ESSENTIAL OILS AND THEIR APPLICATION IN A FOOD MODEL

The aim of the study was to investigate the chemical composition, antioxidant, and antimicrobial activity of essential oils (Canarium luzonicum CLEO, Melaleuca leucadenron MLEO, Amyris balsamifera ABEO). There was Gas chromatographic-mass spectrometric analysis used for the characteristic of the semiquantitative composition of the essential oils. The DPPH method was used to determine the antioxidant activity. Minimum inhibitory concentrations (MIC) of essential oils against Stenotrophomonas maltophilia were analyzed in a 96-well plate. The broth microdilution method was used for the minimal inhibitory concentration. A gas-phase antimicrobial assay was used to determine inhibitory concentrations in a food model. CLEO proved to be the best with the lowest MIC 50 and 90 of 6.67 μL.mL-1 respectively 6.81 μL.mL-1 and antioxidant activity of 33.43% among the tested essential oils. The main volatile compounds CLEO were limonene 36.38%, elemol 16.65%, α-fellandren 12.18% and elemicin 9.59%. It showed inhibition of S. maltophilia growth in the food model at the lowest concentrations among the essential oils.


INTRODUCTION
In recent years, natural substances have come to the fore due to their low toxicity, pharmacological effects, and economic advantage (Dias, Urban, and Roessner, 2012).
Elemi (Canarium luzonicum) essential oil comes from an evergreen tree that reaches a height of more than 30 meters and a trunk diameter of more than 1 meter. C. luzonicum naturally occurs in the Philippines (Barwick, Schans and Claudy, 2004).
Oleoresin is one of the aromatic components. Therefore, it has a wide range of uses in the pharmaceutical and food industries. It is also used for its rubefic, expectorant, antifungal, antibacterial and antirheumatic effects (Nikolic et al., 2016).
Kajeput essential oil (Melaleuca leucadendron) is used for its antifungal, antiviral, antibacterial, antiseptic, and anti-inflammatory effects. The plant occurs predominantly in Indonesia (Pujiarti, Ohtani and Ichiura, 2011). Many of the compounds present in this plant are considered to be bioactive substances (Cleber et al., 2007).
Amyris, essential oil (Amyris balsamifera) comes from always green small trees and it has high flammability. It occurs in the Caribbean and near the Gulf of Mexico (Rohmer, Schwartz and Anton, 2012). Amyris is rich in sesquiterpene alcohol. It has antiseptic effects (Khan and Abourashed, 2009).
Stenotrophomonas maltophilia is a non-fermentative, gram-negative, aerobic bacillus. These bacteria can form biofilm structures. It is most often found in raw milk, vegetables, fruits and fish products regarding the food industry (An and Berg, 2018). We aimed to determine chemical composition, antioxidant activity, and minimal inhibitory concentrations of these essential oils against the bacterium Stenotrophomonas maltophilia.
Another aim was also to evaluate the inhibitory effect of essential oils against S. maltophilia from the surface of carrots, potatoes and apples by using a vapor phase antimicrobial test.

Scientific hypothesis
We assume the presence of biologically active substances and the antioxidant potential of essential oils. Given the available literature, we assume the inhibitory effect of essential oils on the bacteria Stenotrophomonas maltophilia. We believe that essential oils could also have an inhibitory effect in the vapor phase.

Microorganism
Bacteria Stenotrophomonas maltophilia was got from the dairy industry. It was identified by 16S rRNA sequencing and MALDI-TOF MS Biotyper.

Chemical Composition of Essential Oils
There was Gas chromatographic-mass spectrometric (GC-MS, Agilent 7890B, Agilent 5977A, Agilent Technologies Inc., Palo Alto, CA, USA) and CombiPal autosampler 120 (CTC Analytics AG, Zwingen, Switzerland) parsing test of the essential oil used as well as in a previous study (Kačániová et al., 2020). The results were set as the average mean and standard deviation of three repeated measurements.

Radical Scavenging Activity-DPPH Method
The activity of capturing free radicals with essential oil was determined in the same way as in the study of Kačániová et al. (2020) by using 2,2-diphenyl-1picrylhydrazyl (DPPH, Sigma Aldrich, Germany) method.

Minimum Inhibitory Concentration (MIC)
The bacterial culture was cultivated in the Muller Hinton broth (MHB, Oxoid, Basingstoke, UK)) at 37 °C for 24 h. One hundred µL inoculum with a density of 0.5 McF was inoculated into each well of a 96-well microtitration plate. There was 100 µl of the essential oil with a concentration from 0.3125 µL to 10 µL per well added after inoculation. Negative control contended a mixture of MHB with essential oil, while a mix of MHB with bacterial inoculum was used as a control of maximum growth (Hassan et al. 2011). The absorbance was measured and evaluated in the same way as it was in the study of Kačániová et al. (2020). The experiment was carried out in three repeated measurements.

Statistical analysis
The measurements were repeated three times. Statistical variability of the data was processed with Microsoft™ Excel® software.

Minimum Inhibitory Concentration (MIC)
We determined the MIC 50 and 90 CLEO for S. maltophilia to be 6.67 µL.mL -1 respectively 6.81 µL.mL -1 by using an agar microdilution method.

In Situ Antimicrobial Effect on Vegetables and Fruit
The antimicrobial study of essential oils was determined by an in situ method. CLEO inhibited the growth of S. maltophilia on the surface of carrots and potatoes at a concentration of 250 mg/ml. Inhibition for apple was recorded at a concentration of 125 mg.mL -1 (Figure 1). MLEO showed inhibition of S. maltophilia growth on carrots at a concentration of 500 mg.mL -1 , on potato and apple surface at 250 mg.mL -1 (Figure 2). ABEO inhibited bacterial growth at a concentration of 125 mg.mL -1 per carrot, 250 mg.mL -1 per apple and up to 500 mg.mL -1 per potato (Figure 3).

CONCLUSION
The results of our work demonstrated the inhibitory effect of essential oils on S. maltophila in a food model. CLEO proved to be the best with the lowest MIC of 6.67 µL.mL -1 and antioxidant activity of 33.43% among the tested essential oils. It showed inhibition of S. maltophilia growth in the food model at the lowest concentrations among the essential oils.