

Pei et al., (2009) have hypothesized that synergism effect shown by eugenol and carvacrol, could increase the cell membrane permeability, considered it might be due to carvacrol disintegrate the outer cell membrane of S.aureus and facilitates the entry of eugenol into the cytoplasm which finally interacts with proteins and enzymes.

In this study, the results of scanning electron microscopy observation evidenced that the combination of many kinds of EOs could enhance antimicrobial effect by cause more destructive to S.aureus, this might due to the interactions of different active components of EOs.

Conclusion

Using EOs as natural antimicrobial agents is an attractive approach to food preservation. Current research has identified the major constituents of GEO and CEO, explored the vapor-phase antibacterial activities of these two EOs as natural antimicrobial agents, and provided insight into their vapor-phase antibacterial mechanisms on S.aureus. The scanning electron microscopy observations, the change of proteins concentration and CAT activity of S.aureus showed that the EOs volatile gas could destroy the membrane integrity and catalase activity of S.aureus, and then inhibit the growth and led to cell death of S.aureus. Therefore, if the essential oil or essential oils is encapsulated in some breathable materials, such as microcapsules, it can inhibits the bacteria in the meat (and other foods) and keeps the meat fresh when not in contact with meat. At the same time, some problems can be avoided, For example, EOs direct contact with food can affect the sensory qualities such as the color of the food and the consumer's desire to purchase the product, so the elucidation of the non-contact antibacterial mechanisms, such as vapor-phase fumigation, of the EOs will significantly broaden the applicability of EOs and prove the broad potential of the broad potential of the combination of GEO and CEO in non-contact packaging for fresh foods. Future research should be carried out to test and verify the effects of mixtures of EOs in the form of microcapsules for food preservation applications.

Acknowledgments    This work was supported by the ‘13th Five-Year’ scientific research & innovation team of Tianjin University of Science and Technology (green food packaging and intelligent packaging) and the ‘12th Five-Year’ National Science and the Technology Support Project of People's Republic of China (Grant No. 2015BAD16B05-02).

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