GC–MS Profiling of Naturally Extracted Essential Oils: Antimicrobial and Beverage Preservative Actions

The purpose of this study was to demonstrate the antimicrobial effects of natural essential oils (EO) and determine their preservative action. Eight natural essential oils were tested against Staphylococcus aureus, Escherichia coli, and Candida albicans representing gram positive, gram negative, and fungi, respectively. The plant materials were used in this study viz. Thymus vulgaris—thyme (TV), Mentha virdis (MV), Mentha longifolia (ML), Rosmarinus officinalis—rosemary (RO), Lavandula dentata—lavender (LD), Origanum majorana—oregano (OM), which belong to the Lamiaceae family. The other two plants were Cymbopogon citratus—lemon grass (family Poaceae) (CC), and Eucalyptus globulus (family Myrtaceae) (EG). Employing the disc diffusion susceptibility test, minimum inhibitory and minimum bactericidal concentrations were estimated for each oil, followed by the addition of oils to pasteurized apple juice after microbial induction. The results revealed that thyme oil showed the maximum zone of inhibition against all tested microbes enriched with monoterpenes class viz. eucalyptol (24.3%), thymol (17.4%), and γ-terpinene (15.2%). All other tested oils exhibited a concentration-dependent inhibition of growth and their MIC ranged from 0.1 to 100 µL/mL. The recorded minimum bactericidal concentration values were apparently double the minimum inhibitory concentration. The EO of Mentha virdis followed by Mentha longifolia showed maximum antimicrobial activity against the tested organisms in pasteurized apple juice. A gas chromatography–mass spectroscopy (GC–MS) analysis of lemon grass, thyme, and Mentha virdis essential oils showed their enrichment with monoterpenes class recording 97.10, 97.04, and 97.61%, respectively.


Introduction
Food poisoning is a widespread, life-threatening illness that is considered an important public health problem [1]. Its main cause is consuming food or beverages contaminated with viruses, bacteria, fungi, or their toxins. Foodborne disease incidence can be affected by climate factors such as temperature, humidity, and rainfall [2]. Eating raw vegetables, fruits, unpasteurized dairy, and raw meat or fish may increase the risk of contamination. On the other hand, some types of food might be contaminated by microbes through harvesting, improper storage, or even transport, which may lead to cross contamination upon improper cooking [3]. Mild cases of food poisoning may be improved without the need of drug treatments, others may be hospitalized. Pathogenicity of food poisoning illness may differ depending on the source of contamination and individual susceptibility. Population as immunocompromised, children, pregnant women, and the old aged, being more susceptible, show serious and life-threatening symptoms [4]. Food contaminating organisms may be bacteria, such as Staphylococcus aureus and Escherichia coli, or Candida albicans fungus [5]. Food contamination would be via infectious microorganisms and toxins at any stage of processing or production from the farm-to-table causing foodborne illnesses. Food poisoning symptoms appear hours or days after consuming contaminated food and frequently results in vomiting, nausea, watery or bloody diarrhea, and dehydration [6]. from different plant origins were extracted from members of the Lamiaceae family viz. Oregano, lavender, rosemary, lemon grass, Mentha virdis, Mentha longifolia, and thyme, in addition to Eucalyptus (Myrtaceae) and lemon grass (Poaceae) cultivated in Egypt. Further, in this study, the authors compared the activity of the extracted essential oils with limonene and eucalyptol selected as authentic volatiles being potent antimicrobial agents [25].

Preparation of Volatile Oils
The dried grinded plants, each (500 g), were subjected to water distillation (750 mL) for 3-6 h using a Clevenger-type apparatus until plant exhaustion. For each plant, the extraction was carried out in triplets. The EOs yield and their chemical composition variation "in a quantitative not qualitative way" would vary, and so replicates of experimental procedures in EOs extraction and their GC-MS analysis would be encouraged to guarantee the reproducibility and data accuracy [26]. The yield of the volatile oils (expressed as volume mL/500 g weight) per each plant was 6.0 ± 0.10, 5.5 ± 0.21, 4.0 ± 0.12, 4.8 ± 0.11, 5.8 ± 0.14, 5.6 ± 0.30, 6.4 ± 1.20, and 5.0 ± 24 mL for the plant material (TV), (MV), (ML), (RO), (LD), (OM), (CC) and (EG), respectively. The obtained oils were placed in desiccator after collecting and kept at −4 • C in sealed vials the in dark for further analyses. The physical properties of EOs were assessed as per the Egyptian pharmacopoeia 1984 [27].

Microorganisms
The microorganisms studied in this research represent frequent organisms involved in infections related to healthcare and food poisoning. Thus, clinically-pure strains of Escherichia coli representing gram-negative bacteria were isolated on Sorbitol MacConkey agar (Difco TM ), Staphylococcus aureus representing gram-positive bacteria isolated on Mannitol salt agar (MSA) (Difco TM ), and Candida albicans fungus isolated on Sabouraud Dextrose agar (Difco TM ), all acquired from The British University in Egypt.

Juice Sample
Fresh apple juice (pH 6.5-7) was used to assess the preservative potential of the essential oils of the plants. Whole apple fruits were machine-squeezed and then underwent multiple filtration steps through filter paper until obtaining clear juice. Clear apple juice portions of 100 mL were placed in 250 mL bottles. This was followed by pasteurization of the juice by autoclaving at 70 • C for one minute followed by rapid cooling to 7 • C.

The Disk Diffusion Susceptibility Test
The disk diffusion susceptibility test was performed using Mueller-Hinton Agar (MHA) to assess the antibacterial and antifungal effects of the essential oils. Three concentrations of the essential oils were tested: 100% oil, 50% oil (diluted with ethanol with ratio 1:1), and 25% oil (diluted with ethanol with ratio 1:4). Four 6 mm sterile filter paper discs saturated with 20 µL of each of the dilutions were applied on plates on which each of the test organisms were streaked at a concentration equivalent to 0.5 MacFarland standard, and a disc impregnated with 20 µL solvent alone was used as a blank. A standard antibiotic disk was applied as a positive control. The antimicrobial activity was assessed by measuring the diameters of zones of inhibition after a period of incubation (18-24 h for bacteria and 48-72 h for C. albicans). Measuring the diameter of zones of inhibition in millimeters was performed using a Vernier caliper (together with the diameter of the disc). Three readings average were recorded. Zones of inhibition equivalent to or more than 7 mm reflected the antimicrobial activity of essential oils against the test organisms. An activity index (AI) of the tested essential oils was calculated, where the inhibition zone diameter of the tested essential oil was divided by that of the standard antimicrobial agent, and where an activity index greater than 0.5 was regarded as significant antimicrobial activity [28].

Minimum Inhibitory Concentration (MIC) Determination
The MIC of essential oils was estimated by the broth dilution method for the selected test organisms which resulted in diameters of inhibition zones of more than 7 mm. Bacterial and fungal test strains dilutions were prepared using Mueller Hinton Broth (Difco TM Detroit, MI, USA) and Sabouraud Dextrose Broth (Difco TM , Detroit, MI, USA), respectively. Five 2-fold serial dilutions of essential oils were prepared with the highest and lowest concentrations of 2000 µL·mL −1 and 125 µL·mL −1 for bacteria and fungi. The final volume of the prepared concentrations was adjusted to the number of the test organisms. Standard antibiotics (Clotrimazole against fungi and Ofloxacin against gram-positive and gramnegative bacteria) were prepared using the same procedure. To each of the dilution and control tubes containing broth only, a standard inoculum (1.5 × 10 8 CFU·mL −1 ) was added to reach final highest and lowest concentrations of 1000 and 62.5 µg·mL −1 for bacteria and 10,000 and 625 µg·mL −1 for fungi. After the incubation time (18-24 h for bacteria, 5-10 days for fungi), the test tube with the least concentration of essential oils with no visible growth was regarded as the MIC against the test microbe. An average of three readings was recorded as MIC. In this study, a MIC of less than 100 µL·mL −1 was considered as good antimicrobial activity, MICs of 100-500 µL·mL −1 with moderate activity, MICs of 500-1000 µL·mL −1 with weak activity, and MICs greater than 1000 µL·mL −1 with no activity [28].

Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) Determination
MBC and MFC determination were performed by taking 0.1 mL from MIC tubes showing no observable growth was inoculated onto Mueller Hinton agar (Difco TM , Detroit, MI, USA) and Sabouraud Dextrose agar (Difco TM , Detroit, MI, USA) for fungi by the spread plate method. At the end of incubation time (18-24 h for bacteria and 5-10 days for fungi), the lowest concentration of essential oils with no observable growth on subculture was regarded as its MBC and MFC against the test microbe. The ratios of MFC:MIC or MBC:MIC were estimated to determine the antifungal or antibacterial activity of essential oils against the test microbes, respectively. The compound is bactericidal or fungicidal when the ratio is between 1:2 to 2:1, and it is bacteriostatic or fungistatic if the ratio is greater than 2:1 [28].

Induction of Microorganisms in Juice
The concentrations of the 3 different microbial strains under investigation were adjusted to 1.5 × 10 8 CFU.mL −1 (0.5 McFarland) and each were grown in its relevant media.
One mL of each microbial suspension was added to 4 mL sterilized apple juice to form a total volume of 5 mL in the sterile falcon tube. This step was immediately followed by the addition of the essential oils under investigation at concentrations according to their minimum inhibitory concentration (MIC) obtained values and stored at room temperature (Supplementary Table S1). Two control tubes were prepared, one containing juice and microbial strain only and the other contained juice only.
Rigorous antiseptic measures were applied throughout the sample preparation and inoculation to prevent any possible microbial contamination.

Bacterial Count/Viable Count
The original sample was diluted so that a range of 30 to 300 colonies of the test bacterium are grown. A number of dilutions were cultured to be certain that a suitable number of colonies will be grown (500 µL, 200 µL, and 100 µL). Serial dilutions of the sample in sterile water were performed (1:10, 1:100, 1:1000 etc.). This was followed by cultivation on a nutrient agar dish then sealed and incubated. The media used include nutrient agar for the S. aureus count or MacConkey agar to count E. coli gram-negative bacteria or Sabouraud Dextrose agar for C. albicans. One set of dishes were incubated at 22 • C for 24 h and a second set at 37 • C for 24 h. Colonies are counted by eye at the end of the incubation time.

GC-MS analysis of Essential Oils
An analysis of each essential oil was carried out separately via gas chromatographymass spectrometry (GC-MS) where the procedure was adopted from a previous work [29]. A system operating Shimadzu GCMS-QP2010 (Tokyo, Japan) was used with the following conditions: The column (RTX-5 MS) was used with specifications that were (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek Corporation, Bellefonte, Pennsylvania, USA). The starting temperature of the column was 45 • C for 2 min and then increased to 300 • C at a rate of 5 • C/min and kept steady for 5 min. The temperature of the injector was 250 • C. The flow rate of helium (carrier gas) was (1.41 mL/min). The following conditions were applied when recording the mass spectra: (equipment current) filament emission current, 60 mA; ionization voltage, 70 eV; ion source, 200 • C. Automatic injection of the essential oil was at (1 µL, 1% v/v) with a splitting ratio (1:15). The identification of volatile metabolites was performed upon comparing the mass spectra as well as the retention index with those of the National Institute of Standards and Technology's (NIST) chemistry webbook library. In addition, literature data was used to identify n-alkanes series by comparing their mass spectra and retention indices.

The Disk Diffusion Susceptibility Test
The disk diffusion method was used to test the antibacterial and antifungal activities of the essential oils against the selected microorganisms: E. coli as gram-negative bacteria, S. aureus as gram-positive bacteria, and C. albicans representing fungi. The oils were tested in 3 concentrations: 100%; 50%, and 25%. The antibacterial and antifungal activities were estimated according to the American Society for Microbiology, where zones of diameter < 12.00 mm were considered resistant, zones of diameter ranging 13.00-14.00 mm were considered with intermediate activity, and zones of diameter more than 15.00 mm were considered susceptible. The tested oil concentrations showed variable activities according to the results shown in Table 1 and Figure 1. The results showed that thyme (100% and 50% concentrations) and limonene (all concentrations) showed the maximum activity against E. coli, whereas thyme (100% concentration) and lemon grass (all concentrations) showed maximum activity against S. aureus. Against C. albicans, the oils showed decreasing activity according to the following order: thyme (100% and 50% concentration), limonene (100% and 50% concentration), Mentha virdis (100% concentration) and lemon grass (100% concentration), while 25% limonene and 50% lemon grass were the same in showing the least activity.

Minimum Inhibitory Concentration (MIC) Determination
The MIC of oils was determined against the selected strains (E. coli, S. aureus, and C. albicans) where the oils exhibited concentration-dependent inhibition of growth and the MIC of oils ranged from 0.1 to 100 µL/mL as shown in Table 2. The table shows that the least MIC (0.1 µL/mL) against E. coli was shown by lemon grass whereas the highest MIC (25 µL/mL) was shown by oregano and lavender, whereas against S. aureus, the least MIC (0.2 µL/mL) and the highest MIC (100 µL/mL) were shown by lemon grass and limonene, respectively. Lemon grass and thyme showed the least MIC against C. albicans (0.8 µL/mL) and the highest MIC (12.5 µL/mL) was shown with Mentha longifolia.

Minimum Inhibitory Concentration (MIC) Determination
The MIC of oils was determined against the selected strains (E. coli, S. aureus, and C. albicans) where the oils exhibited concentration-dependent inhibition of growth and the MIC of oils ranged from 0.1 to 100 µL/mL as shown in Table 2. The table shows that the least MIC (0.1 µL/mL) against E. coli was shown by lemon grass whereas the highest MIC (25 µL/mL) was shown by oregano and lavender, whereas against S. aureus, the least MIC (0.2 µL/mL) and the highest MIC (100 µL/mL) were shown by lemon grass and limonene, respectively. Lemon grass and thyme showed the least MIC against C. albicans (0.8 µL/mL) and the highest MIC (12.5 µL/mL) was shown with Mentha longifolia. The results in Table 2 shows that the least MBC against E. coli was with lemon grass, Mentha longifolia and rosemary at concentrations 0.2 µL/mL, 0.4 µL/mL, and 0.8 µL/mL, respectively, whereas Mentha virdis, thyme and limonene showed the highest MBC (3.2 µL/mL). Whereas against S. aureus, the least MBC was 0.4 µL/mL and 3.2 µL/mL with thyme and lemon grass, respectively, whereas MBC was highest with Eucalyptus (50 µL/mL). Regarding C. albicans, the least MBC (1.6 µL/mL) was determined with thyme and lemon grass, whereas oregano, rosemary and lavender appeared to have no fungicidal activity at all. Table 2 also shows that the recorded MBC values were apparently double the MICs.

Bacterial Count/Viable Count
Oils were added to apple juice samples according to their calculated MIC. Oils added to apple juice samples containing E. coli showed a decline in the number of bacteria in decreasing order: Mentha virdis, Mentha longifolia and limonene. Regarding the apple juices containing S. aureus and C. albicans, Mentha virdis, Mentha longifolia, and Limonene showed an increased number of bacteria at day 1, which declined by days 5 and 7 to less than 30 CFU (Supplementary Table S2). Other oils did not show any remarkable activity where the bacterial load increased to more than 300 CFU by time. In this study, pasteurized apple juice was expected to have a good initial microbiological quality. All essential oils concentrations added to the juice were selected according to the calculated MIC for each oil. All experiments were assessed at room temperature where the sensitivities of E. coli, S. aureus, and C. albicans to essential oils were in the following decreasing order: Mentha virdis, M. longifolia. and limonene.

Physical and Chemical (GC-MS) Analyses of Essential Oils
The physical properties "specific gravity, relative density, refractive index" in addition to oil appearance, color, and odor of EOs of Cymbopogon citratus "lemon grass", Thymus vulgaris "thyme" and Mentha virdis "mentha" volatiles have been investigated and summarized in Table 3. Table 3. The physical properties of essential oils (average of 3 independent experiments ± SD) of Cymbopogon citratus "lemon grass", Thymus vulgaris "thyme" and Mentha virdis "mentha" volatiles. Upon an GC-MS chemical analysis of volatile oils prepared from Cymbopogon citratus (Poaceae), Thymus vulgaris (Lamiaceae), and Mentha virdis (Lamiaceae), a total of 24, 51, and 43 compounds have been identified in Cymbopogon citratus, Thymus vulgaris, and Mentha virdis volatile oils, respectively. It was observed that in all prepared essential oils, the major class was monoterpenes followed by sesquiterpenes. In both Cymbopogon citratus and Mentha virdis oils, the monoterpenes percentiles were 97.41% and 97.61%, respectively, followed by Thymus vulgaris 93.04%. The total sesquiterpenes in the three analyzed oils were 4.93, 2.4, and 0.89% for Thymus vulgaris, Mentha virdis and Cymbopogon citratus herbs, respectively. In Cymbopogon citratus oil, the monoterpenes class exemplified by dominance of geranial 36.35%, followed by neral 35.00%, representing monoterpene aldehydes, then β-myrcene monoterpene hydrocarbon 11.7% as presented in Figure 2a and Table 4. These results complied with previous studies representing almost the same percentiles where the identification of those monoterpenes as majors in the volatile oil of Cymbopogon citratus herb cultivated in Cameroon [30]. In a previous article, it was proved the potential efficacy of Cymbopogon citratus oil and its main citral "Geranial" being a potent antimicrobial agent against polymicrobial biofilm forming bacteria viz. Staphylococcus aureus and Candida species. The underlying mechanism of action was refereed to its citral volatile ingredient through reducing the biofilm mass and cell viability by interfering of nucleic acids, proteins, and carbohydrates of the biomass that lead to deformity of the biomass matrix in addition to disruptions to the biomass adhesive characters. It worth mentioning that the enrichment of the Egyptian Cymbopogon citratus oil with the citral volatile ingredient of this current study (36.35%) than that mentioned in Gao, et al., 2020 (29.364%) [31]. In Thymus vulgaris volatile oil, the identified major monoterpenes were eucalyptol as monoterpenoid oxide 24.30%, thymol monoterpenoid phenol 17.40%, and γ-terpinene monoterpene hydrocarbon 15.20% shown in Figure 2b and Table 4 where similar percentiles of the major monoterpene class except eucalyptol have been recorded in the previous literature [32]. The enrichment of Thymus vulgaris volatile oil with eucalyptol 24.30% counted for its potent antimicrobial activity against a myriad of pathogens as previously mentioned [33]. In a published article by Sienkiewicz et al., 2011, the authors tested the antimicrobial activity of Thymus vulgaris oil against a myriad of clinically multidrug resistant strains of Staphylococcus, Enterococcus, Escherichia, and Pseudomonas genus. The results revealed the strong antimicrobial activity of the tested thyme oil against multidrug resistant microbes previously mentioned. The authors would relate the bioactivity of thyme oil to its main volatile ingredients p-cymene and thymol recording 29.10 and 38.10%, respectively [34]. The GC-MS analysis of the Egyptian Thymus vulgaris oil in this current study revealed its enrichment with eucalyptol (1,8 cineole) by (24.3%), in addition to thymol (17.4%). Eucalyptol itself possessed potent antimicrobial activity as reported in previous works [34,35]. The volatile oil of Mentha virdis enriched with mainly carvone "monoterpene ketone" 42.50% followed by eucalyptol "monoterpene oxide" 17.40% then finally dihydrocarveol "monoterpene alcohol" 13.00% as in Figure 2c and Table 4 where these data matched with the previous literature by Mkaddem et al., 2022 [36] upon which an analysis of the oil obtained from Mentha virdis collected from Tunisia. In a previous article by Mkaddem et al., 2022, Mentha virdis oil was rich with carvone (50.47%), eucalyptol (9.14%), and limonene (4.87%) which encountered the potent antimicrobial activity of the oil against Listeria monocytogenes and Klebsiella pneumoniae bacteria [36]. It is worth mentioning in this previous study that despite the enrichment of Mentha virdis oil by carvone up to 50.47%, still the oil obtained from the Egyptian Mentha virdis was richer with other oxygenated monoterpenes "eucalyptol 17.40% and dihydrocarveol 13.00%" than the Tunisian one. The major volatile structures of each oil are illustrated in Supplementary Figure S1.

Discussion
E. coli, S. aureus, and C. albicans are common pathogens causing serious systemic infections in humans. Since the continuous development of antimicrobial resistance, natural products and essential oils have been studied as alternatives for the treatment of infections acquired in healthcare [16,37,38].
Essential oils are important sources of new antimicrobial agents particularly against bacterial pathogens. In vitro studies in this research demonstrated that the tested essential oils inhibited microbial growth with variable effectiveness [31].
The tested oils exhibited concentration dependent inhibition of growth and the MIC of oils ranged from 0.1 to 100 µL/mL. MBC/MFC was assessed to demonstrate the least concentration of oils resulting in microbial viability reduction of 99.90% of the initial count. The MBC readings were double the MICs, which indicates that the bactericidal activities of the oils occur at concentrations higher than its growth inhibitory concentrations.
It has been observed the enrichment of the analyzed volatiles of lemon grass, thyme and mentha with oxygenated monoterpenes mainly geranial and neral with a total percent of 74.34%, eucalyptol and thymol with total amount of 41.70% whereas carvone, eucalyptol, and dihydrocarveol with a total content of 72.80%, respectively. Volatile oils enriched with oxygenated monoterpenes encountered the oil as being more potent as antimicrobial rather than monoterpene hydrocarbons [39]. Allenspach et al., 2020 in a recent article for absolute quantification of terpenes in conifer species, the authors implemented a validated simple method for quantification of different terpenes viz. α and β-pinenes, camphene, 3-carene, limonene, bornyl acetate, β-caryophyllene, and borneol. Antibacterial activity of conifer essential oil proved its efficacy on both E-coli and S. aureus as gram-negative and positive bacteria, respectively [40]. In this current study, the GC-MS analysis proved the presence of such terpenes although in low percentile as listed in Table 3, still may be related to the antibacterial activity of the tested oils in this study. It is worth mentioning that previous studies worked on the mechanism of actions being antibacterial for each component in the volatile oil. In a previous work by Oz, et al., 2015, it was mentioned that the presence of an aromatic ring and a polar functional group of a volatile constituent "thymol" could lead to rupture of the bacterial cell membrane leading to release of the vital cell constituents [41]. A previous published review by Wińska et al., 2019 mentioned the effectiveness of essential oils as antimicrobial agents, specifically thyme and mentha in context to their volatile chemical profiling and major phytochemical ingredients. The antimicrobial activity of thyme would be referred to its enrichment with thymol (36-55%) and p-cymene (15-28%) whereas mentha antimicrobial activity referred to its higher percentile of menthol (30-55%) and menthone (14-32%) [42]. Citral "neral and geranial isomers", have been approved by the U.S Food and Drug administration as being safe, so its use as natural preservative and flavoring agent due to its antibacterial activity against gram-negative and positive bacteria as Escherichia coli and Staphylococcus aureus, respectively [43,44]. Eucalyptol "1,8 cineole" antibacterial mechanism was summarized in a previous review where its effect mainly was due to changes in both size and shape of gram-positive and gram negative bacterial cell which ends with apoptosis [45]. α-pinene was detected in both thyme and mentha oils ca. 1.68 and 1.2%, respectively. In a recent published review by Allenspach and Steuer, 2021, the authors summarized the different biological activities of α pinene, of which they mentioned its positive antimicrobial activity on both gram-positive and negative bacteria viz. methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), and antifungal activity against Candida species [46]. Concerning carvone antibacterial mechanism of action, in a previous study, carvone enriched in the oil of Mentha spicata causes instability of phospholipid bilayer structure as well as interacts between the bacterial membrane enzymes and proteins [47].
Finally, the volumes of oils added ranged from 0.8 µL to 400 µL in 5 mL juice (with a range from 0.016% to 8%) (Supplementary Table S1). Concerning the taste of drinks after the addition of the EOs; EOs improve the flavor, odor, and color when added to foods. Many individual EOs are approved food flavorings and impart a certain flavor to foods, as well as delaying food spoilage without changing the organoleptic properties of the food. However, certain strategies could be implemented to decrease the organoleptic effects, if found, by optimizing the food/beverage formulation or by combining the essential oils and/or their active constituents with other means of sterilization such as pH or heat treatment (when applicable) [48].

Conclusions
Chemical preservatives have been utilized as food preservatives, but they turned to have undesirable biological effects on humans and increase in microbial resistance. This study demonstrates that natural volatile oils extracted from plants exhibited a concentrationdependent inhibition of microbial growth and offer potential antimicrobial activity against common food spoilage bacteria and fungi. For future research, the authors would encourage a larger scale comparative study of natural essential oils to chemical ones commonly used in the market as well as preparing a commercial naturally preserved effective product to replace chemical preservatives.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12101587/s1, Table S1: Oils were added with concentrations as shown in the table based on their MIC results. Table S2: Mean readings of colonies count on days 1, 5 and 7. Figure S1: Major identified volatiles in herbs of Cymbopogon citratus, Thymus vulgaris and Mentha virdis.